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by Patricia V.Johnston

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basic lipid methodology

Patricia V.Johnston

Assistant Professor of Food Science

Special Publication 19

January, 1971

College of Agriculture

University of Illinois at Urbana-Champaign

INTRODUCTION

-he size and diverse nature of the group of compounds known as
L the lipids preclude the coverage of their chemistry to any extent in
elementary courses in organic chemistry; this vast group is, in general,
considered to be a field of study in itself. This publication has been pre-
pared as a laboratory handbook for those with little previous experience
in lipid analysis. Therefore, while a knowledge of basic organic chemis-
try is assumed, previous knowledge of lipid chemistry is not. The first
chapter is an introduction to lipid chemistry. It includes definitions,
classification, and basic structures of lipids. The rest of the book deals
with the preparation and care of samples for lipid analysis and pro-
cedures for the analysis of commonly occurring lipids. A knowledge of
newer techniques basic to the study of lipids is not assumed; therefore,
the book also serves as an introduction to column, thin-layer, and gas-
liquid chromatography.

Numerous references to original research papers are not given since
this work is not intended to be an exhaustive account of all the methods
available for the analysis of lipids. Rather, it is a personal account of
methods tried, tested, and known to work. A few references to research
papers are included where pertinent, and a selected bibliography is
designed to lead the interested reader to a wider range of methods
and applications. Analyses of only the more commonly encountered
lipids are described, but the Suggested Further Readings includes some
works dealing with the analysis of the less common lipid subclasses.

Patricia V. Johnston is Research Assistant
Professor in the Children's Research Center and
Assistant Professor of Food Science. She is also
a member of the Nutritional Sciences Faculty.
Developmental neuro chemistry and neurobiology
arc Dr. Johnston's main research interests. She
is especially interested in pre- and postnatal con-
ditions, such as malnutrition and drug therapy,
which may affect the development of the hptd-
rich myelin sheath of nerve cells.
This book was written in part while Dr. Johnston
was supported by funds from the Hatch NC74
committee.
The figure on the cover is a mono sialoganglio side.

TABLE OF CONTENTS

Introduction ii

I. LIPIDS: DEFINITIONS, CLASSIFICATION,
NOMENCLATURE I

Simple lipids 1

Neutral glycerides 1

Esters of fatty alcohols 3

Derived lipids 4

Glyceryl ethers 8

Phosphoglycerides 8

Phosphatidic acids, phosphatidyl glycerols, and

polyglycerophosphatides 10

Phosphatidyl ethanolamines, cholines, serines, and inositols. . . 10

Lysophosphoglycerides 12

Plasmalogens 12

Phosphonolipids 13

Sphingolipids 14

Ceramides 14

Sphingomyelin 15

Glycolipids: cerebrosides and sulfatides 15

Gangliosides 16

II. PREPARATION AND HANDLING OF SAMPLES FOR

ANALYSIS OF LIPID CONSTITUENTS 19

General techniques in lipid chemistry

Prevention of oxidation 19

Elimination of possible contaminants 20

Unwanted emulsions and other hazards in lipid chemistry ... 21

Extraction of lipids from various sources 22

From blood serum 22

From erythrocytes 24

From brain 25

From other sources 26

Removal of nonlipid contaminants from extracts

Aqueous washing 26

Treatment on cellulose and Sephadex columns 27

III. COLUMN CHROMATOGRAPHY 30

Solid-liquid adsorption chromatography 30

Liquid-liquid partition chromatography 31

Preparation of columns 31

Silicic acid columns 34

Initial separation: neutral lipids from phospho-

and glycolipids 35

Separation of neutral lipids 35

Separation of polar lipids 36

Florisil columns 37

Diethylaminoethyl (DEAE) and trimethylaminoethyl (TEAE)

cellulose columns 37

Separation of lipid samples into acidic and

nonacidic fractions 39

A general elution scheme 39

IV. THIN-LAYER AND PAPER CHROMATOGRAPHY 42

Thin-layer chromatography 42

Preparation of thin-layer chromatographic plates 42

Detection of lipids on chromatograms 45

Separation of neutral lipids 48

Argentation thin-layer chromatography 51

Separation of phospholipids and glycolipids 52

Quantitative thin-layer chromatography 53

Paper chromatography 59

V. GAS-LIQUID CHROMATOGRAPHY 61

Instrumentation 61

Qualitative analysis 66

Quantitative analysis 69

Chemical modification of compounds for analysis by GLC 75

Analysis of methyl esters of fatty acids 75

Analysis of fatty aldehydes 78

Analysis of glycerides 81

Analysis of other lipids and components derived from lipids. . . 82

Pyrolysis-GLC 82

VI. PROCEDURES FOR THE DETERMINATION OF SPECIFIC

ELEMENTS, FUNCTIONAL GROUPS, AND LIPID CLASSES 84

Determination of organic phosphorus 84

Determination of cholesterol

By spectrophotometry 85

By gas-liquid chromatography 87

Determination of glycolipid sugars

By using anthrone 89

By gas-liquid chromatography 90

Determination of N-acetylneuraminic acid (to determine gangliosides)

By using resorcinol 90

By gas-liquid chromatography 91

Determination of plasmalogens

By colorimetry 92

By two-dimensional thin-layer chromatography 93

Determination of the amount of trans double bond

By infrared spectrophotometry 94

By gas-liquid chromatography 97

Suggested Further Readings 99

Index 100

I. Lipids: Definitions, Classification,
Nomenclature

The term lipid describes a large group of compounds of a chem-
ically diverse nature. This book employs a simple definition and
classification system, as no system has been universally accepted. Lipids
may be denned as compounds found in living organisms and generally
insoluble in water but soluble in organic solvents. There are exceptions
to this definition since some lipids are sparingly soluble in water while
others are soluble in a very limited number of organic solvents. In
general, however, this definition holds.

Three major lipid classes exist, namely the simple lipids and the two
general groups of polar lipids, the glycerophosphatides and the sphingo-
lipids. We shall consider each of these classes individually. Examples of
the structural formulae of the lipids mentioned are shown.

SIMPLE LIPIDS

These compounds comprise the neutral lipids such as the glycerides,
esters of fatty alcohols, and lipids derived from these compounds by
alkaline or acid hydrolysis.

Neutral Glycerides

The neutral glycerides are fatty acid esters of glycerol, and although
the most abundant are the triglycerides, mono- and diglycerides do occur
naturally. The glycerides are named according to the position of the
fatty acid substituent on the glycerol moiety; thus two isomers of mono-
and diglycerides exist. In a triglyceride all the glyceride hydroxy!
groups are esterified.

0
< H2C-OH H2C-0-C-R

0 "2,

/3 HC-O-C-R HC-OH

I i

<L' H2C-OH H2C-OH

monoglyceride, /?-form monoglyceride, a-form

LIPIDS: DEFINITIONS

H2C - 0 " C " R,

i o

HC - 0 - C - R2

i
H2C-OH

diglyceride, «,/3-form

H2C-0-C- R,
I
HC - OH
, o

H2C - 0 " C " R2

diglyceride, «,«'-form

H2C-0-C-R,
, 0

HC-0-C-R2
i

H0C-O-C-R3
11
0

triglyceride

The carbons of the glycerol moiety are often designated 1, 2, and 3
instead of a, p, and a'. Ri, R2, and R3 refer to the alkyl chain of the
esterifying fatty acids. In general, glycerides are named by the trivial
name for the fatty acids. For example, if stearic acid, CH3(CH2)i6-
COOH, is the esterifying fatty acid, we can have:

H2C "OH
, 0

HC -0- C(CH2),6CH3
1
H2C "OH

2 (or /3)-monostearin

0
H2C-0-C(CH2)l6CH3

HC "OH
1

H2C-0H

1 (or «)-monostearin

0
H2C-0-C(CH2),6CH3

1 0
HC-0-C(CH2),6CH3

1
H2C-0H

1,2 (or a,/?)-distearin

0
H2C-0-C(CH2)l6CH3

1
HC-OH

« 0

H2C-0-C(CH2)l6CH3

1,3 (or a,a')-distearin

H2C-0-C(CH2),6CH3

HC-0-C(CH2),6CH3
1

H2C-0-C(CH2),6CH3
11
0

tristearm

CLASSIFICATION, NOMENCLATURE 3

_ When the fatty acid composition is mixed, the position of each fatty
acid is specified when known. Thus, introducing oleic CH3(CH2)7-
CH = CH(CH2)7COOH and palmitic CH3(CH2)14 COOH acids,
we can have as examples:

H2C-0-C(CH2)l4CH3

HC -0 " C(CH2)7 CH= CH(CH2)7CH3

0
H2C*0H

l-palmitoyl-2-olein

H2C-0-C(CH2),4CH3

HC-0-C(CH2)l4CH3
I

H2C-0-C(CH2)7CH = CH(CH2)7CH3

1,2-dipalmitoyl olein

H2C-0-C(CH2)7CH= CH(CH2)7CH,

HC-OH
• 0

H2C-0-C(CH2),4CH3
l-oleoyl-3-palmitin

H2C-0-C(CH2),4CH3
I 0

HC-0-C(CH2),6CH3

H2C - 0 - C(CH2)7CH= CH(CH2)7CH3
6

l-palmitoyl-2-stearoyl-3-olein, or
(since the 1 and 3 positions are
equivalent) l-oleoyl-2-stearoyl-
3-palmitin

When the fatty acids are more complicated and not generally known
by any trivial name, they must of course be named systematically. The
generally accepted nomenclature for fatty acids is described under
Derived Lipids (p. 4).

Esters of Fatty Alcohols

Alcohols that are relatively insoluble in water but are soluble in
organic solvents fall under the class of fatty (or lipid) alcohols. This
class, therefore, includes long-chain aliphatic alcohols (more than 8 car-
bons), aromatic alcohols such as cholesterol, and other steroids, includ-
ing the vitamin D group. Vitamins A and E are also lipid alcohols, but
because of the vast literature devoted to the vitamins we shall not
discuss them further here. Cholesterol is the alcohol with which we shall
be most concerned. Cholesterol exists in nature in the esterified state and
in the free form. The stearic acid ester of cholesterol is shown (p. 4).

The esters of long-chain alcohols and fatty acids are known as
waxes; natural waxes such as beeswax and wool waxes are mixtures of

LIPIDS: DEFINITIONS

CH3 CH3

CH"CH2"CH2*CH2"CH
CH3

CH3(CH2),6C-0^N/<^

cholesterol ester

these esters. Trace amounts of other components, such as hydrocarbons,
are also found in natural waxes.

Derived Lipids

If the products obtained on hydrolysis of a lipid are soluble in
organic solvents and relatively insoluble in water, they are termed
derived lipids. From our point of view, the most important are the fatty
acids. We shall now discuss their structure and nomenclature in some
detail. For the most part, the fatty acids in mammalian tissues and body
fluids are straight chain and contain an even number of carbon atoms.
Hydroxy, keto, and branch chain fatty acids also occur; indeed, the
nervous system of mammals contains considerable amounts of 2(a)-
hydroxylated fatty acids. Most fatty acids found in tissues are in ester
or amide linkages, but small amounts of free fatty acids (FFA) do
occur and are of great importance metabolically both in supplying
energy and in lipid biosynthesis.

It is usual to divide the fatty acids into two main classes, saturated
and unsaturated. Since aliphatic acids are regarded as derivatives of the
hydrocarbons which have the same number of carbon atoms, the names
of fatty acids are derived from the appropriate parent hydrocarbon.
Saturated fatty acids are named, according to the modified Geneva
system, by replacing the terminal "e" of the parent hydrocarbon name
with the suffix "oic." Thus, the saturated fatty acid that is related to the
hydrocarbon octane, CH3(CH2)GCH3, is known as octanoic acid.
Most fatty acids, however, were known and named before the Geneva
convention, and the use of their non-systematic ("trivial") names per-
sists. The accompanying tables of fatty acids, therefore, include the
trivial names as well as the systematic names. Some commonly oc-
curring saturated fatty acids are shown in Table 1.

The simplest unsaturated fatty acids have the empirical formula
CnH2n_202; that is, they contain only one double bond. They are named
by replacing the "e" of the corresponding unsaturated hydrocarbon with
the suffix "oic"; thus one has octenoic acid, decenoic acid, and so on.
These fatty acids are termed "monoenoic." Fatty acids with two, three,
four, five, and more double bonds are named by taking the stem of the

CLASSIFICATION, NOMENCLATURE
Table 7. — Some Saturated Fatty Acids

Systematic

Trivial name

Formula

Butanoic

Hexanoic

Octanoic

Decanoic

Dodecanoic

Tetradecanoic

Hexadecanoic

Octadecanoic

Eicosanoic

Docosanoic

Tetracosanoic

Butyric

Caproic

Caprylic

Capric

Laurie

Myristic

Palmitic

Stearic

Arachidic

Behenic

Lignoceric

CH3(CH2)2COOH
CHaCCH^COOH
CH3(CH2)6COOH
CH3(CH2)8COOH
CH3(CH2)10COOH
CH3(CH2)12COOH
CH3(CH2)14COOH
CH3(CH2)16COOH
CH3(CH2)18COOH
CH3(CH2)20COOH
CH3(CH2)22COOH

name of corresponding hydrocarbon, octa-, deca-, etc., and adding the
appropriate ending: "-dienoic" (2 double bonds), "-trienoic" (3 double
bonds), etc. Fatty acids with multiple double bonds are referred to
collectively as polyunsaturated fatty acids (PUFA) and individually
as dienoic, tnenoic, tetraenoic, pentaenoic, and hexaenoic fatty acids
(see Table 2). A name must also, of course, designate both the position
of the double bonds along the chain and their geometric configuration:
cis or trans. Double bonds are assumed to be cis, unless a statement is
made to the contrary. This convention has been adopted because most
naturally occurring fatty acids have their double bonds in the cis con-
figuration; moreover, most common trans isomers have a different trivial
name which is used, so the problem is often completely circumvented.
For example, octadecenoic acid with a double bond on the 9th carbon
has both cis and trans forms. The cis form has the trivial name of
oleic acid, and the trans form is known as elaidic acid. Similarly, when

HC(CH2)7CH3
ii

HC(CH2)7COOH
cis form : oleic acid

CH3(CH2)7CH
H

HC(CH2)7C00H
trans form: elaidic acid

both double bonds of octadecadienoic acid are trans, the acid is
termed linolelaidic acid. While trans fatty acids are rare as natural
constituents, they do occur naturally in some plants and animals- more-
over, when included in the diet, they are deposited in body tissues (1).
They are, therefore, nutritionally significant. A further discussion of
trans fatty acids is included in the description of their analysis (see
p. 94) . J v

While the naming of geometric isomers is relatively straightforward,
the designation of double bond position is confused by the fact that

LIPIDS: DEFINITIONS

c
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* — j

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•*»

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CLASSIFICATION, NOMENCLATURE 7

several conventions exist. To be able to follow the literature, one must
know all the conventions. In the simplest procedure the terminal car-
boxyl group carbon is number 1 and the rest of the chain is 2, 3, 4, etc.
Thus palmitoleic acid named systematically becomes 9-hexadecenoic

9 1

acid, CH3(CH2)5CH = CH(CH2)7COOH. However, especially in
older literature, lengthier conventions are used. The Greek letter A is
used to indicate the presence of double bonds, and both carbon atoms
participating in the bonds are indicated. Palmitoleic acid then be-
comes A9»10-hexadecenoic acid. A9-hexadecenoic acid is also used.

There is a growing tendency to use a shorthand designation for fatty
acids. This is very useful especially when long lists of fatty acids are
given as, for example, in the complete gas chromatographic analysis of
the fatty acids of natural lipids. In this convention the Greek letter o>
is used to refer to the terminal carbon atom away from the carboxyl
group. The position of the double bond is then indicated with respect
to the o) carbon. Palmitoleic acid is, therefore, the w 6-hexadecenoic acid.
The length of the carbon chain and the number of double bonds is indi-
cated by the shorthand form C16:l, where the number before the colon
gives the chain length and the number after the colon indicates the
number of double bonds. Palmitoleic acid is, therefore, designated
C16:lo)6.

When there is more than one double bond a question arises ;
namely, is it sufficient to define the position of only one double bond
with respect to the w carbon atom? To answer this, we must con-
sider still another way of classifying fatty acids. Polyenoic acids may
be classified as conjugated or unconjugated (nonconjugated is also
used) depending on the relative position of the double bonds. If the
double bonds are separated by one or more single-bonded carbon atoms,
— C = C— Cn — C = C— , the acid is said to be unconjugated.
When double-bonded carbon atoms are adjacent to each other, — C
= C — C = C —, the acid is termed conjugated. Fatty acids from
mammalian sources are usually unconjugated. Double bonds are usually
separated by one single-bonded carbon atom; that is, by a single meth-
ylene ( — CH2 — ) group. It is, therefore, perfectly correct in such cases
to define double bond position by reference to the first double bond from
the a) carbon atom. Thus, the commonly occurring mammalian fatty
acids, 5, 8, 11-eicosatrienoic acid and 5, 8, 11, 14-eicosatetraenoic
(arachidonic) acid, can be referred to as 20:3 w 6 and 20:4 w 6 respec-
tively. If, however, the double bonds are conjugated, then reference
must be made to this fact. If the acid is unconjugated but double bonds
are separated by more than one single-bonded carbon atom, then clearly
one of the lengthier descriptions of the structure must be used.

The fatty acids listed in Tables 1 and 2 occur widely in nature, as do
numerous others. Linoleic acid is of great importance to mammals: it

8 LIPIDS: DEFINITIONS

must be included in their diets to prevent a deficiency syndrome since
they cannot synthesize it. For this reason linoleic acid is called an essen-
tial fatty acid (EFA). Arachidonic and y-linolenic acids, synthesized in
mammals from linoleic acid, are also sometimes termed essential fatty
acids since they protect and cure the EFA deficiency syndrome.

Hydroxy, keto, branched chain, and cyclic fatty acids all exist. Ex-
amples of such acids and a source of each are shown in Table 3. The
only hydroxy fatty acids of importance in mammals are the 2(a)-
hydroxy substituted fatty acids found in cerebrosides and sulfatides
(see p. 15). Some hydroxy and branched chain acids may be ingested
by man in oils from plant sources ( such as ricinoleic acid in castor oil) ,
and small quantities of hydroxy and keto acids may also be present in
man's diet due to oxidative breakdown of fats (2,3).

Glyceryl Ethers

Neutral lipids with ether or vinyl ether linkages have been isolated
and characterized. In these lipids an ester-linked fatty acid of a glyc-
eride is replaced by a vinyl ether («,/? unsaturated) linkage or an
ether linkage. Thus, one may have a aj-l-(alkenyl) ether of a 2,3-
diacyl glycerol with the general formula:

H2Ct 0 ~ CH = CH 4 R, vinyl ether linkage

R2-C-0-CH

> u

H2C-0-C-R3

and glycerol ethers with the formula :

o ,

H2C TOtR| ether linkage

Rz-C-0-CH Q

H2C-0-C-R3

These lipids are of importance in fish and many invertebrates (4).

PHOSPHOGLYCERIDES

Phosphoglycerides are defined as lipids which, on hydrolysis, pro-
duce derived lipids plus inorganic phosphate and glycerol.

CLASSIFICATION, NOMENCLATURE

'3.

c
c

o
o
o

X X

o-o

CSJ

o
o
o

X X

o-o

CO bo

o
o
o
^cvj

CSJ

cv)

CSJ

as o

O
O

o
o
o

O w
X X

o
o
o

"*J

CSJ <->
X ii

? X

JO LIPIDS: DEFINITIONS

Phosphatide Acids, Phosphatidyl Glycerols,
and Polyglycerophosphatides

The simplest phosphoglycerides are the phosphatidic acids. In these
lipids two of the glycerol —OH groups are esterfied with fatty acyl
groups and the third with phosphoric acid (see also phosphonolipids, p.
13). Closely related to the phosphatidic acids are the phosphatidyl glyc-
erols and the polyglycerophosphatides. Cardiolipin, a polyglycerophos-
phatide, occurs mainly as a mitochondrial lipid.

9 o

* 0 H2C-0-C-R, 0 H2C-0-C-R,

•• ' H2C-OH

r-n-ru ci

a r2-c-o-ch 0 r2-c-o-ch ^ H

A' H2C-0-P-OH H2C-0-P-0-CH2

OH OH

phosphatidic acid phosphatidyl glycerol

H2C-0-C-R,
0 , H2C-0-P-0-CH2 Q

R2-C-0"CH HC'OH 0H HC-O-C-R3

H2C-0" P-0-CH2 H2C-0-C-R4

1 11

OH 0

cardiolipin

Phosphatidyl Ethanolamines, Cholines, Serines, and Inositols

This subclass contains some of the most abundantly occurring phos-
pholipids. In these compounds the phosphoric acid group of the parent
compound, a phosphatidic acid, is linked to either ethanolamine, choline,
serine, or myoinositol. At least three subclasses of phosphoinositide
exist, differing in the number of phosphoric acid residues they contain.

Phosphatidyl choline is known by the generally accepted trivial
name of lecithin. Phosphatidyl ethanolamine, however, should not be
termed "cephalin," as this trivial name originally referred to a mixture
and has since lost its meaning.

It will be noted that the phosphoglycerides are amphiphilic com-
pounds; that is, one end of the molecule consists of a hydrophobic region
of long-chain alkyl groups while the other end is hydrophilic, consisting
of the ionic phosphoryl group and its esterifying molecule. Note also
that the /? carbon of the glycerol moiety is asymmetrical. Phosphoglyc-

CLASSIFICATION, NOMENCLATURE

N+HZ

Q H2C-0-C-R,
R2 " C - 0 - CH n

H2C-0-P-0-CH2-CH2
0

phosphatidyl ethanolamine

0
0 H2C-0-C-R,

R2-C-0"CH

H2C-0-P-0-CH2-CH2-N4(CH3)3

phosphatidyl choline

0
ii

0
H2C-0-C-R,

R2-C-0-CH 0

1 H

H2C - 0 " P - 0 - CH2 -CH - COO'
i i

g nh3

phosphatidyl serine

brides have been found to have the same stereochemical configuration as
^-glycerophosphate. They are, therefore, more correctly termed L-«-
phosphatidyl enthanolamine, and so on. In general, unsaturated fatty
acids estenfy the p position in all phosphoglycerides and saturated fatty
acids estenfy the a position.

The three commonly occurring phosphoinositides are shown below
and on page 12. The monophosphorus compound, phosphatidyl inositol,

H OH R2"C"°-CH2

> L ' °

>h/5h ?Kh

fy h/)-P-q
OU r\Lj 0

hc-o-c-r-

I
-CH,

OH OH

phosphatidyl inositol

RfC-O-CHo
H OH
=°f0) 1 H

hN^ H/O-P-O-CH
OH OH 0

diphosphoinositide

HC"0-C-R2

12 LIPIDS: DEFINITIONS

OH OH

triphosphoinositide

occurs in skeletal and cardiac muscle ; the di- and triphosphoinositides
constitute a considerable proportion of the phospholipids in brain and
occur only as trace amounts in other tissues.

Lysophosphoglycerides

In lysoglycerophosphatides only one alcoholic group is esterified by
a fatty acid; the other one is free. The only common naturally occurring
member of this subclass is lysophosphatidyl choline (lysolecithin),
which usually has the /3-OH group free.

Lysolecithin occurs to an appreciable extent in blood serum. Between
6 and 10 percent of the total lipid phosphorus in human blood serum is
lysolecithin (5). There is evidence for the occurrence of the monoacyl
compounds of all the other phosphoglycerides and for isomeric forms
in which the a-OH group is free.

HgC-O-C-R,

i
HC-OH^

I 9 +

H2C-0-P-CH2-CH2-N(CH3)3

lysolecithin

Plasmalogens

Plasmalogens are monovinyl-ether, monofatty-acyl phosphoglyc-
erides. They are known generally by the trivial name plasmalogens and
occur in a wide range of tissues in most species. Typically, the fatty acyl
group at the a position is replaced by an alkenyl group so that in place
of the ester linkage there is a vinyl ether linkage. The most abundant
plasmalogen in a tissue is usually the ethanolamine derivative.

Plasmalogens are sometimes named by replacing the terminal "yl" of
the phosphatidyl analog by "al," so that we have phosphatidal ethanol-
amine, phosphatidal choline, etc. This method, however, is subject to
error due to the close similarity in the names and has not, therefore,
found universal acceptance. It is preferable when referring to plasma-

CLASSIFICATION, NOMENCLATURE 73

n H2CtO-CH=CHiR,

R2 -C-O-CH

H2C-0-P-0-CH2-CH2-NH2
0

ethanolamine plasmalogen

logens to use either "ethanolamine plasmalogen" or "vinyl ether phos-
phatidyl ethanolamine." These terms clearly distinguish the plasma-
logens from the diacyl phosphoglyceride. Still another type of phospho-
glyceride occurs. This is the relatively rare alkoxy glycerophosphatide
in which the a ester linkage is replaced by an ether linkage.

0 HaC-O-R,
R2 -C-O-CH 0

H2C-0-P-0-CH2-CH2-NH2
OH

alkoxy phosphatidyl ethanolamine

Phosphonolipids

Some years ago an entirely new group of phosphorus-containing
lipids was discovered by two groups of investigators, one studying the
lipids of the sea anemone, Anthopleura elegantissima (6), the other
studying the proteolipid fraction of ciliate protozoa of sheep rumen (7).
This group of lipids contains phosphonic acid in place of phosphoric
acid. The first clues to the existence of these lipids came with the isola-

O
tion of 2-aminoethylphosphonic acid (OH)2 — P— CH2 — CH2 — NH2,

O
suggesting that this replaced 2-aminoethylphosphoric acid (OH)2 — P
— O — CH2 — CH2 — NH2 in some lipids. A typical glycerol phosphono-
lipid would, therefore, have the structure:

0
H2C-0-C-R,

0 .

R2 "C-O-CH

H2C-0-P-CH2-CH2-NH2

dialkyl glyceryl-(2-aminoethyl)-phosphonate

j 4 LIPIDS: DEFINITIONS

Many types of phosphonolipids have now been isolated and char-
acterized, including sphingolipid and plasmalogen classes.

Phosphonolipids have also been synthesized; Baer and Stanacev
(8 9) have prepared glycerol phosphonolipids of the type on page 13
and of a type in which the glycerol is bound directly to the phosphorus
by a carbon-phosphorus bond.

0
0 H2C-0-C-R,

R2-C-0-CH0

H2C-P-0-CH2-CH2-NH2

More recently Chacko and Hanahan (10) have synthesized the
monoether phosphatidyl aminoethylphosphonate and have confirmed
the presence of both mono- and diester phosphonolipids in Tetrahymena
pyriformis (aciliate).

SPHINGOUPIDS

Sphingolipids are compounds which on hydrolysis yield sphingosine
(or a closely related compound), derived lipids, and water soluble
products. Unlike the complex lipids discussed so far, this group does
not contain the glycerol carbon skeleton. Rather, the common link is
sphingosine, an amino alcohol with the following structure:

3 2 I

CH3(CH2)I2CH = CH-CH-CH-CH

OH NH2 OH

sphingosine

Sphingosine carbons are numbered starting with the primary hy-
droxyl group. The configuration of the molecule at C2 is D, and the
relationship of C3 to C2 is erythro. The double bond has the trans con-
figuration. The systematic name for sphingosine is, therefore, D-
ery^ro-l,3-dihydroxy-2-amino-4-*raw.y-octadecene.

Molecules closely related to sphingosine, and which replace it m some
sphingolipids, include dihydrosphingosine (fully saturated sphingosine)
and C20 homologues of sphingosine.

Ceramides

The simplest and most fundamental sphingolipids are called ce-
ramides. In these compounds the amino group of sphingosine (or a
sphingosine-related compound) is in an amide linkage with a fatty acid.
Amide, ether, and vinyl ether linkages are stable to alkali while ester

CLASSIFICATION, NOMENCLATURE 15

CH3 (CH2)|2 CH = CH-CH-CH-CH2
OH NH OH
C^O
R

ceramide

linkages are readily broken by alkali. This difference is used to ad-
vantage in analytical procedures.

Sphingomyelin

Sphingomyelin is a phosphorus- and choline-containing sphingo-
lipid. The primary alcohol group of a ceramide is joined via an ester
linkage to phosphoric acid which is in turn esterified with choline.
Some 10 percent of the lipid phosphorus in brain is sphingomyelin;
kidney, spleen, erythrocytes, and plasma are also rich in sphingomyelin.

CH3(CH2),2CH = CH-CH-CH-CH2

• » • 9 +

OH NH 0- P-0-CH2-CH2-NT(CH3)3

• ■

c = o 9

sphingomyelin

Glycolipids: Cerebrosides and Sulfatides

In the general group of lipids called glycolipids, or glycosyl ce-
ramides, the primary hydroxyl group of a ceramide is linked by a glyco-
sidic bond to a monosaccharide or oligosaccharide chain. A cerebroside
is a ceramide linked to a monosaccharide, usually galactose. Cerebro-
sides are important constituents of nervous tissue; they are major con-
stituents of the myelin sheath. Nervous tissue cerebrosides are of two
types: those containing normal fatty acids and those containing 2-
hydroxy fatty acids. A typical cerebroside containing lignoceric acid is
known by the trivial name "kerasin" (shown below). When the fatty

CH3(CH2),2CH=CH-CH-CH-CH2
OH NH 6

C=0

(CH2)22
CH,

ceramide galactoside (kerasin)

7 6 LIPIDS: DEFINITIONS

acid constituent is 2-hydroxytetracosanoic (cerebronic) acid, the cere-
broside is called "phrenosin." The cerebrosides containing 9-tetraco-
senoic (nervonic) and 2-hydroxy-9-tetracosenoic (oxynervonic) acids
are known as "nervon" and "oxynervon" respectively.

Glycolipids of other types are found in spleen, liver, plasma, and
erythrocytes. These include ceramide-dihexosides, in which a ceramide
is linked to a galactose disaccharide or a glucose-galactose disaccharide ;
ceramide-trihexosides; and ceramide-tetrahexosides.

Sulfatides are sulfate esters of cerebrosides in which the sulfate
group is found on C3 of the galactose moiety. Like the cerebrosides, the
sulfatides are abundant in nervous tissues. These sulfatides also occur
with 2-hydroxy fatty acid constituents, but to a lesser extent than do
nervous tissue cerebrosides. In other tissues, such as kidney, sulfatides
of ceramide dihexosides have been found.

CH3(CH2)J2CH=CH-CH-CH-CH2

OH NH 0

c=o

sulfatide (cerebroside sulfate)

Sulfatides should not be confused with sulfolipids, a group of
plant lipids containing sulfonic acid and having the general formula:

KLC-O-C-R,
0 i OH H

'oh

CH2

0=S=0

I
OH

sulfolipid

Gangliosides

Gangliosides are a complex group of glycosphingolipids which dif-
fer from the other glycolipids in containing sialic acids. Sialic acids are

CLASSIFICATION, NOMENCLATURE 17

COOH

C = 0

CH2
0 H-C-OH
CH3-C-NH-C-H
HO-C-H

H -C-OH

H-C-OH
H2C-0H
D ( — )N-acetylneuraminic acid

N-acyl derivatives of neuraminic acid; the common constituent of
gangliosides is N-acetylneuraminic acid.

High levels of gangliosides occur in nervous tissue; they are be-
lieved to be located specifically in the neurons. Other gangliosides, con-
taining N-glycoylneuraminic acid, have been found in spleen and in red
blood cell stroma.

The gangliosides of brain have been most extensively studied and
have been separated into three main types — mono-, di-, and trisialo-
gangliosides — according to their sialic acid content. The major mono-
sialoganglioside has been shown to have the following structure:

CHoOH _ OH

/ UHoOH „ OH,,, M,

£343-

0
C0'NH/ J CH2OH
C02H '

CH2OH HC"0H

I UM2UM

CH3CO-NH HC-OH

HC-OH

CH2OH

A group of relatively recently discovered compounds, the prosta-
glandins, are also classified as lipids. The study of these substances is
rapidly developing into a separate field, so they are not considered in
this text. A review of the chemistry of the prostaglandins is included
in the list of Suggested Further Readings (p. 99).

78 LIPIDS: DEFINITIONS

REFERENCES

1. Johnston, P. V., O. C. Johnson, and F. A. Kummerow (1957) /. Nutrition
65: 13.

2. Perkins, E. G. (1960) Food Tech. 14: 508.

3. Schultz, H. W., ed. (1962) Symposium on Foods: Lipids and Their Oxida-
tion. Avi Publishing Co., Inc., Westport, Conn.

4. Hilditch, T. P., and P. N. Williams (1964) The Chemical Constitution of
Natural Fats; 4th ed., p. 670. John Wiley & Sons, Inc., New York.

5. Williams, J. H., M. Kuchmak, and R. F. Witter (1965) Lipids 1: 89.

6. Kittredge, J. S., E. Roberts, and D. G. Simonsen (1962) Biochem. 1: 624.

7. Horiguchi, M., and M. Kandatsu (1959) Nature 184: 901.

8. Baer, E., and N. Z. Stanacev (1964) /. Biol. Chem. 239: 3209.

9. (1965) /. Biol. Chem. 240: 44.

10. Chacko, G. H., and D. J. Hanahan (1969) Biochim. Biophys. Acta 176: 190.

11. Kuhn, R., and H. Wiegandt (1963) Chem. Ber. 96: 866.

II . Preparation and Handling of Samples
for Analysis of Lipid Constituents

During the extraction of lipids from tissues and their subsequent
handling during analysis the lipid chemist must constantly battle
his two worst enemies, oxidation and contamination. We cannot over-
stress the need to set up conditions in the laboratory that will prevent
oxidation of the lipids and intrusion by contaminants (lipid and non-
lipid). It is here that the inexperienced lipid analyst most frequently
fails. Before discussing extraction procedures we shall consider some
basic rules to follow when working with lipids.

GENERAL TECHNIQUES IN LIPID CHEMISTRY

Prevention of Oxidation

When exposed to air the unsaturated moieties of lipids rapidly
oxidize, a process that is accelerated by light. It is absolutely essential
that the lipids be manipulated in an oxygen- free environment. This is
generally achieved by carrying out operations under an atmosphere of
nitrogen. A lipid laboratory should have nitrogen supplies at every point
where lipids are to be handled. This can be accomplished by running
several branch lines of polyethylene tubing from a tank (or tanks) of
nitrogen. The nitrogen flow can be regulated by constricting the tubing
with clamps or by inserting Teflon stopcocks or flow regulators at
suitable points. Glass stopcocks should not be used, as the grease from
them may get blown down the lines and contaminate samples. Several
examples of when and how to use nitrogen follow:

1. When filtering a solution containing lipids, attach a line from
the nitrogen to the stem of a funnel and invert over the filtration ap-
paratus, letting a gentle flow of nitrogen continue until filtration is
complete.

2. When placing lipid samples in a dessicator to dry, first flush
out the dessicator with nitrogen, evacuate, and repeat the process. When
releasing the vacuum in the dessicator, do so by letting in nitrogen, not
air. Any apparatus in which lipid is placed should be fitted with three-
way stopcocks so that it can be flushed through with nitrogen.

3. Other manipulations of the lipid can be carried out under a plastic
"tent" within which a slight positive pressure of nitrogen is maintained.
Setups of this type can readily be made by purchasing plastic "glove
bags," which are available from a number of supply houses.

4. When dealing with lipid dissolved in small volumes of solvent,
it is often convenient to remove the solvent under a stream of nitrogen.

20 PREPARATION AND HANDLING

For this purpose a manifold (a tube having several outlets) attached
to a nitrogen line is very useful. The nitrogen stream should be directed
through disposable glass pipets, which can be discarded after use with
one sample.

In general, lipid samples should be blanketed by nitrogen during all
operations. This is not so necessary when the lipid is in an atmosphere
saturated with solvent vapor. If lipid or tissues have to be stored for
a while, they should be frozen rapidly and kept in their containers at
— 20°C (or below) in a plastic bag flushed out with nitrogen.

In addition to using a nitrogen atmosphere it is often convenient to
add to the lipid an antioxidant such as 2,6-di-t-butyl-/>-cresol, more
commonly called butylated hydroxytoluene (BHT). The usual addition
of BHT is 0.1 percent of the weight of the lipid.

Lipids should never be stored in chloroform-methanol solution as
this leads to breakdown of phosphoglycerides.

Additional hazards of breakdown occur during removal of tissues
from animals and during maceration of the sample. Here, lipolytic
enzymes may be activated and break down some of the lipids. It is,
therefore, essential that dissection and maceration are carried out as
rapidly as possible.

Elimination of Possible Contaminants

Lipids and compounds which mimic lipids in their chromatographic
behavior are likely to enter samples in a number of ways. The only
way to avoid artifacts and contaminants is to ensure that all laboratory
personnel abide by a set of rigid rules. The following list of suggested
rules illustrates the vast number of sources of contamination.

1. All reagent grade solvents should be redistilled to remove non-
volatile impurities. Distillation should be carried out in glass apparatus
and solvents stored in glass containers. Chloroform should be stabilized
by adding 0.25 percent (by vol.) methanol and stored in a dark bottle.

2. Corks, rubber stoppers, plastic film wraps, etc., should not be
used to close containers containing lipid samples, especially when these
materials may come in contact with solvent vapors. Rubber tubing
should not be used. Contact of solvent with any tubing should be
avoided.

3. The use of grease on stopcocks and vacuum systems should be
avoided. Teflon stopcocks are the best solution. Good ground glass
apparatus on rotary vacuum evaporators and similar equipment will
usually produce good leakless seals without the use of grease.

4. Personnel should be trained never to put their fingers inside
vessels to be used for lipid work. Fingerprints are a rich source of lipids.

5. Smoking near chromatographic equipment should be forbidden
as tobacco smoke is a rich source of chromatographic artifacts.

OF SAMPLES FOR ANALYSIS 21

6. Vapors from vacuum pumps and other apparatus should be
vented into a hood.

7. If at all possible, glassware should be washed in chromic acid
and rinsed thoroughly in deionized water.

8. Finally, it is good practice in the lipid laboratory to run blanks
of procedures to check for possible artifacts and contaminants.

Rouser et al. (1) have demonstrated the presence of an impressive
number of potential contaminants found in a lipid laboratory.

Unwanted Emulsions and Other Hazards in Lipid Chemistry

The beginner in lipid chemistry encounters hazards in addition to
those of oxidation and contamination. Invariably he will experience
the formation of unwanted and seemingly intractable emulsions. As we
shall see later, procedures in lipid analysis frequently call for the par-
titioning of lipids and nonlipids between aqueous and organic sol-
vent phases. The very property of lipids which makes them important
in the assembly of biological membranes, namely their amphiphilic na-
ture, means that they are frequently good emulsifiers. As a consequence,
the beginner finds that phases refuse to separate; often there is a band
of emulsified material where the interface should be, or the whole sys-
tem becomes emulsified. Emulsions are easier to prevent than to cure.
Prevention is therefore emphasized.

Whenever a procedure calls for the intermixing of aqueous and non-
aqueous phases, this should be achieved by swirling the contents of the
container (usually a separatory funnel) and inverting the vessel several
times. Vigorous shaking, especially when phospholipids are present,
will form emulsions. Emulsions also readily form when soaps are pres-
ent, such as when a soap solution is being extracted with ether or hexane
to remove non-saponifiable lipids. Generally, however, swirling and
inverting the vessel rather than shaking it vigorously will prevent the
formation of hard-to-break emulsions.

If emulsions are formed, there are several techniques for breaking
them. Should the whole system form a very permanent-looking emul-
sion, centrifugation (at 600 to 700 X g) will break it most quickly.
Other emulsion-breaking techniques are generally useless and a waste
of time. If only part of the system is emulsified and the interface can
be detected, the emulsion can often be dispersed by using a disposable
pipet to add a little ethanol at the interface. Adding a salt, such as
sodium sulfate, and gently swirling will also tend to break emulsions.
Generally, however, centrifugation gives clean and quick results and
is the preferred method.

Numerous other tedious difficulties and potential dangers (to per-
sonnel as well as to samples) are encountered in the lipid laboratory.
In general these are discussed in the text as the situations arise. A
common problem should, however, be mentioned immediately. Fre-

22 PREPARATION AND HANDLING

quently, a beginner complains that results are not reproducible. There
are many possible reasons for this, but the adoption of one simple
rule often solves the problem. Lipids must never be sampled from the
solid or semi-solid state unless efforts have been made to homogenize
the sample. Lipids extracted from natural materials are not homoge-
neous: they crystallize and solidify at different rates and different
temperatures so that all parts of the sample are not necessarily the
same. The sample can be homogenized by grinding it under nitrogen
in a mortar, but it is more common and more effective to dissolve the
lipids in a known volume of solvent and to take aliquots from this.

EXTRACTION OF LIPIDS FROM VARIOUS SOURCES

Samples from which lipids are to be extracted should be as fresh
as possible. If tissues dissected from animals cannot be extracted imme-
diately, they should be frozen by plunging them into liquid nitrogen.
They should then be stored at — 20° C or below in closed containers
that have been flushed out with nitrogen. Blood samples should not
be drawn unless it is certain that they can be treated immediately.

It is not possible to describe in detail an extraction procedure that
is applicable to all types of material. In changing from one source to
another, some modification of procedure is usually necessary. Some
general remarks, however, can be made about maceration of samples.

If the sample is no larger than a few grams, it can be ground most
efficiently with a tissue homogenizer of the Potter Elvehjem or Ten-
broech type. These homogenizers consist of ground glass tubes con-
taining closely fitting pestles. They can be used for grinding by hand,
or the pestle can be turned mechanically by attaching it to a high speed
motor. The tube should be placed in ice while grinding. Larger samples
can be handled in Waring Blendors, or by using a mortar and pestle.

The choice of the macerating method depends to a great degree on
the toughness of the sample. When dealing with tough tissues such as
skin or samples rich in connective tissue, it is best to freeze the
sample in a steel mortar in liquid nitrogen. A sharp blow with a steel
pestle or hammer will fragment the material, which can then be ground
to a fine powder in the frozen state and extracted.

From Blood Serum

Blood should be drawn by venipuncture or, in the case of small
animals, by puncturing the heart or a suitable large blood vessel. The
blood should be drawn into hypodermic syringes and immediately trans-
ferred to centrifuge tubes. Centrifugation (at 550 to 700 X g) to obtain
the serum should be carried out in a refrigerated centrifuge at 4°C.
The serum should be straw-colored and not show any signs of hemoly-

OF SAMPLES FOR ANALYSIS 23

sis. Some investigators allow blood to clot before they draw off the
serum, a process that may take several hours. This is not a good
practice, since the lipids will oxidize.

Several procedures for the extraction of lipids from serum have
been described in the literature, all of them similar. The one given
here is a minor modification of the method used by Williams et al. (2).

The serum is extracted with chloroform-methanol using a ratio of
serum: chloroform: methanol of 5:6:12 v/v/v. The appropriate vol-
ume of methanol is placed in a glass-stoppered Erlenmeyer flask. The
serum is then added slowly, with mixing, to the methanol. The chloro-
form is added and the flask is placed in a water bath at 55 °C for 30
minutes. The solution is filtered immediately through a Buchner funnel
and the residue is washed with water: methanol: chloroform 5:6:12
v/v/v, using 1ml of wash solution per ml of original serum. The com-
bined filtrates are allowed to separate into two phases in a separatory
funnel at 4°C. The lower layer contains the lipid. The top aqueous layer
should be free of lipid ; this should be checked by concentrating the
upper layer, running TLC plates (see Chapter 4), and looking for neu-
tral lipids and phospholipids (see p. 45).

The solvent in the lower layer is then removed under vacuum. It is
preferable to carry this out with a good vacuum pump rather than a
water-type pump ; this makes the application of heat unnecessary. If
this is not possible, the heat should be kept to a minimum. The evapo-
rating system (rotary type evaporator, flash evaporator, etc.) should
be fitted so that nitrogen can be introduced into the system for con-
trolling pressure and releasing the vacuum.

The lipid obtained should be redissolved in a minimum amount of
chloroform-methanol and if necessary refiltered. Although the serum
lipid is essentially subjected to a Folch-type washing procedure (3) in
the above routine, some nonlipid material may still be carried through.
Redissolving the sample in chloroform-methanol solution generally
eliminates this.

The lipid sample is then placed in a dessicator over potassium hy-
droxide or some other dessicant and dried under vacuum. As noted
earlier, the dessicator should be flushed out with nitrogen and evacuated
a couple of times. When the weight of lipid is known, an antioxidant
such as BHT can be added at the level of 0.1 percent of the weight of
lipid. This weighing procedure is probably best when only very small
amounts of lipids are being handled and when prevention of waste is
imperative. There is the danger that the lipid may get exposed to air
during weighing. It is essential to flush out containers with nitrogen and
stopper them tightly during this time. When wastage is not an issue,
the weighing procedure outlined by Rouser et al. (4) is recommended:
the lipid is dissolved in a known volume of solvent and an aliquot (50 to
200 fx\) is removed and weighed on a microbalance. This procedure is

24 PREPARATION AND HANDLING

not suitable if the lipid sample contains short-chain volatile fatty acids
or their methyl esters.

Some authorities suggest adding the antioxidant to the extracting
solvents so that the lipid is protected throughout the extraction pro-
cedure. This is a useful technique if one knows approximately the
amount of lipid that will be extracted so that the appropriate amount
of antioxidant can be added to the solvents. This procedure, however,
necessitates either the determination of the amount of antioxidant in
the sample or the accurate measurement of the solvents in order to ob-
tain the weight of lipid.

As always with lipids, analyses should be completed as soon as pos-
sible. Lipids to which antioxidant has been added, however, can be
stored in glass containers and under nitrogen at — 20° C or below with
little decomposition over fairly long periods.

From Erythrocytes

It is somewhat more difficult to extract the lipids from erythrocytes
(or erythrocyte stroma preparations) than from serum. First, there is
a tendency for the erythrocytes to form a rubbery mass that is diffi-
cult to break up, making consequent extraction inefficient. This can
be prevented as follows. Place the appropriate amount of methanol in
any conventional tissue homogenizer, add the cells, and homogenize.
Then add the correct volume of chloroform and homogenize again.
One extraction with this solvent mixture will not remove the lipids
completely. The number of extractions and the variations in the solvent
mixture will depend on the tissue involved. Rouser et al. (4) have pro-
posed an exhaustive extraction sequence (the figures in parentheses
refer to the milliliters of solvent per gram of fresh tissue) :

1. Chloroform-methanol, 2:1 v/v, twice (20ml, then 10ml)

2. Chloroform-methanol, 1:2 v/v (10ml)

3. Chloroform-methanol, 7:1 saturated with 28 percent (by weight)
aqueous ammonia1 (10ml)

4. Chloroform-methanol-glacial acetic acid, 8:4:3 v/v/v (10ml)

5. Chloroform-methanol-concentrated hydrochloric acid, 200:100:1
v/v/v (10ml)

This complete extraction sequence is not required for efficient ex-
traction from most tissues. In most cases the first three systems suffice,
and frequently efficient extraction is obtained by extraction with
chloroform: methanol 2:1 v/v only. In the case of erythrocytes, extrac-
tion with the first three solvents is recommended.

1 Commercial preparations of 28% aqueous ammonia usually contain non-
volatile solids, largely silicates. To avoid this contamination, ammonia can be
bubbled from a cylinder into ice-cold distilled water in a plastic container, in
which the solution is then stored.

OF SAMPLES FOR ANALYSIS 25

The combined solvent extracts will contain nonlipid material. This
can be removed using the Folch washing procedure (3). In this method
0.2 volumes of water (or a salt solution) is added to the combined sol-
vents in a separatory funnel and the nonlipid material passes into the
aqueous phase. This procedure is widely used but is not entirely satis-
factory since some loss of lipid occurs. As an alternative, most nonlipid
material can be removed by one of the other methods described below.

The combined solvents freed of nonlipids and containing the erythro-
cyte lipids are removed under vacuum as previously described, and the
lipid sample is dried and weighed.

When working with erythrocytes, fresh cells must be used, since
in aging erythrocytes the lipids are rapidly oxidized by the catalytic
effect of heme. This is also the reason for removing nonlipid material
from the lipid sample and adding an antioxidant.

From Brain

Brain can be efficiently extracted with chloroform-methanol 2:1
v/v. Fresh or frozen brain should be placed in a suitable homogenizer or
mortar and extracted three times with chloroform-methanol 2:1 v/v,
using 20ml per gram of brain the first time and then 10ml per gram
the remaining two times.

Brain contains many gangliosides that partition mainly into the
top (aqueous) phase in the Folch washing procedure. Some ganglio-
sides usually remain in the bottom phase, however, and any gangliosides
entering the top phase may carry other lipid with them. The method is,
therefore, not reproducible and if a total extract of brain lipids is re-
quired, aqueous washing should not be used.

Redissolving the lipid after removing the solvent is another way of
removing nonlipid material. Provided that the solvent is removed at a
low temperature and two phases exist during evaporation, any carried-
over protein will be denatured. The solvents containing the extracted
lipids plus nonlipid contaminants should be placed in a suitable flask
for the evaporator system being used. If two phases are not present,
add water until two phases are formed. Remove the solvents under
vacuum, applying just enough heat via a water bath to prevent the
formation of ice on the outside of the flask. The dried crude extract
obtained can then be re-extracted three times with chloroform-meth-
anol 2:1 v/v (1ml per lOOmg of lipid) and the insoluble residue re-
moved by filtration through a sintered glass filter (medium). Re-
extraction and filtration can be carried out at room temperature.

Nonlipid material can also be removed by one of the column chroma-
tographic procedures described later (pp. 27-29). When the final solvent
is removed and the lipid dried, great caution must be used in subsequent
handling. Brain lipid is rich in highly unsaturated (penta- and hexa-

26 PREPARATION AND HANDLING

enoic) fatty acids, which oxidize rapidly. An antioxidant should be
used, and the lipid must be carefully protected from air.

From Other Sources

The extraction procedure employed for brain is suitable for ex-
tracting lipids from many soft tissues such as liver, spleen, kidney,
adipose tissue, etc. With other tissue, however, individual problems
are encountered and modifications must be made. Nerves, skin, heart,
and muscle are not easily homogenized in the ground glass homoge-
nizers of the Potter-Elvehjem type. Special handling such as grinding in
a mortar or cutting up in a Waring Blendor may be necessary. The
most useful technique with many tougher tissues is to grind the solidly
frozen tissue to a powder as previously described (p. 22). Plant sources,
particularly some leaves and seeds, frequently present similar problems
and more severe grinding procedures must be employed.

If no established extraction procedure is available for a particular
material, it should be subjected to the exhaustive extraction sequence
previously described. Each solvent system should be evaporated off
separately in order to determine how much lipid it extracted. In any
event, solvent systems containing acid should be evaporated separately
as the presence of acid may hydrolyse labile lipids. Finally, it may be
necessary to subject the homogenized material to acid or alkaline hy-
drolysis and to check the hydrolysate for the presence of fatty acids.
This will ascertain if any very tightly bound lipid remains unextracted.
For example, the fatty acids in feces are present in the form of soaps ;
it is therefore necessary to treat feces with dilute (10 to 20 percent)
hydrochloric acid before extracting. Problems of a similar nature are
associated with some microbial sources in which fatty acids are found
to be associated with glycoproteins. These sources of lipid constitute
separate problems in themselves and, as special cases, are beyond the
scope of this book.

REMOVAL OF NONLIPID CONTAMINANTS FROM EXTRACTS

Aqueous Washing

As noted earlier, hazards are associated with this procedure, and in
certain cases, for instance when total brain lipid is required, its use is
unwise. Although simpler, more reliable methods are now available,
aqueous washing still enjoys wide acceptance, however, and objections
to it are probably somewhat overstated. The method as described here
applies to the combined 2:1 v/v chloroform-methanol extracts.

In this procedure the crude chloroform-methanol extract is washed
with 0.2 its volume of water or a suitable salt solution. It is necessary
to prepare solutions known as "pure solvents upper phase" and "pure
solvents lower phase." These are prepared by mixing chloroform,

OF SAMPLES FOR ANALYSIS 27

methanol, and water (8:4:3 v/v/v) in a separatory funnel. On standing,
two phases separate. These are collected and stored in glass bottles.
The upper phase consists of chloroform-methanol-water, approximately
30:48:47 v/v/v, and in the lower phase the proportions are 86:14:1
v/v/v. The phases may be prepared by using the above proportions if
preferred. A "pure solvents upper phase" that contains 0.02 percent
CaCl2, 0.017 percent MgCl2, 0.29 percent NaCl, or 0.37 percent KC1
serves as the salt solution wash. The salt solutions may be prepared
either by shaking the correct amount of salt with "pure solvents upper
phase" or by replacing the water during the preparation of "pure
solvents upper phase" with the following aqueous solutions: 0.04 per-
cent CaCl2, 0.034 percent MgCl2, 0.58 percent NaCl, or 0.74 percent
KG. Using a salt solution rather than pure water reduces loss of lipids
into the upper phase. Usually a crude lipid extract already contains
salts, which helps conditions somewhat, but additional salts may im-
prove the distribution required. Gangliosides, for example, found
mainly in the upper phase in all systems, will be almost entirely elimi-
nated from the lower phase by a CaCl2 wash. This wash should not be
used, however, if insoluble calcium salts are likely to be formed.

The washing procedure is the same for both the aqueous and the salt
wash. Washing is carried out in a separatory funnel if the phases are
to be allowed to separate by standing, or in a centrifuge tube if phase
separation is to be achieved more rapidly by centrifugation (at approxi-
mately 600 X g).

"The crude extract is mixed thoroughly with 0.2 its volume of water
or one of the salt solutions. When the biphasic system is obtained, as
much of the upper phase as possible is removed by siphoning. The inter-
face is then rinsed three times with small amounts of "pure solvents
upper phase." The rinsing should be done carefully so as to avoid dis-
turbing the lower phase. After the last rinse, the lower phase and any
unremoved rinse solution are combined into one phase by adding meth-
anol. The solution can then be diluted with methanol to any desired
volume, or the solvent can be removed under vacuum and the lipid dried.

Treatment on Cellulose and Sephadex Columns

Rouser et al. (5) describe a cellulose column chromatographic pro-
cedure which removes water-soluble nonlipid contaminants from crude
lipid extracts. Gangliosides are eluted with the nonlipid fraction.

The column size chosen will depend on sample size. The procedure
described below is suitable for a 100 to 500mg sample.

A glass column 2.5cm internal diameter (i.d.) X 20 to 30cm long is
packed with 20g of Whatman standard grade ashless cellulose powder
suspended in methanol- water 1:1 v/v (see p. 31 for hints on packing
glass columns). The packed bed is washed at a flow rate of 3 ml/min
with the following set of solvents: methanol-water 1:1 v/v (7 column

28 PREPARATION AND HANDLING

volumes2), chloroform-methanol 1:1 v/v (3 column volumes), and
chloroform-methanol 9:1 v/v saturated with water (4 column volumes).

The sample, 100 to 500mg, is added to the column in 5 to 10ml of
the last washing solution. Two fractions are then eluted: the first, with
chloroform-methanol 9:1 v/v saturated with water (20 column vol-
umes), contains lipids minus the gangliosides, if present; the water-
soluble nonlipid contaminants and the gangliosides are then eluted with
12 column volumes of methanol-water 9: 1 v/v.

Sephadex, a cross-linked dextran gel, can also be used to separate
lipids and water-soluble nonlipids. Procedures have been described by
Wells and Dittmer (6) and by Siakotos and Rouser (7). The latter
method is described below.

Four solvent systems are required (all proportions are by volume) :

1. Chloroform-methanol 19:1, saturated with water

2. 850ml chloroform-methanol 19:1 plus 170ml glacial acetic acid
plus about 25ml water (to saturate)

3. 850ml chloroform-methanol 9:1 plus 170ml glacial acetic acid
plus about 42ml water (to saturate)

4. Methanol-water 1 : 1

When preparing solvent mixtures 1, 2, and 3, shake them vigor-
ously and allow them to separate in a separatory funnel. The lower
phase of each is used for chromatography. When adding the water to
mixtures 1, 2, and 3, do so slowly so that the mixtures are slightly
undersaturated.

Sephadex (G-25, coarse, beaded, Pharmacia Fine Chemicals, New
Jersey) is placed in methanol-water 1:1 v/v. The "fines" in the material
are removed by decantation and dissolved gases are removed by gentle
suction from a vacuum pump. This takes about 1 minute. After
equilibrating at room temperature for several hours, the gel is again
degassed and poured into a suitably sized column. For samples of 50 to
250mg, a column 1cm i.d. X 10 to 30cm is appropriate. A packed
2.5cm i.d. X 30cm column will accommodate several grams of sample.
To prevent the gel from floating in the solvents add a 2.5 to 5cm layer
of clean sand above it.

Transfer the sample onto the column in solvent mixture 1 and cover
the top of the sand with a plug of washed glass wool. For the smaller
samples on the 1cm i.d. X 10cm columns elute with the following vol-
umes of the solvents: 1, 25ml; 2, 50ml; 3, 25ml; and 4, 50ml. The flow
rate should be 3ml per minute. Larger samples up to 5g on the 2.5cm
i.d. X 30cm columns should be eluted with 20 times the above volumes.

2 A column volume (sometimes called the bed volume) is the volume of the
column occupied by the adsorbent.

OF SAMPLES FOR ANALYSIS 29

Depending on the complexity of the lipid-nonlipid mixture, the four
fractions may contain a variety of substances. The following is a com-
prehensive list of the substances found after chromatography of
samples from various sources (5).

Fraction 1: Hydrocarbons; mono-, di-, and triglycerides; sterols;
sterol esters; waxes ; all phosphoglycerides (including lyso compounds) ;
sulfatides; cerebrosides; sulfolipids; free fatty acids; glycosyldiglycer-
ides; unconjugated bile acids; conjugated bile acids (glycine, taurine) ;
and uncharacterized nonlipid compounds. The unconjugated bile acids
are eluted whether they are applied as salts or free acids and the
conjugated when applied in the free acid form (4). The bile acids and
related steroids are not covered in this text; any major biochemical
text (such as 8) gives accounts of the steroids.

Fraction 2: Gangliosides; glycine conjugated bile acids (applied as
salts) ; some acidic phosphatides (in "altered" form) ; urea; and other
materials soluble in organic solvents.

Fraction 3: Traces of gangliosides; taurine-conjugated dihydroxy-
cholanic (deoxy and chenodeoxy) acids (when applied as salts); some
amino acids; and other uncharacterized organic substances.

Fraction 4: Taurocholate and most water soluble nonlipids such as
salts, amino acids, sugars, etc.

Note that, for the purposes of the types of analyses covered in this
text, Fraction 1 contains the bulk of the lipids, while Fraction 2 is rich
in gangliosides.

This procedure is the most efficient and reliable method now avail-
able for the removal of water-soluble nonlipids from crude lipid ex-
tracts. It is also relatively simple to set up and use. Columns can be
reused after allowing them to stand in methanol-water 1:1 (solvent
mixture 4) for about 48 hours, then washing with 100 to 500ml of sol-
vent mixture 1 immediately before use.

REFERENCES

1. Rouser, G., G. Kritchevsky, M. Whatley, and C. F. Baxter (1965) Lipids
1 : 107.

2. Williams, J. H., M. Kuchmak, and R. F. Witter (1965) Lipids 1: 89.

3. Folch, J., M. Lees, and G. H. Sloane-Stanley (1957) /. Biol. Chem. 226: 497.

4. Rouser, G., G. Kritchevsky, and A. Yamamoto (1967) in Lipid Chromato-
graphic Analysis, ed. G. V. Marinetti ; vol. 1, p. 99. Marcel Dekker, Inc.,
New York.

5. Rouser, G., C. Galli, and G. Kritchevsky (1965) /. Am. Oil Chem. Soc. 42:
404.

6. Wells, M. A., and J. C. Dittmer (1963) Biochem. 2: 1259.

7. Siakotos, A. N., and G. Rouser (1965) /. Am. Oil Chem. Soc. 42: 913.

8. Mahler, H. R., and E. H. Cordes (1966) Biological Chemistry. Harper &
Row, New York.

III. Column Chromatography

The chromatographic technique has been used by organic
chemists as a separation method for many years. Early develop-
ments were confined to column and paper chromatography and the
types of separations achieved were limited. However, in 1952 the first
gas-liquid chromatograph (1) was introduced, and in 1958 Stahl (2)
described his development of thin-layer chromatography. Rapid devel-
opment of these two techniques, together with improved applications
of column chromatography, has revolutionized the field of lipid
analysis. Xew and improved methods are still being reported in some
abundance, from which we have selected a few well-tested procedures
in each area of chromatography. These can be applied with good re-
producibility to the analysis of mixtures of neutral, phospho-, and
glycolipids. Separations of the rarer lipids and of complex lipids such as
gangliosides still present some difficulties, as does the separation of
individual molecular species of lipid classes. The major lipids of blood
serum, say, can be analyzed, including their fatty acid and fatty alcohol
patterns, provided that the indicated procedures are followed strictly
and the lipids are carefully protected from oxidation and contamination.
This introduction to chromatographic techniques is geared to that level
of achievement. It should, however, be stressed that in many respects
chromatography is very much an art, requiring practice before effective
separations can be obtained.

Chromatography includes any technique in which compounds are
physically separated by differential distribution between two phases,
one of the phases being stationary and the other moving. Stationary
phases are either solid or liquid and moving phases either liquid or
gaseous.

In column chromatography the stationary phase is packed in a glass
column onto which the sample is introduced ; the moving phase is
liquid. Separation is achieved by percolating suitably constituted sol-
vents through the bed of stationary phase. Column chromatography
can be subdivided into two main types according to the nature of the
stationary phase: solid-liquid adsorption chromatography and liquid-
liquid partition chromatography.

SOLID-LIQUID ADSORPTION CHROMATOGRAPHY

This type of chromatography is based on the differences in affinity
of compounds for the solid adsorbent that serves as the stationary phase.
The relative affinity of compounds for the phase depends upon the na-
ture of their polar groups and to some extent upon the van der Waals
forces exerted by their nonpolar groups. The greatest forces involved

COLUMN CHROMATOGRAPHY 31

in the affinity for the adsorbent are those due to polar and ionic groups.
This means that this type of chromatography is useful for separating
lipid mixtures which differ in the number and type of their polar groups.
Thus, broad separations of the neutral lipids from the main types of
polar lipids can readily be carried out. Separations within lipid classes
can also be achieved. Many adsorbents have been employed, among
them alumina, magnesium oxide, urea, Florisil (a magnesia silica gel),
silicic acid, diethylaminoethyl cellulose (DEAE), and others. The most
efficient and versatile separations to date have been obtained using
DEAE. Silicic acid is, however, very useful for separation of lipid
classes, and Florisil is especially useful for the separation of cerebro-
sides. The procedures given utilize these three main adsorbents.

A typical adsorption chromatography set-up is shown in Figure 1.
This is the simplest form of apparatus which should be employed. Note
the Teflon stopcocks (to avoid use of grease). Also note the nitrogen
line so that a slight positive pressure of nitrogen can be maintained over
the system.

LIQUID-LIQUID PARTITION CHROMATOGRAPHY

Liquid-liquid partition chromatography is essentially a counter-
current distribution process. In this method, separation depends on the
relative solubility characteristics of the compounds with respect to the
stationary and moving phases of the column. Two types of liquid-liquid
columns are in general use, one in which the more polar liquid is station-
ary (partition type) and the other in which the less polar liquid is
stationary (reversed-phase partition type). Liquid-liquid partition chro-
matography has been used largely for the separation of fatty acids.
Separations have been obtained according to unsaturation, chain length,
and geometrical isomerism. One of the major uses of this method (es-
pecially when preparative gas chromatography is not available) is the
large-scale preparative separation of fatty acids.

We shall be concerned only with adsorption chromatography. Before
describing some separations it is essential that we consider the basic
principles of column packing and preparation.

PREPARATION OF COLUMNS

Separation and reproducibility depend on a number of factors, the

most important of which are packing of the column, pretreatment of the

; adsorbent, shape and dimensions of the column, rate of elution, quality

I of the eluting solvents, and load of sample in relation to weight of

adsorbent. Temperature and humidity also influence reproducibility.

It is usually best to introduce the adsorbent into the column in the

form of a thin slurry or suspension. The slurry is prepared by adding

the desired weight of packing material to a suitable volume of solvent.

The solvent used is generally the one with which the adsorbent is to be

COLUMN CHROMATOGRAPHY

nitrogen line

paper disc
or sand

receiver

solvent reservoir

— Teflon stopcock

solvent (moving phase)

column packing
(stationary phase)

glass wool

Teflon stopcock 'H f""^

eluate

Apparatus for adsorption column chromatography.

(Fig. 1

COLUMN CHROMATOGRAPHY 33

prewashed or pretreated; in some instances, the first eluting solvent
may be used. A plug of glass wool should first be placed in the shoulder
of the column (Fig. 1). As the adsorbent is poured in and allowed to
settle, the flow of solvent through the column is kept constant. This pro-
cedure prevents the formation of "channels" in the column, a common
hazard when columns are packed dry. Channel formation is quite dis-
astrous in column chromatography, since it leads to very inefficient
separation and overlapping of fractions. Applying pressure to pack
columns is a dangerous practice and generally does not prevent channel-
ing. If pressure is applied to a glass column, which is hardly designed
as a pressure vessel, the glass may break and possibly cause injuries.

Pretreatment of the adsorbent varies according to the adsorbent
being used. In the examples which follow, therefore, pretreatment is,
dealt with individually. It is essential that a recommended pretreatment
is carried out, as frequently this step endows the adsorbent with its
particular characteristics.

The shape and length of columns affect separation; column dimen-
sions, therefore, should always be stated. It has been demonstrated that
longer, narrower columns usually give better separations than shorter,
wider ones (3). If a column is tried and it is found that some fractions
overlap, increasing the column length often solves the problem. Ex-
tremely long columns are, of course, inconvenient; increasing the length
beyond 40cm is not recommended.

Once elution is started, a flow of nitrogen through the column
(preferably via a proper flow control) can be maintained as an anti-
oxidation precaution.

While investigators invariably recommend a particular sample load
for best resolution, the same load may not be optimal in all laboratories.
Slight differences in the adsorbent as well as in environmental factors
such as laboratory temperature and humidity may affect resolution.
Therefore, even though a method may be well established, it may be
necessary to change some dimensions of the procedure if resolution is
not satisfactory. This is done by applying different loads to a particular
column until the best resolution is obtained. Ideally, and in the interests
of saving time, one should strive to ascertain the maximum load that
can be applied to the smallest column (that is, the column with the
smallest elution volume) to give satisfactory resolution of fractions.
The composition of fractions and the degree of resolution obtained are
very readily checked by thin-layer chromatography. This technique and
its use as an adjunct to column chromatography are discussed in the
next chapter.

The quantitative analysis of samples by column chromatography can
be accomplished in a number of ways, depending upon the composition
of the sample and the needs of the investigator. It is usual to analyze
each tube of eluate for a suitable element or functional group and to

34 COLUMN CHROMATOGRAPHY

plot its concentration versus tube number. Each tube should contain a
constant volume of eluate (usually 5 or 10ml), which may be collected
manually or by one of the many available automatic fraction collectors.
In making a preliminary separation of, say, cholesterol and glycolipids
from phospholipids, each tube would be analyzed for phosphorus.

Once the elution pattern has been established, an appropriate large
volume of eluate may be collected, concentrated to a known volume,
and checked by thin-layer chromatography for composition. A known
aliquot can then be analyzed for the appropriate element or compound
and the total amount in the fraction calculated. Contents of each lipid
can be reported in a variety of ways, such as mg lipid phosphorus per g
of total lipid, or per g of wet — or dry — tissue. Examples of the cal-
culation of such expressions are given in the final chapter. Sometimes
merely a weight of a fraction may be required ; this may be done di-
rectly by evaporating the solvents to a known volume, taking an aliquot,
removing the solvent, and weighing on a microbalance. Thin-layer
chromatographic checks of fraction content should still be made for
every run.

Silicic Acid Columns

For some time silicic acid and alumina were the only adsorbents used
for the chromatography of lipids. Both adsorbents have to some extent
been supplanted by others, but silicic acid is still used, especially for
initial broad separations. Fine-degree resolution requires rechroma-
tography on other adsorbents.

Early separations on silicic acid were done using fine mesh prepara-
tions. As a consequence, flow rates were very slow and the procedures
time-consuming. Reproducibility of results on silicic acid columns is
not easily achieved since different batches of the adsorbent may vary
considerably in their properties. To some extent, these disadvantages
can be overcome by using one of the commercially available silicic acids
that have been manufactured with a view to maintaining a uniform
standard grade (for example, Adsorbosil, Applied Science Laboratories,
and Unisil, Clarkson Chemical Company). Using these preparations
in coarser mesh sizes overcomes the problem of slow flow rates. Using
standardized silicic acid preparations also gives better reproducibility,
although reproducibility between one laboratory and another remains
difficult. The procedures that follow are, therefore, generalized. Each
investigator must standardize procedures according to his individual
needs, and each lot of adsorbent must be separately assessed.

Usually the better grades of silicic acid do not require pretreatment,
but they should be activated at 120° C if they have been in a moist
atmosphere. Activation is necessary to remove some of the absorbed
moisture, which could affect the adsorption properties of the silicic
acids.

COLUMN CHROMATOGRAPHY 35

The column in general use is 2.5cm i.d. and 10 to 20cm long; the
sample loading for separations of neutral from phospholipid classes is
about 10 to 30mg per g adsorbent.

The mechanisms involved in silicic acid chromatography and the
elution patterns of lipids on this adsorbent have been discussed in great
detail by Wren (4). Wren gives an expanded version of Trappe's
"eluotropic series" (solvents listed in order of their eluting power).
The solvents as listed by Wren are: methanol; ethanol; 1-propanol;
acetone; methyl acetate; ethyl acetate; ether; dichloromethane; ben-
zene; toluene; 1,1-dichloroethane ; 1,1,2,2-tetrachloroethane; chloro-
form; trichloroethylene; carbon tetrachloride; cyclohexane ; and
petroleum ether of various boiling ranges. Of these solvents those most
frequently used are methanol, ether, chloroform, and petroleum ether.
Wren (4) also listed the approximate order in which one may expect
lipids to be eluted from a silicic acid column: hydrocarbons, esters
(other than sterol esters and glycerides), sterol esters, fatty aldehydes,
triglycerides, long-chain alcohols, fatty acids, quinones, sterols, di-
glycerides, monoglycerides, glycolipids, lipamino acids, bile acids, gly-
cerophosphatidic acids, inositol lipids, phosphatidyl ethanolamines,
lysophosphatidyl ethanolamines, phosphatidyl cholines, sphingomyelins,
lysolecithins.

Initial separation: neutral from phospholipids and glycolipids.
When attempting to separate naturally-occurring lipids, it is best to
start by carrying out a crude separation into neutral lipids, phospho-
lipids, and glycolipids. Like all the column chromatographic procedures
described here, this is done using a stepwise elution.1 The sample (100
to 150mg per g of adsorbent) is added to a column of coarse mesh
silicic acid. Elution is carried out first with chloroform (10 column
volumes for a 2.5cm i.d. X 5cm column). This elutes neutral lipid.
Ten column volumes of chloroform-methanol 1:1 v/v or of methanol
alone will elute the phospholipids. If the lipid sample contains a lot of
glycolipids, an intermediate elution with acetone (30 to 50 column
volumes) will elute these as a separate fraction before the phospho-
lipids. Cardiolipin will be largely eluted in an acetone fraction along
with the glycolipids. The three-step elution is very useful for the pre-
liminary separation of nervous tissue lipids, in which the first fraction
will consist almost entirely of cholesterol.

Separation of neutral lipids. Numerous solvent systems have
been used for the separation of neutral lipids on silicic acid. The choice
of procedure will depend on the composition of the fraction and the

1 Better resolutions can often be obtained by using gradient elution, which
frequently overcomes tailing of one lipid fraction into another; however, step-
wise elution is simpler and needs no special equipment. Automatic equipment for
the mixing of solvent gradients can be obtained commercially. Some references
to gradient elution procedures occur in the text.

36 COLUMN CHROMATOGRAPHY

relative proportions of the constituents. A procedure to suit most needs
should be found among the following:

Hydrocarbons (saturated) can be eluted first with light petroleum
ether, hexane, or Skelly B. Unsaturated hydrocarbons such as squalene
will usually be the tail of this fraction.

Esters of fatty acids, cholesterol esters, and wax esters are eluted
together with 1 percent diethyl ether in petroleum ether (5). Esters
containing hydroxylated fatty acids require a more polar solvent for
their elution, a fact that can be used to separate nonhydroxylated and
hydroxylated fatty acids and their esters. For example, pentane with
3 percent diethyl ether elutes nonhydroxylated methyl esters, and
pentane with 20 percent diethyl ether elutes the hydroxylated form (6).
Free acids can be separated into nonhydroxylated and hydroxylated
groups by eluting first with benzene, then with benzene-diethyl ether
9:1 v/v (7).

Fatty acid esters which differ in degree of unsaturation can be
separated on silicic acid by various procedures. Goldfine and Bloch (8)
and Erwin and Bloch (9) achieved separation of fatty acids according
to degree of saturation by treating the methyl esters with mercuric
acetate and chromatographing the mercuric adducts on silicic acid. The
saturated esters were eluted with pentane-ether 95 : 5 v/v, the monoenoic
esters with ethanol-ether 50:50, the dienoic with ethanol-acetic acid
99:2, the trienoic with ethanol-acetic acid 99:1, and the tetraenoic and
polyenoic esters with ethanol-acetic acid 95:5.

Silicic acid impregnated with silver nitrate has been used by DeVries
(10) to separate saturated and unsaturated fatty acid methyl esters and
their geometric isomers. DeVries (11) also employed this absorbent to
separate triglycerides according to their degree of unsaturation. See
also pages 51-52.

Triglycerides separate from wax and sterol esters on silicic acid
by eluting with 3 to 5 percent ether in petroleum ether. Free fatty acids
and fatty alcohols are eluted after triglycerides with a slightly more
polar solvent, but tailing into the triglyceride fraction is usual. Diglyc-
erides elute with 20 to 60 percent ether in petroleum ether, and mono-
glycerides elute with ether or chloroform (5). Monoglyceryl ethers
accompany the monoglyceride fraction.

A hazard is encountered when chromatographing monoglycerides on
silicic acid: both the 1- and 2-isomers isomerize. Diglycerides do not
isomerize (5, 13). Isomerization of monoglycerides is prevented by
using silicic acid impregnated with 10 percent w/w boric acid (12). On
this packing, the 1- and 2-monoglycerides can be efficiently separated.

Separation of polar lipids. Polar lipids cannot be completely
separated on silicic acid, but some separations are possible by eluting
with increasing amounts of methanol in chloroform. Much better reso-

COLUMN CHROMATOGRAPHY 37

lutions, however, can be obtained using diethylaminoethyl cellulose
(DEAE), described below.

Fiorisil Columns

Florisil (Floridin Co., Pennsylvania Glass Sand Corp., 2 Gateway
Center, Pittsburgh, Pennsylvania) is a synthetic magnesium silicate.
It has a number of uses in lipid separation, the chief one being the sepa-
ration of cerebrosides, sulfatides, and ceramides. Florisil has the dis-
advantage, however, that its chromatographic properties are changed
when traces of water are present. This problem may be overcome by
drying the adsorbent and by using very dry solvents. Solvents may be
dried by adding 5 percent 2,2-dimethoxypropane to them as described
below. A useful procedure for the preparation of ceramide, cerebrosides,
and sulfatides, described by Rouser et al. (14), is outlined here.

One pound of Florisil is washed on a sintered-glass filter (medium
porosity) with 8 bed-volumes of water. It is then activated at 100°C
for 6 hours and cooled without exposure to air. The appropriate
amount of Florisil is quickly weighed and 1 percent (by weight) water
is added. Let the mixture stand for an hour or more in a closed con-
tainer so that the water and adsorbent will equilibrate. Mix the equili-
brated adsorbent with chloroform containing 5 percent (by volume)
2,2-dimethoxypropane. This slurry is then poured into a chromato-
graphic tube.

A 10.2cm i.d. column is used for the separation of ceramides,
cerebrosides, and sulfatides from about 3g of brain lipid. The lipid
sample is placed on the column in chloroform containing 5 percent 2,2-
dimethoxypropane. The eluting solvents, which must all also contain
5 percent 2,2-dimethoxypropane, are:

1. Chloroform, 10 column volumes

2. Chloroform-methanol 19:1 v/v, 10 column volumes

3. Chloroform-methanol 70:30 v/v, 20 column volumes

The first fraction will consist of cholesterol and any less polar lipids.
The second will be ceramides, and the third, cerebrosides and sulfa-
tides. The flow rate of solvents should be about 50ml per minute. If the
phosphatides are to be collected, they can be eluted last with 20 column
volumes of chloroform-methanol 2:1 v/v, saturated with water. The
ceramides can be purified by preparative thin-layer chromatography
(see next chapter), and the cerebrosides and sulfatides can be sepa-
rated on a DEAE column.

Diethylaminoethyl (DEAE) and Triethylaminoethyl (TEAE)
Cellulose Columns

DEAE cellulose gives the most satisfactory column chromatographic
separations of complex lipid mixtures. Separation is achieved either

38 COLUMN CHROMATOGRAPHY

by ion exchange or by differences in polarity of nonionic groups. The
elution characteristics of DEAE are most easily understood if lipids are
considered as being in three groups — nonionic, nonacidic ionic, and
acidic. Nonionic lipids are eluted according to relative polarity. The
least polar lipids such as the sterols, sterol esters, glycerides, and hydro-
carbons are eluted with chloroform. Three to 5 percent methanol in
chloroform elutes cerebrosides and glycosyl diglycerides. All of the
nonionic and ionic nonacidic lipids are eluted with increasing amounts
of methanol in chloroform. The acidic lipids, however, are not eluted
with chloroform-methanol or methanol unless an acid, base, or salt is
added to the solvent.

The development of DEAE cellulose column chromatography is
due largely to Rouser and his co-workers, who have compiled an ex-
tensive review and detailed description of all aspects of this method
(14). Here we shall deal with preparation and pretreatment of columns
and typical elution sequences for the more common lipids. For varia-
tions and separations of more complex mixtures, the Rouser article
should be consulted.

The DEAE cellulose usually recommended is Selectacel DEAE
cellulose regular grade (Brown Co., Berlin, New Hampshire). This is
a coarse grade, which is usually best for lipid separations. The DEAE
must be washed before use, as it contains impurities. Washing can be
done on a sintered-glass or Buchner funnel, using about 3 bed volumes
of acid or base. The DEAE is washed first with IN hydrocloric acid,
rinsed with water to a neutral pH, then washed with 0.1N aqueous
potassium hydroxide followed by a water rinse to neutral pH again.
After washing, the DEAE is converted to the acetate form by washing
it with 3 bed volumes of glacial acetic acid. The adsorbent is air-dried,
then dried to constant weight in a vacuum dessicator over potassium
hydroxide. Thorough drying is important. The dry DEAE is left over-
night in glacial acetic acid and finally is added to the column as a slurry
in glacial acetic acid. The excess acid is blown through the column with
nitrogen, and the adsorbent is patted down gently with a glass rod. A
30cm column, 1.0 to 4.5cm i.d., is a useful size. The packing should be
about 20cm high. After packing, the bed of adsorbent is washed with
3 bed volumes of methanol, 3 bed volumes of chloroform-methanol 1:1,
and 3 to 5 bed volumes of chloroform. The packing should not be al-
lowed to run dry at any time. The packed, washed column should then
be tested. The recommended testing procedure is as follows:

To a column (2.5cm i.d. X 20cm) add a solution of 10 to 30mg of
cholesterol in 5 to 10ml of chloroform. Collect 10ml volume fractions
using chloroform as the eluting solvent (flow rate about 3ml/min.) and
test each fraction for cholesterol. The best test is to add 5 drops of
acetic anhydride and 1 drop of concentrated sulfuric acid to 1ml of
fraction. A green-blue color indicates cholesterol. The column is judged
satisfactory if cholesterol first appears in tube 7 to 9. If satisfactory,

COLUMN CHROMATOGRAPHY 39

the remainder of the cholesterol can be eluted with chloroform and
the sample applied.

There are many possible elution schemes depending on the separa-
tions desired. Many elution sequences have been described by Rouser
and co-workers (14, 15). Two of more general use are described below.

Separation of lipid samples into acidic and nonacidic fractions
(14). Using a 6cm long and 4.5cm i.d. column of DEAE prepared in
chloroform-methanol 2:1 v/v, apply the sample in the same solvent,
then elute with the following sequence at a flow rate of 10ml/min.:

1. Chloroform-methanol 2:1 v/v (4 column volumes)

2. Methanol (10 column volumes)

3. Chloroform-methanol 4:1 v/v made 0.01 to 0.05M with respect to
ammonium acetate, to which is added 20ml of fresh 28 percent w/w
aqueous ammonia2 per liter (10 column volumes)

4. Methanol (10 column volumes)

The first two fractions will contain all nonacidic lipids plus salts. The
third fraction will contain acidic lipids plus salts, and the fourth will
contain salts and possibly traces of lipids. The nonacidic lipids include
all the neutral lipids and nonionic glycolipids (like the cerebrosides and
phospholipids) that lack a negative charge. Acidic lipids include all those
having only negatively charged groups, such as fatty acids and cere-
broside sulfates, and those lipids that have at least one more negative
group than positive groups, such as phosphatidyl serine.

A general elution scheme (14). This procedure utilizes a 20cm
high, 2 to 5cm i.d. column of DEAE prepared in a slurry with choloro-
form. The sample is applied in chloroform, and the following solvents
are used at a flow rate of 3ml/min.

1. Chloroform (10 column volumes)

2. Chloroform-methanol 9:1 v/v (9 column volumes)

3. Chloroform-methanol 7:3 v/v (9 column volumes)

4. Chloroform-methanol 1:1 v/v (9 column volumes)

5. Methanol (10 column volumes)

6. Chloroform-glacial acetic acid 3:1 v/v (10 column volumes)

7. Glacial acetic acid (10 column volumes)

8. Methanol (4 column volumes)

9. Chloroform-methanol-ammonium salt (as described in step 3 of
the preceding procedure) (10 column volumes)

10. Methanol (10 column volumes)

Composition of the resulting fractions can be expected to be:

1. Neutral lipids

2. Cerebrosides, lysophosphatidyl choline, phosphatidyl choline,
sphingomyelin, and mono- and diglycosyl diglycerides

2 See footnote, p. 24.

40 COLUMN CHROMATOGRAPHY

3. Ceramide, aminoethylphosphonate, ceramide dihexosides and
polyhexasides, and phosphatidyl ethanolamine

4. Ceramide polyhexosides, lysophosphatidyl ethanolamine, and un-
characterized oxidation products

5. Oxidation products and salts

6. Free fatty acids, glycine conjugated bile acids, and unconjugated
bile acids

7. Phosphatidyl serine

8. Lipid-free fraction

9. Cardiolipid, phosphatide acid, phosphatidyl glycerol, phos-
phatidyl inositol, sulfolipid, sulfatides, and salts

10. Salts and traces of lipid

Of course, not all the lipids listed above are likely to be present in one
sample, and in most cases fractions will contain only one or two major
components. Thus, a sample of serum lipid would yield the following
fractions:

1. Neutral lipids

2. Lysophosphatidyl choline, phosphatidyl choline, and sphingo-
myelin

3. Phosphatidyl ethanolamine

4. Phosphatidyl serine

5. Phosphatidyl inositol

The first fraction could readily be separated on a silicic acid column
into glycerides, sterol esters, and free fatty acid fractions. Separation of
the second fraction would be best achieved by chromatography on a
silicic acid-silicate column. The latter column is prepared by treating
silicic acid with aqueous ammonia. Silicic acid (100 to 200 mesh) is
placed on a coarse sintered glass filter and washed first with 3 bed
volumes of 6N hydrochloric acid, then with 5 bed volumes of water.
The adsorbent is then heated at 120°C for 6 hours and cooled without
exposure to air. Using a 2.5cm i.d. column, a slurry of 25g of the
silicic acid is prepared in 40 to 100ml chloroform-methanol 1:1 v/v and
4ml of 28 percent aqueous ammonia. The chromatography tube is filled
to a height of 10cm. Before the sample is added, the column is washed
with 4 column volumes of chloroform. The fraction containing phos-
phatidyl choline, lysophosphatidyl choline, and sphingomyelin (50 to
75mg) is added to the column in about 5ml of chloroform.

The following elution sequence is then applied:

1. Chloroform-methanol 4:1 plus 1.0 percent (by volume) water
(8 column volumes)

2. Chloroform-methanol 4:1 plus 1.5 percent (by volume) water
(11 column volumes)

3. Methanol plus 2 percent (by volume) water (5 column volumes)

Fraction 1 will contain the phosphatidyl choline, fraction 2 the sphin-
gomyelin, and fraction 3 the lysolecithin.

COLUMN CHROMATOGRAPHY 41

Even the most complex mixtures of lipids can be separated by
chromatography on DEAE when followed by suitable rechromatog-
raphy of mixed fractions on silicic acid, silicic acid-silicate, Florisil,
DEAE, or TEAE columns. Rouser et al. (14, 15) should be consulted
for elution sequences suited to separation of complex mixtures.

The triethylaminoethyl (TEAE) cellulose differs from DEAE in
having a greater capacity for lipids that have carboxyl groups as their
only ionic group. Thus, it is an ideal choice for the separation of mix-
tures containing large proportions of fatty acids, gangliosides, and bile
acids. TEAE is usually used in the hydroxyl form, and elution se-
quences similar to those used with DEAE are generally employed (14).

REFERENCES

1. James, A. T., and A. J. P. Martin (1952) Biochem. J. 50: 679.

2. Stahl, E. (1958) Chem. Ztg. 82: 323.

3. Hagdahl, L. (1961) in Chromatography, ed. E. Heftmann ; p. 56. Reinhold
Publ. Corp., New York.

4. Wren, J. J. (1960) /. Chromatog.4: 173.

5. Hirsch, J., and E. H. Ahrens, Jr. (1958) /. Biol. Chem. 233: 311.

6. Fulco, A. J., and J. F. Mead (1961) /. Biol. Chem. 236: 2416.

7. Kishimoto, Y., and N. S. Radin (1963) /. Lipid Res. A: 130.

8. Goldfine, H., and K. Bloch (1961) /. Biol. Chem. 236: 2596.

9. Erwin, J., and K. Bloch (1963) /. Biol. Chem. 238: 1618.

10. DeVries, B. (1963) /. Am. Oil Chem. Soc. 40: 184.

11. (1964) /. Am. Oil Chem. Soc. 41 : 403.

12. Thomas, A. E., J. E. Scharoun, and H. Ralson (1965) /. Am. Oil Chem. Soc.
42 : 789.

13. Mattson, F. H., and R. A. Volpenhein (1962) J. Lipid Res. 3: 281.

14. Rouser, G., G. Kritchevsky, and A. Yamamoto (1967) in Lipid Chromato-
graphic Analysis, ed. G. V. Marinetti; vol. 1, p. 99. Marcel Dekker, Inc.,
New York.

15. Rouser, G., G. Kritchevsky, D. Heller, and E. Lieber (1963) /. Am. Oil
Chem. Soc. 40 : 425.

IV. Thin -Layer and Paper Chromatography

THIN-LAYER CHROMATOGRAPHY

Although the basic principle of thin-layer chromatography (TLC)
was first described many years ago, it did not become a popular tech-
nique until Stahl (1) reported his standardization of procedures.

In TLC the adsorbent is spread in a thin, even layer on a glass
plate, and the components of an applied sample are separated by allow-
ing a suitably composed solvent mixture to move through the adsorbent.
The technique is simple and, in many cases, more rapid than others.
The speed with which many separations can be achieved make TLC a
most useful adjunct to column chromatography. It is relatively simple
to apply a concentrated aliquot of solvent from a column chromato-
graphic fraction to a TLC plate, develop the plate, and by suitable de-
tecting agents determine the composition of the fraction. TLC is also
useful in many instances as a purification procedure. Moreover, TLC
enjoys wide popularity as an analytical tool in its own right; frequently
the analysis of very small samples, which would be extremely tedious
or impossible by other procedures, can be readily achieved by TLC.
The technique is not without hazards, however, and they will be noted
as each stage of TLC procedure is discussed.

Preparation of Thin-Layer Chromatographic Plates

Two sizes of glass plates are in general use: 4cm X 20cm and 20cm
X 20cm. While plates can be obtained commercially as matched sets,
it is usually more economical to have plates cut from plate glass and
smooth their edges. Plates should then be marked, on the basis of
evenness of thickness, as matched sets (5 large plates per set is useful
for most spreading devices).

The plates should be thoroughly cleaned. Just prior to use they
should be cleaned with a suitable solvent, such as chloroform or ben-
zene, to remove any traces of grease that would interfere with the
spreading of the adsorbent.

Several types of adsorbent are available. Some satisfactory ones
for universal use are the Merck Silica Gel G (with and without calcium
sulfate as binder), Merck Silica Gel H, and Supelcosil 12 B (Supelco,
Inc.). Rouser and co-workers (2) recommend the use of Silica Gel G
plain (no binder) plus 10 percent by weight magnesium silicate (Al-
legheny Industrial Chem. Co., P.O. Box 786, Butler, New Jersey) for
chromatography of polar lipids.

Sometimes it may be preferable to wash adsorbents before use since
impurities in them may interfere with subsequent analyses. A bulk

THIN-LAYER CHROMATOGRAPHY

washing procedure for Silica Gel H, as described by Parker and Peter-
son (3), is recommended. The Silica Gel H (125g) is weighed and
placed in a Buchner funnel on 2 pieces of Whatman No. 2 filter paper.
Over this is poured 1 liter of a mixture of formic acid (98 to 100 per-
cent), chloroform, and methanol 1:2:1 v/v/v. This is sucked through
by water pump vacuum, and the bed of Silica Gel H is then washed
with 500ml of deionized water. The adsorbent is then spread out on
an enamel or heavy aluminum foil tray and dried in an oven at 110 to
120° C for 48 hours. The lumps should be broken up after 24 hours.
When preparing a slurry from washed adsorbent, mix thoroughly in
a Waring Blendor.

The one disadvantage of washing procedures of this type is that
some characteristics of the gel change, and frequently it is difficult to
prepare smooth, even plates. Usually it is more convenient to wash the
adsorbent when it has already been plated. This is done by allowing a
solvent system to develop beyond the planned solvent front. Chloroform-
methanol-water, 65:25:4 v/v/v is a suitable solvent in most cases.

While TLC plates can be prepared by dipping and spraying, by far
the most successful and reproducible method is spreading, using a com-
mercial spreader and tray such as the Desaga ( Brinkmann Instruments,
Inc., 115 Cutter Mill Road, Great Neck, Long Island, N.Y.). The plates
are aligned on the tray and a slurry of the adsorbent is placed in the
spreader at one end of the board (Fig. 2). The slurry will vary in con-
sistency according to the thickness of the layer desired. A suitable
thickness for general use is 0.25mm. A fixed thickness 0.25mm
spreader is available; otherwise, the variable thickness spreader is set
accordingly. A slurry is prepared by mixing 20g of adsorbent
thoroughly in about 65ml water. Note (Fig. 2) that a small plate is
placed at each end of the board to allow for run-out from the spreader

small plate
(4cm x 20cm)

A thin-layer chromatography tray and spreader.

small plate

(Fig. 2)

44 THIN-LAYER CHROMATOGRAPHY

when it is opened and closed. These end plates are unevenly spread and
therefore are not used. The lever on the spreader is turned through 90°
and the spreader is moved steadily across the plates to the end, where
the lever is again turned through 90° to close. The speed with which
the spreader moves across the plates affects the thickness of the layers,
and some practice is required before plates of uniform thickness are
obtained. After the slurry has been applied, the spreader is removed
and washed. The plates are then vibrated slightly by bouncing the tray
up and down several times. This helps to even out the layers. The plates
are air dried and then dried at 120°C for 30 minutes to an hour in a
clean atmosphere free of vapors. It is best to prepare and use TLC
plates under standardized conditions ; otherwise, separations will not
be reproducible from one day to the next. If the atmosphere is very
humid, it may be necessary to treat plates in a humidifying chamber
to ensure a more constant water content.

Before applying samples to plates, the edges should be made uni-
form by stripping the first 4 or 5mm of adsorbent in a straight line. If
plates are to be developed in only one dimension, it is wise to divide the
plate into lanes by scoring through the adsorbent. This prevents lateral
spread of components towards each other.

Samples may be applied either as spots or streaks. They are usually
applied with Hamilton Syringes of 10 to 50/xl capacity (Hamilton Co.,
P.O. Box 307, Whittier, Calif.). It is usual to apply samples as a short
streak (1cm) made by overlapping several small spots or as a single
discrete spot. If TLC is being used to prepare pure samples ("prepara-
tive TLC"), the sample is applied as a large streak made by overlapping
spots straight across a large (20cm square) plate to within 1cm of the
edges, or by using a commercially available "Streaker" (Applied Sci-
ence, Inc., State College, Penn.). The sample load may vary consider-
ably and good results still be obtained. One may plate as little as 0.5/xg
of a single component (on a 0.25mm plate) and still be able to detect
it. A load of 1500/xg on a 0.25mm plate (to be developed in 2 dimen-
sions) is not uncommon, although loads of 250 to 500/ig are more usual.
With preparative TLC plates (layer thicknesses of 0.5 to 1mm), several
milligrams may be plated.

As a precaution against oxidation, samples should be plated under a
flow of nitrogen. Application chambers through which nitrogen can be
passed are available commercially; however, one can easily be made
from Plexiglas (Fig. 3). A box about 25cm square and 3cm deep is
made with an inlet and outlet for a nitrogen line. A narrow (1cm or
less) slot is made in the lid to coincide with a line about 1cm from the
bottom of a plate placed in the chamber. A sliding cover is fitted over
the slot so that the plate can gradually be covered as the sample is ap-
plied. A good flow of nitrogen is allowed to pass through the chamber
during plating.

THIN-LAYER CHROMATOGRAPHY 45

nitrogen out <*—mmm' " 77

/ / /■■^■r-^ — nitrogen in

// / sliding cover

Chamber for applying samples to TLC plates under a flow of nitrogen.

(Fig. 3)

Chromatograms are developed in glass chambers, in wide-mouth
Erlenmeyer flasks (for small plates), or in special "S-chambers." The
most commonly used are glass chambers 10^" X 2j4" X IO1/2"
(Brinkmann Instruments, Inc., 115 Cutter Mill Road, Great Neck, Long
Island, N.Y.; Analabs, Inc., P.O. Box 1501, North Haven, Conn.
06473; and other suppliers). The sides of the chambers should be lined
with Whatman No. 3 paper; just before use, 250ml of the solvent to
be used for developing the chromatogram should be placed in the
chamber, tilting the chamber back and forth to saturate the paper
with the solvent. A heavy glass lid is used without a grease seal.

For routine checking of column chromatography fractions, it is
often convenient to have a series of 1 -liter wide-mouth Erlenmeyer
flasks covered with one-half of a Petri dish. Each flask will accom-
modate two 4cm X 20cm plates. S-chambers are commercially available
vessels which sandwich plates within a narrow space, thus providing an
area uniformly and highly saturated with the solvent vapor. This type
of chamber is frequently used with the commercially available pre-
coated plates ("Prekotes," Applied Science, Inc.) or adsorbent coated
plastic sheets (Eastman Kodak). The S-chambers generally give the
most consistent results.

Detection of Lipids on Chromatograms

Treatment of developed plates with a variety of reagents allows
visualization of separated components and possible identification of
specific lipid functional groups. When combined with the chroma-
tography of standards developed under the same conditions, detection
of specific groups in most cases allows the identification of individual
lipids.

There are three general ways in which separated components may
be visualized. These methods show the position of the spots but give
little or no further information about the compound. These procedures
are as follows:

46 THIN-LAYER CHROMATOGRAPHY

1. Spray1 with a fluorescent reagent such as rhodamine or 2,2-
dichlorofluorescein and view under ultraviolet light. Protective glasses
must be worn when using UV. The rhodamine reagent is either
Rhodamine 6G or B, 0.05 percent by weight in 96 percent ethanol, and
the dichlorofluorescein is 0.2 percent by weight in 90 percent ethanol.
The plate must be viewed while still wet with the spray reagent.

2. Spray with a solution of 55 percent by weight sulfuric acid and
0.6 percent by weight potassium dichromate and char in an oven at
120°C.

3. Expose to iodine vapor in a closed chamber.

Method 2, the charring procedure, is fairly specific for lipids,
but carbohydrates also react. As little as 0.5/xg of a compound may be
detected in this way. However, there is the disadvantage that the lipids
are destroyed and cannot be recovered for further investigations.
Method 1, especially the dichlorofluorescein, is the method of choice
if the lipids are to be recovered for further analysis. Unfortunately,
visualization of small amounts of phospholipids is difficult with this
method. Method 3 is excellent in most cases but depends upon
the presence of unsaturated lipids, the reaction involved being that of
iodine with double bonds. Some saturated nitrogenous lipids (for ex-
ample, phosphatidyl serine) will take up iodine, but most other satu-
rated lipids will not. Since most natural lipids contain some unsaturated
moieties, this procedure can be widely used. Iodine vapor should not be
used when lipids are to be recovered for fatty acid analysis since some
iodine may remain associated with double bonds and lead to erroneous
results.

Sprays that can be used to detect the presence of specific elements
or groups and, therefore, the presence of lipid classes are listed below.

Acid-molybdate spray for detection of phosphorus-containing lipids.
This spray consists of 5ml of 60 percent perchloric acid, 10ml IX
hydrochloric acid, and 25ml of 4 percent w/v ammonium molybdate.
Make up to 100ml with distilled water. Spray and heat in oven at 110°C.
Blue spots on gray background are given by phosphorus-containing
lipids. Sensitivity is about 1/xg.

Ninhy drin spray for detection of lipids containing free amino groups.
A positive reaction to this spray is obtained with phosphatidyl ethanol-
amine and serine, their lyso derivatives, and any other lipid with a
primary amine group.

The spray is a 0.2 percent solution of ninhydrin (1,2,3-indantrione-
hydrate) in n-butanol-10 percent aqueous acetic acid 95:5 v/v. Spray
and heat in oven at 110°C. Purple spots indicate the presence of free

1 A number of spraying devices are available commercially and prepacked
sprays can be purchased. One useful spraying aid is the Sprayon-jet pack power
unit (Sprayon Products, Inc., Cleveland, Ohio). Note: Use all sprays in a hood.

THIN-LAYER CHROMATOGRAPHY 47

amino groups. Phosphatidyl serine does not always react well unless
some acetic acid (from a developing solvent) remains on the plate;
hence, the plate should be sprayed soon after development.

Dragendorff reagent for choline-containing lipids. Positive results are
given by phosphatidyl choline, its lyso derivative, sphingomyelin, and
related compounds.

A solution of 8.0g bismuth subnitrate in 20 to 30ml 30 percent nitric
acid (density 1.18) is added slowly, with stirring, to a solution of 28g
potassium iodide and 1ml 6N hydrochloric acid in approximately 5ml
water. The dark precipitate obtained will redissolve giving an orange-
red solution. Cool this to 5°C and filter. Make up to 100ml with dis-
tilled water. This is the stock solution. If stored in a dark bottle and
refrigerated, the stock is stable for 1 to 3 months. To make the spray
reagent, combine the following in the given order: 20ml water, 5ml
6N hydrochloric acid, 2ml stock solution, and 5ml 6N sodium hydrox-
ide. If the precipitate of bismuth hydroxide does not redissolve even on
shaking, add a few drops of 6N hydrochloric acid. The spray solution
is stable for 10 to 14 days in a refrigerator. Spray heavily. Positive is
a dark orange on a yellow background. Sensitivity is lO^g.

2,4-dinitrophenylhydrazine sptay for free aldehyde and keto groups.
A positive reaction is given by plasmalogens, but the spray is not very
sensitive. The SchifT reagent spray is better (see below).

Spray heavily with a 0.5 percent solution of 2,4-dinitrophenlhydra-
zine in 2N hydrochloric acid. Yellow or orange spots are positive.

Schiff reagent spray for plasmalogens. SchifT reagent is prepared by
dissolving lg basic fuchsin and lOg sodium metabisulnte in 10ml con-
centrated hydrochloric acid and 100ml water. This solution is treated
for 1 hour with charcoal, filtered, and made up to 500ml with distilled
water. This stock should be colorless. It is stable in a refrigerator for
several months. The spray reagent consists of 250ml 0.05 percent
sodium metabisulnte, 2.5ml 0.05 M mercuric chloride (1.35g in 100ml
water), and 2.5ml stock SchifT reagent. Spray heavily and allow several
minutes for the reaction. Mauve spots are positive.

Diphenylamine spray for glycolipids. Positive results with this spray
are given by all glycolipids, such as the cerebrosides.

The spray consists of 20ml of a 10 percent (by weight) ethanolic
solution of diphenylamine, 100ml concentrated hydrochloric acid, and
80ml glacial acetic acid. Spray and heat for 5 to 10 minutes at 100 to
120° C. Blue-gray spots on a light gray background are positive. Sen-
sitivity is within the range of 4.0 to 10.0(ug.

a-naphthol spray for glycolipids. This spray gives positive results with
sterols and steroid conjugates, as well as with glycolipids.

The spray consists of 10.5ml of a 15 percent (by weight) solution
of a-naphthol in ethanol, 6.5ml concentrated sulfuric acid, 50.5ml

48 THIN-LAYER CHROMATOGRAPHY

ethanol and 4ml water. Heat for 3 to 6 minutes at 100 to 110°C. Spots
of various colors are positive.

Resorcinol spray for gangliosides. Two grams of resorcinol are dis-
solved in 100ml distilled water. Ten ml of this solution are added to
80ml of concentrated hydrochloric acid containing 0.25ml of 0.1M
copper sulfate, and the resulting solution is made up to 100ml with
distilled water. This spray reagent is stable for about one week in the
refrigerator. Spray and heat in oven at 110 to 120°C in closed jar.
Gangliosides give violet-blue reaction; other glycolipids go yellow.

Other spraying methods. Many other sprays for specific groups can
be applied to TLC plates. Generally any spray reagent used in paper
chromatography can be applied with little or no modification to TLC
plates. The TLC plate has an advantage over the paper chromatogram
in that corrosive sprays can be used. The very corrosive procedure of
spraying with the sulfuric acid-chromate solution has the advantage that
observation of the spots during charring may provide additional infor-
mation. Sterols, such as cholesterol, and various bile acids undergo bril-
liant color changes, from green through red-purple, before blackening.

As with paper chromatograms, if radioactively labeled lipids are
used, spots may be detected by exposing the plates to X-ray film. In
these cases a permanent record is obtainable in the form of an X-ray
print. Otherwise, results can be documented by copying the patterns
into a notebook or by taking photographs with a Polaroid camera.

Whatever visualization and detection techniques are employed, it
is absolutely essential that standard lipids are run under the same con-
ditions. Runs should also be made using more than one solvent system
before identifications are made. Identification should never be made
on the basis of RF values2 alone since separations may vary with dif-
ferences in humidity, batch of adsorbent, etc. If the sample contains
rare lipids or is derived from a source not previously examined by
other investigators, any unusual lipids should be assigned an identity
only when additional aids have been employed. In such cases it is
necessary to isolate enough of the component in pure form to be ex-
amined by mass spectrometry, infrared spectroscopy, and other ana-
lytical aids.

Separation of Neutral Lipids

The choice of solvent systems for the development of chromato-
grams is quite wide and depends on the adsorbent used, the tempera-
ture and humidity conditions in the laboratory, and the complexity
of the mixture to be resolved.

In general, mixtures of low boiling petroleum hydrocarbons con-
taining various amounts of diethyl ether and benzene will separate

distance from origin to component spot

Rf value =

distance from origin to solvent front

THIN-LAYER CHROMATOGRAPHY

most neutral lipid mixtures. Neutral lipids are readily separated from
the polar in a number of ways. In many cases, development of the
chromatogram with a more polar solvent mixture such chloroform-
methanol-water 65:25:4 (all mixtures are given by volume) will move
the neutral lipids to or near the solvent front, whereas the polar lipids
will lag behind to varying degrees.

If free fatty acids, cerebrosides, and cardiolipin are present, how-
ever, there will be fraction overlap. In these cases the technique of
multiple development is useful. With this procedure, the plate is de-
veloped with the chloroform-methanol-water system to a height of
about 10cm, instead of the usual 15cm employed with average plates.
The plate is then dried and redeveloped in the same direction with a less
polar solvent, such as hexane-ether 4:1, to a height of 15cm (Fig. 4).
This solvent will hardly move the polar lipids, but will redistribute the

neutral
lipids

polar
lipids

<^- 2nd solvent front
(15cm from origin)

r |i

1st solvent front
(10cm from origin)

- 2nd solvent system

1st solvent system

origin

Separation of polar from neutral lipids by multiple development technique.
(1) Developing solvent allowed to rise to height of 10cm (chloroform-metha-
nol-water 65:25:4). (2) Developing solvent developed to height of 15cm
(hexane-ether 4:1). Adsorbent, Silica Gel G. (Fig. 4)

THIN-LAYER CHROMATOGRAPHY

•

solvent front

origin

Separation of some neutral lipids. Solvent system hexane-diethyl ether-gla-
cial acetic acid 90:10:1.

(1) 9-octadecene, (2) oleyl alcohol, (3) oleylaldehyde, (4) oleic acid, (5)
methyl oleate, (6) cholesterol oleate, (7) monolein, (8) diolein, (9) triolein,
(10) cholesterol, (11) tristearin, (12) phosphatidyl choline. Note that the
polar lipid (12) and monolein (7) do not move from the origin. Adsorbent,
Silica Gel G. (Fig. 5)

neutral lipids in the 5cm band above the polar lipids. It should be noted
here that if the lipids are to be recovered for further analysis, especially
for fatty acid and fatty alcohol analyses, the plates must be dried in a
box through which there is a flow of nitrogen.

Figure 5 shows the separation of some neutral lipids. The solvent
system is hexane-diethyl ether-glacial acetic acid 90:10:1. The glacial
acetic acid in the system prevents tailing of the more acidic components.
Similar separations can be obtained using a system without the acid and
with differing proportions of hexane-ether. The more polar the system
(that is, the more diethyl ether, or acid, or both, it contains), the further
it will move the relatively more polar materials.

Isomeric mono- and diglycerides can be separated by TLC. As we
previously noted (p. 36), 1- and 2-monoglycerides can be separated on
columns impregnated with boric acid. Similarly, these isomers may be
separated on Silica Gel G TLC plates which have been impregnated
with ammonium borate. The solvent system is chloroform-acetone 96:4.
The 1,2- and the 1,3-diglycerides may be separated on Silica Gel G by

THIN-LAYER CHROMATOGRAPHY 51

using petroleum hydrocarbon (boiling range 40 to 60° C) -diethyl ether
70:30.

Numerous other systems are available for special separations of
neutral lipid mixtures. Many of the earlier ones were reviewed by
Mangold and Malins (4), and more recently by Renkonen and
Varo (5).

Argentation Thin-Layer Chromatography

Fatty acids, their methyl esters, cholesterol esters, and glycerides
can be separated according to degree of unsaturation by TLC on silica
gel impregnated with silver nitrate. The cis and trans isomers of some
fatty acids can also be separated in this way.

Silver nitrate plates can be made in several ways. Some investiga-
tors prepare plates by spraying silica gel plates with a 10 percent
solution of silver nitrate in aqueous ethanol. "Prekotes" or other com-
mercially available plates can be impregnated with silver nitrate by
dipping the plates into a 10 to 12 percent solution of silver nitrate.
The most reliable procedure, however, is to prepare the silica gel
slurry in silver nitrate solution rather than water and to spread the
plates in the usual manner. The following formula will generally give
satisfactory plates, although it may be necessary to adjust to the pre-
vailing humidity conditions.

Weigh 25g silver nitrate and dissolve in 200ml distilled water.
Weigh 60g silica gel and prepare a slurry using 136ml of the silver
nitrate solution. Make sure that the slurry is very thoroughly mixed
by using a Waring Blendor or by shaking vigorously in a stoppered
flask for about 10 minutes. Prepare plates in the usual way; dry and
store plates in a dark place.

These plates can be spotted in dull light ; however, they should be
developed in a dark jar or the developing chambers should be placed
in a dark place. Depending upon the separations desired, hexane-diethyl
ether in varying proportions will be found suitable in most cases. Figure
6 illustrates the separation of several cholesterol esters on silver ni-
trate impregnated Silica Gel G. The plates were developed with hexane-
ether 93:7 for monoenoate separation and 93:17 for dienoates.
To separate the more unsaturated compounds (trienoates, tetraenoates,
etc.), increasing quantities of ether in hexane must be used. For
example, a mixture of saturated, monoene, diene, triene, tetraene-
pentaene-hexaene (polyene fraction) of fatty acid methyl esters can
be separated into 5 bands by using 60:40 hexane-ether, although there
may be slight overlap. Lipids can be recovered from the scraped off
silica gel by extraction with moist diethyl ether. Rechromatography on
silver nitrate impregnated plates will yield purified lipid fractions.

Solvent systems similar to those described above can be used to
separate various glyceride mixtures on the basis of unsaturation. Each

THIN-LAYER CHROMATOGRAPHY

Plate 1 : monoenoates

Plate 2: dienoates

Thin-layer chromatography of cholesterol esters on Silica Gel G impregnated
with silver nitrate. Plate 1: (a) mixture of cholesterol palmitate, elaidate,
oleate; (b) stearolate [CH3(CH2)7C = C(CH2)7COOH is stearolic acid];
(c) mixture of elaidate, oleate, stearolate; (d) trans and cis vaccenate;
(e) trans and cis erucate; (f) trans and cis petroselenate. Plate 2: (a)
linoleate; (b) 9-cis- 12- trans octadecadienoate; (c) linelaidate; (d) mixture
of a, b, and c; (e) 9-trans-12-c/s octadecadienoate. (After Sgoutas, IS.)

(Fig. 6)

investigator will, however, find some trial runs necessary in order to
ascertain the particular solvent systems suited to his conditions.

Separation of Phospholipids and Glycolipids

Numerous solvent systems for one-dimensional TLC of phospho-
lipids and glycolipids have been reported. Among those that give
satisfactory separations of mixtures containing most of the common
phospholipids and cerebrosides are: (a) chloroform-methanol-28 per-
cent aqueous ammonia 75:25:4 (on Silica Gel G) ; (b) chloroform-
propionic acid-n-propanol-water 10:10:10:4 or 2:2:3:1 (on Silica Gel
G plates prepared in 5 percent ammonium nitrate); (c) chloroform-
methanol-acetic acid- water 25:15:4:2 (on Silica Gel G plain prepared in
0.001M sodium carbonate).

For routine checking of column chromatography fractions and for
many analyses, unidimensional systems are suitable. If, however, it
is desired to separate all the phospholipids and glycolipids of a multi-
component mixture or to characterize a lipid mixture from a new
source, then two-dimensional TLC must be used. Furthermore, it is
highly desirable that the number and identity of components in a
complex mixture be established with more than one set of developing
solvents. Several such systems have been developed by Rouser et al. (2).

THIN-LAYER CHROMATOGRAPHY 53

In two-dimensional TLC the sample is spotted in one corner of a
20cm X 20cm plate and developed in one direction with one solvent
system; then, after removal of the first solvent, the plate is turned
through 90° and developed in the second direction with solvent 2. The
adsorbents used may be any commonly used TLC adsorbent. The
chromatogram maps shown in Figure 7 (p. 54) illustrate separations
obtained by using pairs of solvent systems.

In the separations shown in Figure 7, the gangliosides are present
as one spot. However, gangliosides can be separated into mono-, di-,
tri-, and tetrasialogangliosides by using several solvent systems. The
system described by Kuhn and Wiegandt (<5) gives good separation of
the major gangliosides. The solvent system is propanol-water 7:3 (on
Silica Gel G). Chloroform-methanol-water 60:35:8 (on Silica Gel G)
also gives good separation of gangliosides (7). Leedeen (8) has de-
scribed a multiple development system that gives good resolution of
major gangliosides; double length (40cm) plates and two sucessive
runs of chloroform-methanol-2.5N ammonia 60:40:9 are used.

Quantitative Thin-Layer Chromatography

There are several possible approaches to quantitative analysis by
TLC. These approaches can be divided into four main groups.

1. Direct analysis on the TLC plate.

a. Charring under standard conditions and subjecting the plate
to photodensitometry.

b. Direct radio scanning (when radioactively labeled lipids are
being handled).

c. Neutron activation analysis.

2. Gravimetric analysis by recovery of the lipids from each spot.

3. Analysis for specific elements or functional groups on lipids
extracted from the adsorbent.

4. Analysis for specific elements or functional groups on lipid
plus adsorbent.

In the first group, charring followed by densitometry has been used
successfully by a number of investigators. This method is, however,
often difficult and tedious to standardize. Standard lipids must be
charred and separate curves prepared for the densitometry of each.
The size of spots for each lipid should be fairly consistent from day
to day, which is often difficult to achieve. Moreover, charring should
ideally convert the lipids quantitatively to carbon, but this is not easily
achieved in practice. Other detection methods besides charring have
been successful. Such methods are usually specific for lipids containing
one specific group; for example, staining with chlorox-benzidine re-
agent is specific for amide groups. Sphingolipids have been analyzed
by densitometry after having been stained by this method (9).

THIN-LAYER CHROMATOGRAPHY

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THIN-LAYER CHROMATOGRAPHY 55

Radio scanning of TLC plates, using a proportional gas-flow counter
tube, is generally successful but is of limited value.

Neutron activation analysis has been tried and promises to be of
great value in the analysis of very small quantities of lipids. This
approach awaits further development.

Gravimetric analysis is not generally a reliable approach. It is
often adequate on a preparative TLC scale when large quantities are
being chromatographed and recovered. At lower levels, however, it is
often difficult to recover lipids completely from the adsorbent, par-
ticularly polar lipids. It is not recommended as an analytical procedure.

The third approach suffers the same disadvantage as gravimetric
analysis in that complete or consistent recovery of lipids from the
adsorbent is frequently difficult and tedious.

The fourth approach is less cumbersome than other methods and,
in general, is the best procedure. It is essential, however, that pre-
cautions are taken against interference with the analysis by the adsor-
bent itself, by impurities in it, or by reagents used for localization of
the spots. Exposing plates to iodine vapor and outlining spots with a
needle is ideal in many cases. The iodine resublimes fairly rapidly and
the spots can then be removed either by scraping or aspirating off.
This method cannot be used if the lipids are being removed for analysis
of their fatty acid moieties, since some iodine may remain irreversibly
added to double bonds. In such cases, it is best to localize spots by
spraying with one of the fluorescent dyes like 2,7-dichlorofluorescein.

After the spots are removed and put into suitable containers, many
analyses can be carried out directly on the adsorbent plus lipid. For ex-
ample, phosphorus analyses for the relative proportions of phospho-
lipids can be carried out in the presence of adsorbent directly after
digestion of the lipid. Detailed instructions for determination of organic
phosphorus and other analytical procedures are given in Chapter 6.
Procedures for the analysis of fatty acids and fatty aldehydes of lipids
recovered from TLC plates are given in the chapter on gas-liquid
chromatography.

A scheme for analysis of phosphoglycerides. Lipids from natural
sources are complicated mixtures of different molecular species. A
triglyceride preparation that contains only two different fatty acids
could actually be a composition of six different species. Thus, with
fatty acids A and B, we can have triglycerides AAA, BBB, AAB,
BBA, ABA, and BAB. Similarly, with a phosphoglyceride containing
only two fatty acids, we could have four species: AA, BB, AB, BA.
With increasing frequency, answers to the role in biological systems
demand that we know the pattern of the existing molecular species.
In the case of glycerides, there is already a vast amount of knowl-
edge about determining structural patterns, and references to pertinent
methods and results are given in Suggested Further Readings. In the

56 THIN-LAYER CHROMATOGRAPHY

case of polar lipids, however, only in the last few years has progress
been made in the analysis of molecular species. Renkonen (10)
demonstrated that much could be learned about the molecular species of
many natural phosphatides by examining the diglyceride parts of these
molecules by thin-layer chromatography. Kuksis and Marai (11) ex-
tended this approach to include examination of the derived diglyceride
acetates by gas-liquid chromatography. They first applied this technique
to the determination of the complete structure of Qgg yolk lecithins.
More recently Kuksis et al. (12) have shown that the method can be
applied successfully to the determination of the molecular species of
the lecithins from rat heart, kidney, and plasma. The outline of their
procedure (summarized in Fig. 8) is as follows:

1. The phosphoglycerides to be studied are isolated and purified
by column or preparative thin-layer chromatography. Aliquots of the
isolated pure phosphoglycerides are taken for immediate transmethyla-
tion. The methyl esters of the fatty acids thus obtained are analyzed
by gas-liquid chromatography (see Chapter 5 for details of GLC).

2. Separate aliquots of the pure phospholipids are converted directly
to the diglycerides by dropping the removed silica gel bands into a buf-
fered phospholipase C solution (10). The incubations are continued
for 30 to 60 minutes at 28 to 30 °C under a layer of diethyl ether.

3. At the end of digestion the phases are separated and further
extractions with ether are made. The combined solvents are evaporated.

4. The diglycerides obtained are purified by TLC on Silica Gel
H (Merck & Co.) using petroleum ether-diethyl ether- formic acid
120:40:3 v/v/v as the developing solvent.

5. The diglycerides are recovered from the scraped-ofr* bands (using
2,7-dichlorofluorescein for location) by elution with chloroform.

6. The diglycerides are dissolved in pyridine and converted to the
diglyceride acetates by treatment with acetic anhydride at room tem-
perature for 12 hours. If the original lipid sample is 400 to 500mg,
the diglycerides obtained should be dissolved in about 0.1ml pyridine
and treated with 0.25 to 0.50ml acetic anhydride.

7. The diglyceride acetates are separated according to degree of
unsaturation by TLC on 0.25mm thick Silica Gel G plates (20cm X
20cm) containing 20 percent silver nitrate. About 10 to 15mg of ma-
terial is applied as a band and the plate is developed twice in the same
direction with a solution of 0.7 or 0.8 percent methanol in chloroform.
The bands are located using the fluorescein reagent. The individual
bands are recovered by eluting with chloroform-methanol 9:1 v/v to
which 5 percent water has been added.

8. The eluates are reduced to dryness under nitrogen and subjected
to gas-liquid chromatographic analysis.

THIN-LAYER CHROMATOGRAPHY

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58 THIN-LAYER CHROMATOGRAPHY

9. The mixture of diglyceride acetates (from step 6) is analyzed by
gas-liquid chromatography in order to obtain the overall molecular
weight distribution.

10. Further information is obtained by subjecting the diglyceride
acetates to hydrolysis by pancreatic lipase, which selectively removes
the fatty acid in the a(l) -position. The free fatty acids and mono-
glycerides obtained are removed separately after thin-layer chroma-
tography as described for the diglycerides (step 7).

11. The positional distribution of the fatty acids in the original
mixture is determined by hydrolysis with phospholipase A, which re-
moves the fatty acids in the f3 (2) -position ; the fatty acids are then
converted to their methyl esters and analyzed by gas liquid chroma-
tography. This allows for verification of the positional distribution of
the fatty acids derived from the lipase digestion.

The two enzymic incubations in this procedure are carried out as
follows :

The lipase digestion of the diglyceride acetates. Fifty to lOOmg of
sample is added (in a 5ml screw cap vial) to a predetermined weight
of pancreatin (Steapsin, Nutritional Biochemical Corp.). The amount
of enzyme chosen is that which hydrolyses 50 percent of a given weight
of diglyceride acetate in 1 to 2 minutes. One ml of 1M trishydroxy-
methylamino methane (pH 8.0), 0.1ml 22 percent calcium chloride, and
0.25ml 0.1 percent bile salts solution are added to sample plus pancreatin.
The vial is warmed in a water bath at 40° C for 1 minute. The cap is
then screwed on tightly and the vial is shaken for the time required;
for 50 percent hydrolysis, this is usually 1 to 2 minutes. At the end of
reaction the contents are acidified with 6 N HCL and extracted with
ether.

The phospholipase A digestion of the phosphoglyceride. From each
sample, 150 to 200mg of phosphoglyceride is dissolved in 100ml diethyl
ether and treated with 1ml of 0.1 percent rattlesnake venom (Crotalus
adamenteus) in 0.005M CaCl2 solution. The solution is allowed to
stand overnight, after which the precipitate is removed by centrifuga-
tion and washed once with diethyl ether. The ether phase should be
free of phosphorus (it should contain only free fatty acid) if the
reaction is complete.

This scheme, therefore, supplies the following information:

1. The total fatty acid composition of the phosphoglyceride (step
A in Fig. 8).

2. The overall molecular weight distribution of the diglyceride
acetates derived from the phosphoglyceride (steps B, C, D).

PAPER CHROMATOGRAPHY 59

3. The overall molecular weight distribution and proportional
contribution of the diglyceride acetates according to their degree of
unsaturation (steps B, C, E).

4. The overall composition and positional placement of the fatty
acids in the derived diglyceride acetates (steps B, C, G, H).

5. The fatty acid composition of position 2 in the original phospho-
glyceride (steps I & J), a verification of the information derived in 4.

From the above information it is, therefore, possible to deduce the
position of the fatty acids in the phosphoglyceride and to describe the
molecular species present. The method allows for cross-checking in
that the total fatty acids composition of the original phospholipid
(under 5 above) should be the same as that of the derived diglyceride
acetates (under 4 above) .

The conditions suitable for the GLC analysis steps in the above
scheme are to be found in the next chapter (see pp. 76-78, 82).

PAPER CHROMATOGRAPHY

Early attempts to apply paper chromatography to the resolution
of naturally occurring lipid mixtures were only partially successful.
It was not until chromatography on impregnated papers was introduced
that more successful separations were made. Papers have been treated
in various ways, including acetylation and impregnation with tetralin
and formalin. The most successful and widely used procedure is chro-
matography on paper impregnated with silicic acid, a technique de-
veloped mainly by Marinetti (14) and his co-workers. This approach
enjoyed wide popularity for some time until the development of TLC
procedures, which are more rapid and more versatile than paper chro-
matography and allow a wider range of sample loading. Paper chroma-
tography is, however, still popular in a number of laboratories for
some special purposes. A number of reviews give details of paper
chromatographic procedures (14, 15, 16).

One special use of paper chromatography in lipid analysis should
be mentioned here, the method of phosphatide analysis introduced by
Dawson in 1954. Very mild hydrolysis is used to deacylate phosphatides,
after which the water soluble phosphorus-containing compounds are
analyzed by paper chromatography. Again, after the introduction of
TLC this method declined in popularity. It does, however, offer su-
perior resolution of the acidic phosphatides. If a detailed analysis of
previously uncharacterized lipids is being undertaken, this method pro-
vides a valuable adjunct to TLC and column procedures for the analy-
sis of intact lipids. Dawson (17) has recently reviewed this procedure
in detail and discusses those areas in which it offers special advantages.

60 THIN-LAYER CHROMATOGRAPHY

REFERENCES

1. Stahl, E. (1958) Chem. Ztg. 82: 323.

2. Parker, R, and N. F. Peterson (1965) /. Lipid Res. 6: 455.

3. Mangold, H. K., and D. C. Malins (1960) /. Am. Oil Chem. Soc. 37: 383.

4. Renkonen, O., and P. Varo (1967) in Lipid Chromatographic Analysis, ed.
G. V. Marinetti; vol. 1, p. 41. Marcel Dekker, Inc., New York.

5. Rouser, G., G. Kritchevsky, and A. Yamamoto (1967) in Lipid Chromato-
graphic Analysis, vol. 1, p. 99.

6. Kuhn, R., and H. Wiegandt (1963) Chem. Ber. 96: 866.

7. Wagner, H., L. Horhammer, and P. Wolff (1961) Biochem. J. 334: 175.

8. Leedeen, R. (1966) /. Am. Oil Chem. Soc. 43: 57.

9. Austin, J. H. (1966) /. Neurochem. 10: 921.

10. Renkonen, O. (1966) Lipids 1: 160.

11. Kuksis, A., and L. Marai (1967) Lipids 2: 217.

12. Kuksis, A., W. C. Breckenridge, L. Marai, and O. Stachnyk (1969) /. Lipid
Res. 10 : 25.

13. Sgoutas, D. S. (1968) Biochim. Biophys. Acta 164: 317.

14. Marinetti, G. V. (1962) /. Lipid Res. 3: 1.

15. (1964) in New Biochemical Separations, ed. A. T. James and L. G.

Morris ; p. 339. Van Nostrand, Princeton, NJ.

16. Kates, M. (1967) in Lipid Chromatographic Analysis, vol. 1, p. 1.

17. Dawson, R. M. C. (1967) in Lipid Chromatographic Analysis, vol. 1, p. 163.

V. Gas-Liquid Chromatography

So far we have considered chromatographic techniques in which
the moving phase was a liquid and the stationary phase a solid.
In gas-liquid, or gas-liquid partition, chromatography (GLC), the
moving phase is a gas and the stationary phase is a liquid. The liquid
is spread over an inert solid support, usually a flux-calcined, diato-
maceous earth of fairly small particles. Separation takes place in a
series of partitions whereby the sample goes into solution in the liquid
phase and is subsequently revaporized. The length of time it takes a
component to emerge from a GLC column depends upon the affinity
it has for the liquid phase and upon its boiling point. Thus components
are separated because of differences in their affinity for the adsorbent
and differences in their boiling points.

INSTRUMENTATION

A typical GLC apparatus consists of the following units:

carrier gas — » injector — -> column packed — > detector — > signal
(moving system with solid sup- system recorder

phase) port coated with

liquid phase;

column enclosed

in oven

The various components of an injected sample separate into discrete
bands in the gas flow and move through the column at different veloc-
ities. As they emerge, they pass through a detector system and a signal
is sent to a millivolt recorder. The record so obtained is termed the
chromatogram. A typical chromatogram is shown in Figure 9.

The time lapse measured between sample injection and the apex
of a component peak is termed the retention time. Frequently, a small
positive or negative recorder response at the point of injection makes
a suitable start for measurement. If this response is not obtained,
measurements may be made starting at the apex of the solvent peak.
The distance may be measured in any units (cm., inches); the reten-
tion time is then calculated from this measurement and the speed of
the recorder chart. Before discussing the routines for making qualita-
tive and quantitative analyses, each unit of the typical GLC instrument
will be considered in a little more detail.

GAS-LIQUID CHROMATOGRAPHY

solvent

injection — i ^ ^j

of sample i | I

retention
time for A

A typical gas chromatogram.

time'

(Fig. 9)

Carrier gases. The selection of carrier gas is dependent upon the
type of detector, as shown here:

Detector

Flame ionization
Thermal conductivity
/3-ionization
Electron capture

Carrier gas

Nitrogen, helium, argon
Hydrogen, helium
Argon
Argon

The carrier gas must be pure, inert, and dry. It is best to dry the
gas by passing it through a molecular sieve, which can be reacti-
vated periodically by heating at approximately 350°C for four hours
with the carrier gas flowing through it. Impure or wet carrier gas will
lead to such problems as the appearance of "ghost" peaks, decreased
column life, baseline drift, and noise.

The carrier gas should pass through a flow regulator, as carrier
gas flow rates must be adjusted to achieve optimum efficiency. Optimum
flow rates will vary from column to column.

GAS-LIQUID CHROMATOGRAPHY 63

Injection system. The usual injection system consists of a heated
injection port with a self -sealing rubber or silicone septum through
which the sample can be introduced into the gas stream with a micro-
liter syringe. Most injection ports allow for projection of the column
into the injection area so that on-column injection is possible.

The injection port temperature must be controlled and must be
high enough to vaporize samples. The injection system should be leak-
proof and must be so designed as to prevent back flow into the carrier
gas inlet. The velocity of gas flow through the injection port must
sweep the vaporized sample rapidly into the rest of the system.

Column technology. This is the most important part of the chroma-
tograph. Problems can often be traced to a faulty column. The column
consists of three parts: the container, which may be metal (copper,
stainless steel) or glass; the solid support; and the stationary phase.

For some analyses, including the analysis of sterols, a glass column
and injection port are essential, since metal, especially copper, may
catalyze sample breakdown. In general, however, stainless steel is
satisfactory.

The purpose of the solid support is to hold the liquid phase. The
support can, however, influence events occurring within the column
by adsorbing components at active sites or by catalyzing reactions due
to trace metal impurities. The former problem is alleviated by treating
the support with a silylating reagent, such as bis-trimethylsilylacetamide
(BSA). Acid washing will remove metals.

The choice of column, solid support, and liquid phase varies with
the separations being attempted. The catalogs of all leading suppliers
generally recommend specific packings for various types of separations.
In the sections that follow, suggestions for column packings are in-
cluded in the discussions of the various separations.

Length of column may vary from a few inches to about one mile.
The latter extreme is the case for capillary or Golay columns, in which
the support and liquid phase are merely coated on the inside of capil-
lary tubing. Such columns are especially useful in some tricky situations
involving geometric isomer separations. For general use, columns are
18 inches to 6 feet long and U-shaped, straight, or coiled, depending
on the instruments.

The temperatures at which columns are used depends on the com-
pounds being analyzed but above all on the stability of the stationary
phase. The maximum temperature at which various phases are stable
is reported by suppliers. This is a most important consideration since
excessive bleed of liquid phase from the column or its breakdown will
lead to problems, including fouling of the detector system. The per-
centage of liquid phase used varies from as little as 1 percent to as
much as 30 percent; most analyses, however, are achieved with con-
centrations of 1 to 10 percent. Before use, a new column is always

64 GAS-LIQUID CHROMATOGRAPHY

"bled out" (usually overnight or for 24 hours) to rid the column sup-
port of excess phase. This is generally done with the column detached
from the detector.

Columns may be operated isothermally for analysis at a predeter-
mined optimum temperature, or the temperature may be programmed
to rise from a given lower temperature to a given maximum tempera-
ture during each run. The rate of rise of temperature may vary from
3 to 18°C per minute. Temperature programming is especially useful
when samples with a wide range of boiling points are being analyzed
or when samples contain components that elute over a long period of
time. Programming is not merely a time saver, however; it also im-
proves the general symmetry of the recorded peaks. As we shall see,
this is a big advantage in quantitative analysis. The longer a com-
ponent remains on a column, the broader its band becomes as it dif-
fuses during passage through the column. The result is loss of peak
response, since small amounts of the diffused band (with and against
the direction of carrier gas flow) are not detected. Instead, the peak
may appear as a long hump on the baseline. Temperature programming
reduces the length of time that high-boiling and slow-eluting compo-
nents remain on the column, and the decrease in diffusion leads to
sharp, well-defined peaks.

Detector systems. The separation achieved by the column must be
translated into an electrical signal which can be fed into a recorder
and used for qualitative and quantitative analysis. The translation is
done by a detector. The "perfect detector," one which responds linearly
and identically to any amount of any type of compound, does not exist.
Each detector has a finite range within which response changes linearly
with the quantity of components. This is called the linear dynamic
range (LDR) of the detector. If the detector is used outside its LDR,
the peak shapes become distorted and reproducibility decreases. Detec-
tor responses also depend on the functional groups on the component
being analyzed. An equal number of moles of a hydrocarbon and a
fatty acid methyl ester will not give the same signal. Some calibration
of the detector response is required for every compound being analyzed
quantitatively. These and other factors involved in detector technology
will be discussed for each type of detector considered.

Of the many kinds of detectors, the most commonly used are the
thermal conductivity cells and the ionization-type detectors. Thermal
conductivity (TC) detectors detect changes of thermal conductivity in
the carrier gas when it becomes diluted by components in the sample.
The more highly conducting the carrier gas is, the greater will be the
thermal conductivity change when a component enters the TC cell.
Hydrogen and helium are recommended carrier gases. When the
thermal conductivity of the component is less than that of the carrier
gas, sensitivity is good but will diminish as the thermal conductivity

GAS-LIQUID CHROMATOGRAPHY 65

approaches that of the carrier gas ; response will be a negative peak if
the thermal conductivity of the carrier gas is exceeded.

A filament heated by direct current is usually the varying resistance
element. Two of these elements or two matched pairs of elements are
placed in two streams of carrier gas; they are called the reference and
the sensor elements. The temperature of the elements depends on the
carrier gas conductivity, its flow rate, and the temperature of the detec-
tor block. To decrease noise and increase cell life, filament detector
cells must be run as cool as possible, consistent with the boiling points of
the sample components.

The typical circuitry for a TC detector is a Wheatstone bridge used
as a temperature sensing bridge. When a component dilutes the carrier
gas and passes with it over the sensor element, the element heats up
and its resistance increases. As a consequence, there is an out-of-
balance signal between the sensor element and the reference element
(over which pure carrier gas flows). This signal is transposed to a
voltage output and fed to the recorder.

The most popular type of TC detector employs the hot wire element.
This type of detector has a wide temperature range, does not destroy
the component (which can, therefore, be collected and fed into other
instruments such as infrared spectrophotometers or mass spectrom-
eters), and has no selectivity so far as any particular type of com-
pound is concerned. If conditions are favorable, a hot wire detector
can measure as little as 0.1 fig.

The thermistor, another type of varying resistance element, is in-
frequently used now except for gas analysis. It is very efficient in this,
measuring in the parts per million range. Sensitivity decreases above
150°C.

Ionization detectors have more complicated circuitry than TC detec-
tors, are more trouble to maintain, and, since they show selectivity of
response, require more detailed calibration and sample handling. They
are, however, very sensitive, giving in some cases full-scale recorder
deflection in response to 1 picogram (10~12 g) of component.

The flame ionization detector (FID) is one of the most popular
detectors. It measures the minute current developed when a combustible
material in carrier gas enters a hydrogen-air flame. A d.c. potential
is applied across the flame by a pair of electrodes. As the component
burns, the electrodes collect the charged species and the resulting cur-
rent is amplified by an electrometer and led to a recorder. Routine use
allows analysis of 10 nanograms (1 nanogram = 10~9 g), and under
favorable conditions 0.1 nanogram can be measured. Since response
is a function of the number of carbon atoms and the groups in which
they appear, the detector must be standardized and calibrated to handle
a specific type of sample. This detector has some degree of selectivity
since poorly combustible materials such as carbon tetrachloride give
weak signals; the detector is also not suitable for the analysis of many

66 GAS-LIQUID CHROMATOGRAPHY

gases. However, the FID detector is not so easily damaged as the TC
detector and is readily cleaned.

Another type of ionization detector is the Argon, Beta Ray, or
Alpha Ray type. The usual name is the Argon Ionization Detector,
since argon is the commonly used carrier gas for these detectors; Beta
Ionization Detector is also frequently used, whether the ionizing source
emits /3- or a-particles. In this detector, radiation from an isotope
source ionizes a small percentage of the carrier gas (argon) atoms,
giving rise to an "ionization" or a "standing" current. The isotope
sources are strontium 90 or tritium (/3-particle emitters) or radium
226 (an a emitter). A few argon atoms receive less than enough energy
to be ionized but enough to displace an electron from its normal orbit
or shell — about 11.6 electron volts. Such atoms remain in their meta-
stable state until something collides with them. If one collides with a
sample component that needs less than 11.6 ev to be ionized, ionization
will take place and another electron is set free in the ionization cell.
If a polarizing voltage is applied across the cell, these ionized sample
molecules will increase the ionization (or "standing") current. Increas-
ing the cell voltage increases the number of metastable atoms as fol-
lows: the electrons composing the ionization current accelerate more
rapidly; the kinetic energy of the electrons is transferred to the argon
atoms during collisions, resulting in more transfers of 11.6 ev ; and
more metastable atoms are formed which, in turn, ionize more sample
molecules. Therefore, raising the cell voltage (and hence the number
of metastable argon atoms per unit volume) results in a higher ioniza-
tion efficiency for sample molecules. As with other detectors, changes
in ionization current are fed to a recorder.

Still another type of ionization detector is in common use for
certain types of analysis: the Electron Capture (or Attachment)
Detector (ECD). Here /^-particles generate an atmosphere of electrons
and positive ions. The electrons are collected at the cell anode. There
is a low voltage on the electrodes when only carrier gas is in the cell.
If sample molecules which readily accept electrons enter the cell, the
low-energy electrons may attach themselves to the molecules. Negative
ions of sample components and positive argon ions combine much
faster than electrons and positive ions, hence components which have
a high electron affinity reduce the primary ionization current. This
detector is much more selective than any we have discussed so far,
particularly, for example, towards halogenated compounds. For this
reason, the ECD is much used by investigators measuring small quan-
tities of chlorine-containing pesticides. ECD sensitivities are in the
picogram range.

QUALITATIVE ANALYSIS

If it is desired to analyze completely unknown samples, GLC is
not a suitable technique when used alone ; other techniques such as

GAS-LIQUID CHROMATOGRAPHY 67

infrared and mass spectroscopy must also be used. One must, there-
fore, have some idea of the type of compound being investigated. The
only definitive property of a compound measured by GLC is its reten-
tion time, and that only to the extent that all conditions are carefully
standardized during the chromatographic run. Temperature, gas flows,
etc., should be controlled carefully; if temperature is programmed,
the rate of rise should be uniform. Also, the retention time should be
measured from some reproducible point, for example, as we previously
noted, from the solvent peak. The start may be taken as the leading
edge of the solvent peak or its apex, whichever is more convenient.
With a TC detector the air peak is frequently a suitable starting point.
Retention time (RT) itself varies with several instrument parameters,
including column temperature, column length, flow rate of carrier gas,
type of liquid phase, loading of sample, and weight of column packing.
This makes RT not too reliable a figure for correlating data obtained
at different times or in different laboratories. A more reliable param-
eter is relative retention time (RRT), the ratio of the RT of a known
standard component to the RT of the sample.

If one has some preliminary information about the unknown ob-
tained by other means (having determined functional groups by spot
tests, infrared analysis, etc.), one may proceed with some GLC analy-
sis. For example, suppose that preliminary analysis indicates that the
sample is a mixture of aliphatic alcohols: then the number of com-
ponents, their chain length, etc. can be determined by GLC. Chain
lengths are determined by utilizing the fact that for various members
within a chemical class (the n-alcohols, the ethers, the ketones, the fatty
acids, etc.) log RT varies linearly with molecular weight. Thus, if one
chromatographs a series of saturated n-alcohols on a suitable phase
(silicone or carbowax), measures retention time, and plots log RT
against the number of carbon atoms in the alcohol, a straight line is
obtained (Fig. 10). A series of straight line graphs can be obtained
from the mono-, di-, and tri-unsaturated species, and so on. Thus, the
possession of a series of standards enables one to test the unknown
for fit on one of the curves. Ideally, unknowns and standards should
be run on the same day and, of course, under standardized conditions
of column, flow rate, and temperature.

In addition to determining functional groups by conventional means,
it is often possible to use GLC data to find out what type of compound
one is dealing with. This requires collecting RRT data for a large num-
ber of functional group classes on at least two standard columns, such
as silicone and carbowax. Because of the linearity of homolog retention
on the two columns, a plot similar to that in Figure 11 can be obtained.
Thus, by injecting an unknown on two columns, a compound can be
identified that fits several of the prepared standard plots.

The utility of GLC is enhanced when combined with a number of
other analytical procedures; in particular, chromatographing an un-

GAS-LIQUID CHROMATOGRAPHY

c
o

u
bJD

number of carbon atoms
Results of chromatography of homologs.

(Fig. 10)

c
o

esters

ketones

alcohols

RRT on carbowax column
Chromatography of homologs on two columns.

(Fig. 11)

known before and after a specific chemical treatment designed to remove
or modify specific groups yields information in the form of missing and
enlarged peaks. Treating the sample vapor with sodium metal will, for
example, leave only hydrocarbons and ethers; hydrogenation (with
platinum oxide as a catalyst) will saturate unsaturated compounds ;
treatment with hydrochloric acid will leave only neutral and acidic
compounds.

In summary, GLC alone is never useful for the characterization of
complete unknowns; used in conjunction with other analytical tech-

GAS-LIQUID CHROMATOGRAPHY 69

niques, it can be a useful aid. For qualitative analysis, GLC is most
useful when linked to such analytical tools as infrared and mass
spectrometers. In such cases, characteristic "fingerprints" of each
GLC peak (on different columns) can be obtained, and frequently
quite complex unknowns can be characterized.

QUANTITATIVE ANALYSIS

Gas chromatography is an extremely powerful tool for separation
and quantitation of complex mixtures. It has revolutionized lipid chem-
istry in the last 15 years. It is, for example, possible with GLC to
analyze mixtures of dozens of fatty acids in a relatively short time,
an analysis which in the past would have taken months and even then
probably would not have included trace components.

Certain rules must be rigidly observed during quantitative analysis
by GLC, and time must be taken to standardize conditions. A little
time spent in the beginning will pay dividends later in the form of
smooth-running, routine, accurate analyses.

The first essential is investment in a set of highly purified standards.
These standards should be analyzed to determine the linear dynamic
range and response of the instrument. Correction factors should then
be calculated for all types of columns to be used. Standards should be
run periodically to check for changes in the instrument; they should
always be run whenever a new column, new detector, or any new
electronics part is fitted.

Before standardization of methods can begin, a method of peak
measurement must be selected. The two methods available are peak
height and peak area.

The measurement of peak height is the easier method provided
that clear, sharp peaks are routinely obtained. In practice, this is seldom
achieved for all compounds except when programmed temperature is
employed. Peak height measurement under other conditions requires
much standardization and is not recommended.

A number of techniques for measuring peak area are available:

(a) Disc integrator. This is an electromechanical device attached to the
recorder. Over the bottom 10 percent of the recorder chart paper, the
integrator pen travels with a speed proportional to the displacement of
the recorder pen. This technique gives an accuracy slightly better than
that in method (d) described below. It is especially useful when peaks
are asymmetrical. It has the added advantage that a permanent record
of quantitation is available on the original chromatogram. Peaks must
stay on scale with this method, so integrator settings must be adjusted
during a run or must be predetermined.

(b) Electronic integrator. These integrators usually integrate peaks
and print out peak area and retention time. This is the quickest and most

70 GAS-LIQUID CHROMATOGRAPHY

accurate method of peak area measurement. Usually these devices
correct for baseline drift, determine areas of peaks that are incom-
pletely resolved, and operate independently of the recorder so that off-
scale peaks are still measured. Not unexpectedly, these devices are
quite costly.

(c) Planimetry. Peak area may be determined by tracing the peak
periphery with a planimeter, a device that mechanically integrates the
peak and records the area digitally on the planimeter dial. This tech-
nique can be quite accurate, depending upon the operator's skill.

(d) "Height times width at one-half height" method. Using this pro-
cedure one must first construct a baseline by drawing a line with a
ruler across the bottom of a peak, making the best connection between
the peak's leading and trailing edge. The peak's height, half-height, and
width at one-half height are determined. The area is the product of
the height and the width at one-half height. In other words, it is as-
sumed that the peak is essentially a triangle. The usual formula for
area of a triangle (1/2 bh) is not used, since the length of the base
of a broad peak is often difficult to determine. This method works best
when peaks are well resolved and fairly sharp and symmetrical. In-
creasing the speed of the recorder chart often improves peaks for
this method.

(e) Triangulation. In this method a triangle is constructed by drawing
tangents to the peak sides. The apex of the triangle will appear above
the peak apex, but this allows for the area lost by drawing tangents to
the sides. The base of the triangle is defined by the intercept of the
tangents with the base line drawn across the bottom of the peak. The
area of the peak is determined by the usual formula for area of a
triangle, J/2 bh. Trouble is encountered with this, as with the previous
procedure, when peaks are asymmetrical or when peaks are incom-
pletely resolved (the recorder pen does not return to the baseline be-
tween peaks). One way to overcome this is to measure the peak width
at two points and calculate the area according to the formula:

area = l/2 X height X (width at 0.2 height + width at 0.8 height).

This procedure is said to compensate for peak asymmetry and provide
a more accurate area.

(f) "Paper doll" technique. In this method the peaks are cut out and
weighed. This gives good reproducibility and is excellent for asymmetric
peaks. The disadvantages are that the chromatogram is destroyed and
the method is time consuming.

Standardization Procedures for Quantitative Analysis

Purity of standard compounds should first be assessed. As with
all analyses, the standards should be run on two columns, one with a

GAS-LIQUID CHROMATOGRAPHY 71

polar liquid phase (such as ethylene glycol succinate, EGS) and one
with a nonpolar phase (SE-30 or an Apiezon). If the component gives
a full-scale deflection on the recorder chart without the appearance
of impurity peaks and if it has the correct RRT (on both columns),
checked by referring to the literature, then it is about 99 percent pure.
This, of course, assumes that impurities separate from the main com-
ponent and is why at least two columns should be used to assess purity.
If this condition is satisfied, then 10 times the amount can be chro-
matographed to establish 99.9 percent purity. Generally speaking, 99 per-
cent purity is acceptable and the above procedure is adequate, provided
that the standards have been purchased from one of the established
suppliers. If the source of the standard is in some doubt, however,
then establishing purity by GLC alone is insufficient. It must be re-
membered that the impurity may not be revealed by GLC. In such
cases, it is advisable to use other approaches; for example, TLC in
two different systems may be used to determine if only a single com-
ponent is detected. If a lot of material is available, the compound
should be examined by some other procedure such as characteristic
infrared spectra, etc.

In the case of lipids it is seldom necessary to go to other analytical
aids to assess purity when the source is one of the well-known lipid
suppliers (such as Applied Science Laboratories, Mann Chemical Cor-
poration, Sigma Chemical Corporation, Supelco, Inc.). These suppliers
state the purity of their products and are on the whole reliable. Often
chromatographic proof of purity is supplied with the more expensive
standards. However, spot GLC checks should be run. If the compound
is rare, detailed proof of purity should be obtained.

In place of the check on RRT's from the literature, another prop-
erty, called the Carbon Number (CN) may be used. Within reasonable
limits CN's are accurately reproducible for a particular liquid phase.
The CN of a compound is found in the following way. Using fatty
acid methyl esters as an example, assume that at least four standard
esters have been run on a polar (EGS) and nonpolar (SE-30) column
and that the retention times have been plotted on semilog graph paper
versus the number of carbon atoms (here called the CN) in the ester.
Figure 12 shows two typical graphs. If another ester, say methyl
oleate, is now run on a column and its RT calculated, its CN can be
read off the graph as shown by the dotted line. Lists of CN's are
found in the literature and can be used both to help establish purity
of standards and to identify fatty acids in a series being analyzed. It
is advisable to establish the CN on both a polar and a nonpolar column.

Once purity of standards is established, the Detector Response
(DR) and Linear Dynamic Range (LDR) should be determined.

Detector response must be determined so that the relationship
between the amount of sample injected and the response of the detector

GAS-LIQUID CHROMATOGRAPHY

bJD
O

carbon number

(a) on EGS

fbJ on SE-30

Determining carbon numbers (CN's) by chromatography on two columns.

(Fig. 12)

is known. Unless good DR is obtained, analysis of small samples is
not possible. DR varies with detector type, column, instrument make,
and type of compound. To determine DR, a weighed mixture of stan-
dards A, B, C, and D is chromatographed. The weight injected must
be known, and dilution of the mixture in solvent and measurement of
injected volume (using a 10/aI Hamilton Syringe) must be accurate.
The injection should be repeated several times to check for repro-
ducibility. The injection should be done steadily, but quickly, and the
septum on the injection port must be leak free. After obtaining the
chromatogram, the area of each peak is determined. The response of
the detector to each compound per unit weight is then determined. The
adjusted area of detector response is calculated from the relationship
DR = area -4- weight. The response ratio is then calculated by selecting
one compound as having a response of unity. Suppose compound B is
our response standard. If one microgram of B gave peak area of 352
units (352 units per /xg), then we divide the other DR's by 352 in order
to obtain the ratio of response per jug to compound B. Alternatively, a
factor can be obtained by dividing the other DR's by the DR of the
compound selected as unity. Peak areas obtained on the chromatogram
can then be corrected for variation in detector response by multiplying
by the correction factor (or the response ratio) obtained.

The determination of response factors is time consuming, but in
lipid chemistry some short cuts are usually possible. For example,
many compounds of the same carbon length will have the same response
factor. While the response factor for stearic (C18:0) acid may differ
from that of oleic (C18:l) acid, other unsaturated acids of the same
chain length may give the same response factor correction as oleic acid.

Linear Dynamic Range refers to the sample sizes that can ap-
propriately be used on a given instrument, from the minimum detect-

GAS-LIQUID CHROMATOGRAPHY

concentration
Typical linear dynamic response curve for a given instrument.

(Fig. 13)

able quantity to the point of overload. Ideally, if 1 jug of a sample
gives a peak area of 10 units, 10 /xg should give 100 units. Also, a plot
of response versus concentration should pass through zero. In most
cases these requirements are met, but the instrument's LDR must be
checked. A linearity curve will show the concentrations at which the
system becomes nonlinear. In addition, any interaction of the sample
with the system (sample loss) will show up.

A typical LDR curve is shown in Figure 13. Point B is the mini-
mum detectable level (response X 5 times the noise level); point C is
the upper limit of linearity (frequently due to column overload); D
is a typical point at which the concentration will not give valid results.
An LDR curve which approximates an S-shape and has a narrow
range (say from A to B) indicates sample adsorption by the column
or other parts of the instrument. This type of LDR curve indicates a
need for silylation of the column, use of a glass rather than a metal
column, or both. If the curve crosses the Y-axis above zero, this can
indicate either response when no sample is injected or sample measure-
ment error on the part of the operator. If more than one effect is present
at one time — say adsorption plus sample measurement error — a very
distorted curve will be produced, and all possibilities must be con-
sidered. It is usually wise to consider sample measurement error first
before changing column material.

Now that DR and LDR have been determined, one of the following
standardization techniques can be chosen: internal normalization, ex-
ternal standardization, or internal standardization.

Internal normalization. In this procedure the quantity of a com-
ponent in a mixture is expressed as a percentage of the total area of
the chromatogram. Thus, if the sum of the areas of all the peaks is
100 and component A gives an area of 10, then the area percentage

74 GAS-LIQUID CHROMATOGRAPHY

for A is 10 percent. In calculating areas, the response factors for each
component should be used to correct the areas. This technique is in
common use and has a number of advantages — mainly, speed and
independence from sample size. There are, however, several disad-
vantages: first, all peaks must be measured; second, absolute measure-
ments rely on the assumption that the entire sample chromatographs ;
and third, for complete accuracy response factors for all peaks should
be known. In practice these disadvantages are not very serious. Often
in lipid chemistry one is attempting to completely analyze a series of
monoglycerides, fatty acid methyl esters, etc., so one wishes to measure
all peaks anyway. Such samples are usually prepurified (by TLC or
some other method), and all components usually chromatography In-
deed, it is common practice to convert peak area percentages into moles
using the molecular weight — which assumes that everything chro-
matographs. Usually this is safe, but the investigator should ascertain
that no adsorption is occurring.

Internal normalization is the most commonly used technique in
GLC of fatty acid methyl esters and of dimethyl acetals (and other
derivatives) of fatty aldehydes; it is also used in other lipid GLC work.

External standardization. This technique demands that a number of
analyses of both the standard and sample be made and averages deter-
mined. The concentration of the standard should be near that of the
sample or should be the same compound as the sample so that no
response factor is required. Samples of known size must be injected;
in practice this is hard to achieve, and the procedure is generally used
only when a single peak is being analyzed. It is not recommended as
a quantitative procedure for any of the lipid analyses discussed later
in this section.

Internal standardization. This method is the best for obtaining
accurate determinations by GLC and it is highly recommended when-
ever conditions permit its use. An example of internal standardization
in cholesterol analysis by GLC is discussed in Chapter 6 (p. 87).

In this method, a known amount of a substance that is not present
in the sample is added to an aliquot of the sample. The area of the
peak of the added standard is determined and compared with the area
of the sample component of interest. When the component is 1 to 100
percent of the sample, it is usual to use a weight of internal standard
of about 10 percent of the weight of the component; for trace amounts,
however, the standard should be in the same concentration range as
the components.

It is not always possible to use internal standardization since suitable
substances for standards are not always available. The standard chosen
must fit certain specifications. It must elute from the column well
separated from all sample components but near to them. It must have

GAS-LIQUID CHROMATOGRAPHY 75

functional groups similar to that of the sample component or be an
appropriate hydrocarbon. It must be stable under the analytical con-
ditions and not react with sample components. Finally, it must be
sufficiently nonvolatile and stable to permit storage in solution for a
significant period of time.

The first steps in internal standardization are preparing the
standard solution and determining the response factor (RF) for the
sample compound relative to the internal standard. A mixture of the
component to be analyzed and the internal standard is prepared, and
the RF is calculated as follows:

_ weight of internal standard X area of component peak
weight of component X area of internal standard peak

Next, a sample mixture is prepared by adding a known amount
of the internal standard to a known amount of the sample containing
the component of interest. This mixture is then chromatographed
under the same conditions as the standard and the percent of the
component by weight is calculated by the equation:

component area X internal standard weight X 100
^ RF X internal standard area X sample weight

Following the selection of an internal standard, it is necessary to
determine the response characteristics of standard mixtures. A response
versus concentration curve should be reproducible and preferably
linear. The linearity plot should be linear over a wide concentration
range and should finally plateau. In preparing this curve, the concen-
tration of the internal standard is kept constant and only the concen-
tration of the sample is varied.

CHEMICAL MODIFICATION OF COMPOUNDS FOR ANALYSIS
BY GAS-LIQUID CHROMATOGRAPHY

Many lipids can now be analyzed by GLC in their unmodified state.
Frequently, however, the preparation of a more volatile derivative,
or one which has certain functional groups blocked, or both, leads to
more efficient chromatography. Furthermore, chromatography of both
the original and the chemically modified form will provide additional
information about a sample and may serve as a check on quantitative
procedures.

Analysis of Methyl Esters of Fatty Acids

Fatty acids are generally analyzed as their methyl esters,1 although
free fatty acids can be chromatographed. The method of methyl ester

1 Other derivatives are employed ; however, not enough is known about the
quantitation of yields to rank these as competitors with methyl esters.

76 GAS-LIQUID CHROMATOGRAPHY

preparation varies with the source of material. Transesterification with
acidic methanol is in common use. This approach gives the methyl
ester of all the fatty acids in lipids from biological sources whether
they are in amide (sphingolipid) or ester (glyceride and phosphoglycer-
ide) linkages. Numerous procedures have been reported and a partial
list of suitable reagents follows.

1. 1 to 10 percent sulfuric acid in methanol at 60 to 100°C for 1
to 16 hours.

2. 1 to 10 percent hydrochloric acid I . -- ,. Al ,
or 5 percent sulfuric acid j + 2,2-dimethoxypropaue (as

a water scavenger) in methanol at 50 to 70°C for 2 to 4 hours.

3. 3 to 14 percent boron trifluoride in methanol at 60 to 100°C for 1
minute to 16 hours.

Some workers carry out reactions by refluxing (under nitrogen),
while others seal the reaction mixture in screw cap vials or glass
ampules and heat in a water bath. Definitely avoid using hydrochloric
acid-methanol, since this reacts to yield artifacts which mimic fatty
acid esters on GLC (1).

The method we recommend uses 4 percent sulfuric acid in methanol
in a sealed ampule at 90°C for 2 hours. Ten to 500mg of sample are
placed in a 10ml glass ampule and an appropriate volume (0.5 to 10ml)
of H2S04-methanol is added. The ampule is cooled in dry ice-acetone
and sealed in an oxygen flame. It is then heated for 2 hours in a water
bath at 90°C (Caution! Do not heat above this temperature or in an
oven as the ampules will explode). This procedure can be used directly
on spots removed from a TLC plate if a noninterfering detection spray
has been used. After heating, the ampule is again cooled in dry ice-
acetone, opened, and 2 to 5ml water are added dropwise until two
phases appear. The methyl esters are extracted with hexane or diethyl
ether (3X3 volumes).

If there is doubt about the purity of the methyl ester preparations,
they should be purified by TLC (in the presence of an antioxidant).
For example, if total lipid from an animal source is transmethylated,
the methyl esters must be purified by TLC since cholesterol derivatives
that mimic fatty acid esters on GLC may appear ; moreover, aldehydes,
derived from plasmalogens, will form dimethylacetals under acid
methanolysis conditions. These must be separated from the methyl
esters by TLC or other means (see pages 78-79).

Generally, a good instrument will give a good resolution of the
methyl esters of most natural fatty acid mixtures, and numerous column
packings are available for this analysis. If good standards are avail-
able and semilog plots of RT versus carbon number are drawn (see
pages 71-72), all major components can usually be identified. The ideal
way to characterize complex mixtures is to make a preliminary separa-
tion by TLC first and then gas-chromatograph each fraction separately ;

GAS-LIQUID CHROMATOGRAPHY 77

for example, mixtures may be separated into saturated and unsaturated
fractions on silver nitrate impregnated plates as described earlier. If
only small quantities of material are available, however, this approach
may involve too many manipulations, and some losses may occur.

A previously uncharacterized mixture should be chromatographed
on both a polar column, such as ethylene glycol succinate, and a non-
polar one, such as one of the Apiezon or SE-30 greases. On a polar
column, it is usual for unsaturated fatty acid esters to elute after the
saturated ones of the same carbon chain length, and branched chain
esters are eluted just before the straight chain ester of the same carbon
chain length. On nonpolar columns, unsaturated esters precede the
saturated esters of the same carbon chain length.

Some coincident elutions often occur, and on many columns some
unsaturated acids, especially those with many double bonds, may elute
at the same time as a long-chain saturated acid. Uncharacterized mix-
tures must be checked for this possibility. If such mixtures were not
initially separated by TLC, the sample should be treated in methanol
with platinum oxide as a catalyst2 to hydrogenate the fatty acid esters.
The mixture should be chromatographed both before and after hydro-
genation and on more than one kind of column. The appearance of
new peaks, the change in size of old peaks, the disappearance of old
peaks, and so forth will provide information about the presence of
any unsaturated species.

The presence of hydroxy fatty acid methyl esters, eluted from
many types of columns under certain conditions, can confuse identi-
fication of peaks. Mixtures containing hydroxylated fatty acids (for
example, cerebroside and sulfatide fatty acids) should be separated
into hydroxylated and nonhydroxylated ester fractions by one of the
TLC or column chromatographic procedures described on pages 36
and 48. Derivatives of the hydroxylated esters can be prepared in
which the free hydroxy groups are blocked. GLC of these derivatives —
as trifluoroacetates, methyl ethers, or trimethylsilyl ethers — gives a
much more rapid analysis and better peak symmetry. The third of
these derivatives is highly recommended and easily prepared. Most
suppliers of GLC accessories now offer a variety of "silylating kits,"
and while these are relatively expensive, they generally pay for them-
selves in the form of time saved. Usually all the investigator does is
add a small volume of the silylating agent; after a short waiting period,

2 Preferably, the hydrogen should be supplied via a manometer so that hy-
drogenation can be continued until there is no further uptake of hydrogen. The
mixture should be stirred with a magnetic stirrer during the reaction. A simple,
safe, and inexpensive hydrogenation apparatus has been designed by Applied
Science Laboratories, Inc., State College, Pa. 16801. The apparatus is not offered
for sale, but it is described in Applied Science's Gas -Chromatography Newsletter
10, No. 3 (1969). Further details can be obtained from the Biochemicals Depart-
ment at Applied Science.

78 GAS-LIQUID CHROMATOGRAPHY

the mixture can be chromatographed directly without having to extract
the derivative. If the hydroxy groups are sterically hindered, it may
be necessary to heat the reaction mixture.

While many positional isomers of fatty acid esters separate under
average conditions, geometric isomers do not. Cis and trans isomers
can be separated efficiently with Golay (capillary) columns (see p. 97).
Another approach is to convert the isomers to hydroxylated acids by
oxidizing with osmium tetroxide (2). The hydroxylated compounds
are then derivatized by one of the procedures mentioned above and
chromatographed. After such treatment, the erythro and threo deri-
vatives separate on the usual columns used for methyl ester GLC.

Quantitation of methyl ester analyses may be carried out using any
of the peak measuring techniques previously described. Generally, it
is wise to obtain response correction factors for all the saturated esters
and for at least one unsaturated ester of each chain length.

For details of the GLC of some rare fatty acids, such as those con-
taining acetylenic bonds, Lipid Chromatographic Analysis, vol. 1,
should be consulted (see Suggested Further Readings).

Fatty Aldehydes

Usually the lipid chemist encounters fatty aldehydes in the form
of their dimethyl acetals (DMA's), derived from the acid hydrolysis of
plasmalogens. While aldehydes can be analyzed by GLC in their underi-
vatized form, this is not a wise procedure. On standing, even for
short periods, aldehydes readily undergo condensation and polymeriza-
tion, reactions that are accelerated under alkaline conditions. It is prefer-
able to obtain aldehydes in a stable form, such as the DMA, or to
convert them to this or other stable derivatives as soon as possible. The
DMA is the most satisfactory derivative since it is readily formed in
quantitative yield and is very stable under neutral and alkaline condi-
tions. Furthermore, if lipid mixtures are subjected to acid hydrolysis,
fatty acids (whether in ester or amide linkages) are converted to
methyl esters and plasmalogen aldehydes are converted to DMA's all
in one step. Only a simple separation procedure must be employed, and
both sets of derivatives can be subjected to GLC.

H+ /0CH3

R-CH0+2CH30H-*R-CH +H20

^0CH3

dimethyl acetal (DMA)

The methyl esters and DMA's may be separated either by TLC on
silica gel using benzene as the developing solvent or by taking advan-
tage of the fact that DMA's are stable in alkaline solution whereas

GAS-LIQUID CHROMATOGRAPHY 79

methyl esters are not. In the latter procedure, the methyl ester-DMA
mixture is refluxed with 0.5N methanolic sodium hydroxide for two
hours. The methyl ester fatty acids will be converted to sodium salts
(soaps) and the DMA's will remain unchanged. On cooling, the solu-
tion is diluted with water (1 vol.) and the DMA's are extracted three
times with hexane (1 vol.). The extract is washed with water-ethanol-
3N sodium hydroxide 40:10:1 v/v/v (5). The combined hexane ex-
tracts are dried over sodium sulfate, the solvent is removed, and the
acetals are reserved for GLC. The soaps in the water layer are con-
verted to free fatty acids by acidifying the solution with IN hydro-
chloric acid. The free fatty acids are then extracted with hexane, and,
after drying, the solvent is removed. The fatty acids may then readily
be reconverted to the methyl esters by any of the usual procedures
(see p. 76).

Other derivatives of long-chain aldehydes that are useful for
GLC analysis are the alcohol, the alcohol acetate, and the acid. Far-
quhar (3) used all these derivatives, in addition to the DMA's, in his
detailed study of the fatty aldehydes of erythrocyte plasmalogens. He
prepared the free aldehydes by dissolving the DMA's in 90 percent
acetic acid 1:30 w/v and adding 1 drop of a saturated solution of
mercuric chloride. The mixture was heated in a sealed ampule (under
nitrogen) for 8 to 24 hours at 37 °C. The aldehydes were recovered as
follows: 1 volume of water was added; the solution was neutralized
with 3N sodium hydroxide and extracted four times with petroleum
ether; the extracts were washed once with water-ethanol-3N sodium
hydroxide 40:10:1 v/v/v and dried over sodium sulfate. The solvent
was then removed and the aldehydes were converted to derivatives as
soon as possible.

The alcohols were prepared by reducing 3 to 30mg of the free
aldehyde with 10ml of 3 percent lithium aluminum hydride anhydrous
ether kept at — 20°C for 2 hours. ''Anhydrous" is stressed as the re-
action of lithium aluminum hydride with water is extremely violent.
The reaction was stopped by adding 5ml of water drop by drop to
the reaction mixture which was still at — 20°C. Five ml of ethanol
were added, the supernatant ether was removed, and the lower layer
was extracted 5 times with petroleum ether at room temperature. The
combined extracts were washed with water-ethanol 4:1 v/v, dried, and
the solvent removed.

One to lOmg of the fatty alcohols were converted to the acetates by
dissolving in 9ml of acetic anhydride-pyridine 3:6 v/v in a glass-
stoppered tube. The tube was kept at 37 °C for 15 minutes and shaken
occasionally. Five ml of water were added and the acetates were re-
covered by extracting 3 times with 5ml petroleum ether.

Conversion of the free aldehydes to the fatty acid is of limited
usefulness since the usual oxidation procedures destroy the unsaturated

80 GAS-LIQUID CHROMATOGRAPHY

aldehyde. Farquhar (3) employed alkaline silver oxide as the oxidizing
agent. Some information, however, can be derived from the fatty acids
obtained from the aldehydes derived from the DMA mixture before
and after hydrogenation. The DMA's can be hydrogenated in methanol
using platinum oxide as a catalyst. It is usually necessary to add chloro-
form to the reaction mixture (up to 20 parts by volume) to keep the
DMA's in solution. The fatty acid derivatives are generally prepared
only when additional structural information is sought. For most pur-
poses, the preparation of the DMA's or the alcohol acetates suffices
for the GLC analysis of fatty aldehydes. If fatty acid derivatives are
used, they can be converted to their methyl esters for GLC analysis.

A number of liquid phases are suited to the GLC analysis of DMA's
and alcohol acetates. It is most important to remember that acidic
columns must not be employed, as the DMA's will readily be hydrolyzed
in acid conditions. Farquhar used ethylene glycol adipate (EGA) and
Apiezon M for the analysis of the acetates, the DMA's, and the methyl
esters. Other useful phases are ethylene glycol succinate (EGS),
ethylene glycol succinate-silicone copolymer (EGSSX), Reoplex 400,
and Apiezon L. Usually 10 to 15 percent EGA, EGS, EGSSX (polar)
packings or 10 to 12.5 percent nonpolar packings is employed. Alkaline
washed supports are recommended. Temperatures used with average
columns (6 to 10 ft. X 1/6 -inch) are about 150°C for polar phases and
190° C with nonpolar phases.

Aldehydes are not readily available in pure form, so standards for
these analyses can present a problem. It is usual to use relative retention
data and to refer all retention times to the normal C18 saturated hydro-
carbon (octadecane) or, preferably, the corresponding 16-carbon deriv-
ative of hexadecanal (palmitaldehyde). The palmitaldehyde is available
in a relatively pure state as its bisulfite. The bisulfite is converted to the
DMA by heating with 4 percent sulfuric acid in methanol, or the free
aldehyde is obtained by aqueous acid hydrolysis. The DMA preparation
can be freed from aldehyde by shaking the extract with a saturated solu-
tion of sodium metabisulfite. The DMA extract should also be washed
with an alkaline wash solution (water-ethanol-3N sodium hydroxide
40:10:1 v/v/v) according to Farquhar's procedure to prevent acid-
catalyzed breakdown. Retention times of components are stated as rela-
tive to hexadecanal, hexadecanal acetate, hexadecanal DMA, or methyl
hexadecanoate, according to the derivative chromatographed, since
long-chain aldehyde GLC data is generally so reported in the literature ;
this permits ready comparisons with the work of others.

On polar columns, the order of elution of fatty aldehydes and their
derivatives of the same carbon chain length (unbranched) is: free
aldehyde, DMA, methyl ester of fatty acid, alcohol acetate, free alcohol.
On a nonpolar column the order is: free alcohol, methyl ester, DMA,
and alcohol acetate. Free aldehyde is omitted from the last series be-

GAS-LIQUID CHROMATOGRAPHY 81

cause according to Farquhar, it was not eluted from an alkaline treated
nonpolar column. More recently, Gray (4) has reported aldehyde
chromatography on untreated Apiezon L, but under these conditions
breakdown of DMA's occurred.

Peak area calculations may be made by any of the methods previ-
ously described.

Glycerides

The beginner in gas chromatography can, after a short practice
period, expect to achieve reasonably successful analysis of uncompli-
cated fatty acid and fatty aldehyde mixtures. This is not so, however,
in the case of glyceride analysis since the situation is complicated by the
high molecular weights of the compounds and by the complexity of
the molecular species found in naturally occurring glycerides. Glyceride
analysis by GLC is still in its infancy. The first practical demonstration
of glyceride analysis (5) by GLC was shown one decade after the intro-
duction of GLC analysis. Although glyceride GLC analysis is beyond
the scope of this book, we can appropriately include a brief account of
the difficulties and approaches to success in this area.

So far we have considered GLC analyses that can be readily ac-
complished using isothermal conditions. In general, the efficiency
achieved under isothermal operation is not conducive to satisfactory
analysis of glycerides. The efficiency of a GLC column isothermally
operated is given by the number of "theoretical plates" (a term allied
to distillation theory), for which the expression is: N = I6(tr/w)2.
N is the number of theoretical plates, tr is the residence time of the
component in the column, and w is the width of the peak. An efficient
column gives peaks of narrow width. If band diffusion is large and
broad peaks result, programmed temperature is called for. A column
operated isothermally under linear temperature programming conditions
may often give 5 or 6 times the apparent number of theoretical plates.
This improvement is very important to the chromatographer of
glycerides. The best conditions determined so far for glycerides are
low liquid phase concentrations; narrow, short columns; and tempera-
ture programming from 200 to 350°C. At temperatures above 350° C
thermal cracking may result.

Triglycerides can be chromatographed directly, but in the interests
of increased stability and volatility, mono- and diglycerides should be
converted to suitable derivatives such as acyl esters or silyl ethers.
Isopropylidene and benzylidene derivatives have also been used.

The usual columns employed for glyceride analysis (triglycerides
or derivatized mono- and diglycerides) are the silicone polymers (Se-
30), polysiloxane polymers (JXR, Applied Science Laboratories, Inc.),
and fluoroalkyl silicone gums (Dow-Corning). The percentage of
liquid phase employed does not usually exceed 3 percent and is fre-
quently below 1 percent.

82 GAS-LIQUID CHROMATOGRAPHY

Because column "bleed" at high temperatures is considerable, instru-
ments with "dual column operation" are preferable. Dual column opera-
tion usually compensates for baseline drift due to bleed by splitting the
effluent stream and using dual compensating flame ionization detectors.

Unless some preliminary separations are performed, chromatography
of glycerides yields peaks of mixed molecular species, generally classi-
fied according to the total number of carbon atoms they contain. If
detailed molecular species analyses are sought, then mixtures must be
subjected to preliminary separations by column and thin-layer chroma-
tography. Mixtures must be separated not only into mono-, di-, and
triglyceride fractions, but also into fractions based on the degree of
unsaturation. This may be achieved by argentation chromatography.

Acetate derivatives. The determination of phosphoglyceride struc-
ture has been discussed previously (Chapter 4, p. 55). This analytical
procedure included the preparation and analysis of the diglyceride
acetates derived from the phospholipids. These acetate derivatives are
readily prepared at room temperature by dissolving about 5mg glyceride
in 1ml dry pyridine (distilled over barium oxide) in a tight screw
cap vial and adding 0.5ml acetic anhydride. The mixture is allowed to
stand overnight, after which excess reagent is removed by evaporation
under vacuum. Alternatively, diglyceride analysis may use silyl ether
derivatives prepared with one of the available silylation reagents.
Whichever derivative is chosen, the analysis can be carried out (iso-
thermally or by programmed temperature operation) on column pack-
ings previously mentioned (for example, 1 to 3 percent SE-30,
3 ft. X i/8-inch column) at temperatures between 180 and 300°C.

The preceding comments merely cover some of the major points re-
garding GLC of glycerides. The reader interested in pursuing this field
is referred to the Suggested Further Readings.

Other Lipids and Components Derived from Lipids

Other nonpolar lipids, such as the glyceryl ethers, can also be sub-
jected to GLC analysis usually as trifluoracetate or silyl ether
derivatives.

Glycerol, amino alcohols such as sphingosine and related compounds,
and carbohydrates derived from lipids can also be analyzed by GLC
generally in derivatized form. References to the GLC analysis of non-
lipid moieties of lipid molecules are given in Suggested Further
Readings.

PYROLYSIS-GLC

The GLC analysis of the products of pyrolysis has been used ad-
vantageously in the study of hydrocarbons. Recently, several investiga-
tors have started to explore the use of pyrolysis-GLC as an approach
to the determination of phospholipid structure. Kuksis, Marai, and

GAS-LIQUID CHROMATOGRAPHY 83

Gornall (6) noted that serum lecithins undergo pyrolysis in a flash
evaporator (at 280 to 300°C) attached to a GLC apparatus. They
tentatively identified the chromatographed peaks as the propenediol
diesters, which under most conditions have the same retention times as
the corresponding diglycerides. Perkins and Johnston (7) subjected
a number of phosphoglycerides to pyrolysis-GLC and made a mass
spectral study of the products. All the phosphoglycerides were found to
cleave at the phosphate ester bond, and the GLC peaks obtained had
retention times the same as those of the corresponding diglycerides.
The mass spectral data confirmed the elimination of the phosphate ester
group and showed that the products obtained were the dehydrated di-
glycerides, that is, the diacylesters of propenediol. A different approach
has been used by Horning, Casparrini, and Horning (8), who subjected
phospholipids to silylation with bis-trimethylsilylacetamide (BSA) and
trimethylchlorosilane (TMCS) and injected the derivatives into the
gas chromatograph. They identified the products of the phosphoglycer-
ides as the corresponding dehydrated trimethylsilyl derivatives of the
diglycerides. These investigators also found that the phosphate ester
group was eliminated from silylated sphingomyelin on thermal cracking,
and that trimethylsilyl derivatives of ceramides amenable to GLC and
GLC-mass spectrometry studies could be obtained.

Further studies are needed to explore the possibility of adapting
these findings to the analysis of polar lipids by GLC.

REFERENCES

1. Johnston, P. V., and B. I. Roots (1964) /. Lipid Res. 5 : 477.

2. Wood, R., E. L. Bever, and F. Snyder (1966) Lipids 1 : 399.

3. Farquhar, J. W. (1962) /. Lipid Res. 3 : 21.

4. Gray, G. M. (1967) in Lipid Chromatographic Analysis, ed. G. V. Marinetti ;
vol. 1, p. 401. Marcel Dekker, Inc., New York.

5. Kuksis, A., and M. J. McCarthy (1962) Can. J. Biochem. Physiol. 40: 679.

6. Kuksis, A., L. Marai, and D. A. Gornall (1967) /. Lipid Res. 8: 352.

7. Perkins, E. G., and P. V. Johnston (1969) Lipids 4: 301.

8. Horning, M. G., G. Casparrini, and E. C. Horning (1969) /. Chromatogr. Sci.
7 : 267.

VI. Procedures for the Determination

of Specific Elements, Functional Groups,
and Lipid Classes

In this chapter the determination of specific elements, functional
groups, and individual lipids are described in detail. These pro-
cedures allow the investigator to determine the amount of specific lipids
(cholesterol, gangliosides) and specific lipid classes (total phospholipid,
glycolipid, etc.) in lipid mixtures. They can also be applied to the
quantitative analysis of lipids separated by column, thin-layer, and
paper chromatographic procedures.

In keeping with all former accounts in this text, this chapter is not
a compilation of all available methods for these determinations. The
methods presented are selected on the basis that the author or a col-
league has had considerable experience with them and has found them
to be satisfactory.

DETERMINATION OF ORGANIC PHOSPHORUS

The method described is a modification of Bartlett's (1) procedure.
Reagents: concentrated sulfuric acid

70 percent perchloric acid

ammonium molybdate, 0.26 percent aqueous solution

disodium hydrogen phosphate (for standard curve)

Fiske-Subbarow Reagent
Equipment: pyrex test tubes

water bath and sand bath

spectrophotometer reading at 800 to 820m/x

Fiske-Subbarow Reagent: To 80ml of 15 percent sodium bisulfite
add 0.2g purified l-amino-2-naphthol-4-sulfonic acid and 0.4g an-
hydrous sodium sulfite, while stirring. Filter and store in brown bottle.
Make fresh solution each week.

Procedure: To a test tube containing 0.5 to 20.0/xg of lipid phos-
phorus add 0.5ml concentrated sulfuric acid. Digest the lipid by placing
the tube in a sand bath at 250° C for 3 hours. Complete the digestion by
adding 3 drops (approx. 0.15ml) 70 percent perchloric acid and heat
at 250°C for a further 30 minutes. Mix the contents of the tube oc-
casionally. After digestion is complete (solutions should be clear),
cool the tubes and add 9.1ml of 0.26 percent ammonium molybdate and
0.4ml Fiske-Subbarow reagent. After the solution has been mixed

FUNCTIONAL GROUPS AND LIPID CLASSES 85

thoroughly (preferably with a Vortex mixer), heat the tubes in a bath
of boiling water for 10 minutes, then cool. Read the blue color developed
at 800 or 820mjn; the latter is preferred if available. Zero the instrument
with a prepared reagent blank.

Prepare a standard curve 0.2 to 30/ig of phosphorus, using potassium
dihydrogen phosphate.

Application of method to spots on TLC plates. This procedure can
also be applied directly to adsorbent plus lipid removed from a TLC
plate. The digestion will go to completion in the presence of adsorbent
(preferably Silica Gel H) and remaining traces of solvents such as n-
butanol. The solution will clear when digestion with perchloric acid
is complete.

Before deciding on a loading for a TLC plate, the total lipid phos-
phorus should be determined and a trial TLC plate should be run to
determine how many phospholipids are present. The relative concen-
trations of the phospholipids may be judged by charring. A suitable
loading can then be selected. A load which gives about 0.5 to 15.0/xg
lipid phosphorus per spot is ideal ; however, the method will detect
amounts from as little as 0.1 to 0.2/xg up to 25.0/xg.

After developing the plate, spot an amount of total lipid containing
5 to 10/Ag phosphorus on a part of the plate untouched by solvent, and
remove the spot to make a total lipid phosphorus check. Also remove a
clear area of equal size to the blank. Spots are best visualized by ex-
posing the plate to iodine vapor in a closed jar, after the solvents have
evaporated. Spots of interest can be outlined with a needle, removed
either by aspiration or scraping, and placed in test tubes. Remove clear
areas of equal size and place in test tubes to use as sample blanks. The
procedure is carried out as described above, except that after color
development the tubes must be centrifuged at about 600 X g and the
solutions decanted into clean tubes to free them of the silica gel. The
color is read in the usual way, subtracting adsorbent blank optical
density from the sample before reading off the standard curve. Blank
readings above optical density of 0.03 to 0.04 are undesirable and indi-
cate contamination of either the adsorbent or reagents with a phos-
phorus containing compound. To avoid contamination, wash all glass-
ware in chromic acid and rinse thoroughly in deionized water. It may
prove necessary to wash the Silica Gel H as described on pp. 42-43.

DETERMINATION OF CHOLESTEROL

By Spectrophotometry

The procedure is that of Zak, Luz, and Fisher (2).

Reagents: ferric chloride stock solution — 700g ferric chloride
(FeCl3 * 6H20) in glacial acetic acid in a 100ml
volumetric flask; dilute to mark and mix well.

86 DETERMINATION OF SPECIFIC ELEMENTS

ferric chloride working solution — stock solution diluted
1:10 v/v with glacial acetic acid.

cholesterol stock standard — lOOmg pure cholesterol
in glacial acetic acid in 100ml volumetric flask; dilute
to mark.

digitonin solution — lg digitonin in 50ml ethanol, di-
luted with distilled water to 100ml in volumetric flask,
acetone-alcohol 1 : 1 v/v
acetone

concentrated sulfuric acid
Equipment: spectrophotometer and 1cm cuvets
conical centrifuge tubes, 15ml
test tubes, 15ml

Procedure: Prepare a standard curve by diluting 1ml of the ferric
chloride stock solution and 1ml of the cholesterol stock standard with
glacial acetic acid to 10ml in a volumetric flask. Pipet 1.0, 2.0, and 3.0ml
of this standard into test tubes, diluting the 1.0 and 2.0ml fractions to
3.0ml with the ferric chloride working reagent. Prepare one blank tube
of 3ml ferric chloride working reagent. Carefully layer 2ml of concen-
trated sulfuric acid over each solution, then mix well. When the solu-
tions have cooled to room temperature, measure absorbancies at 560m//.
against the blank.

To determine serum cholesterol levels, pipet 0.2ml of serum into a
10ml volumetric flask containing approximately 5ml of acetone-alcohol
solution. Dilute to mark with acetone-alcohol and shake vigorously
to extract the cholesterol. Filter mixture through Whatman Xo.
41 filter paper, keeping a watch glass over the funnel. Pipet 5ml of
filtrate into conical centrifuge tube for determination of free cholesterol
and 2.5ml of filtrate into a test tube for total cholesterol determination.
Evaporate contents of test tube to dryness and contents of centrifuge
tube to 0.5 to 1.0ml.

To test tube add 3ml of ferric chloride working solution to dissolve
residue. Layer in 2ml of concentrated sulfuric acid and mix solution
well. Wait 15 minutes for color development, then measure the absor-
bance against a blank at 560m/,t.

To the centrifuge tube add 0.5ml of digitonin solution, wait 15
minutes, then centrifuge at 600 to 700 X g for 10 minutes. Decant the
supernatant. Add 4ml of acetone to disperse the precipitate, tapping the
tube until the precipitate is homogenous, then centrifuge again for 10
minutes. Decant the wash solution. Invert tube on absorbent paper
to drain. Add ferric chloride (3ml) and sulfuric acid (2ml) as de-
scribed above, wait 15 minutes, and read absorbance at 560m/x.

The method has been shown to give stoichiometric final color re-
action with amounts of cholesterol up to lOOOmg per 100ml. The range
of analysis can be extended by appropriate dilution of the original

FUNCTIONAL GROUPS AND LIPID CLASSES 87

sample. While the method has been described for analysis of serum
cholesterol levels, it can, of course, be adapted to the analysis of the
cholesterol content of any sample of total lipid. All that is required is
dilution of the original sample to give a cholesterol content within the
range 0 to lOOOmg per 100ml.

By Gas-Liquid Chromatography

The method described utilizes cholestane as an internal GLC stan-
dard (3).

Reagents: cholesterol

cholestane

95 percent ethanol

diethyl ether

hexane

chloroform

potassium hydroxide
Equipment: 18mm X 150mm test tubes

serum caps to fit test tubes

centrifuge tubes, 15ml, screw cap

20ml vials

5 and 10ml pipets

graduated pipets

gas chromatograph equipped with glass column, glass
inlet system, and flame ionization detector

Procedure: Prepare the following standard solutions:

1. Ratio Solution. Weigh lOOmg cholesterol and lOOmg cholestane.
Dilute to 100ml with chloroform. Mix and store in refrigerator. These
compounds are stable for several weeks.

2. Potassium hydroxide. Weigh out 3.3g potassium hydroxide and
dilute to 10ml with distilled water. Prepare fresh daily.

3. Internal standard. Weigh lOOmg cholestane and dilute with
chloroform to 100ml. Subdivide solution into 10ml portions. Store in re-
frigerator in tightly stoppered containers.

Operating conditions for gas chromatograph :

Column. 18-inch glass packed with 3.8 percent SE-30 on Diatoport

S. (Note: This column is recommended in the original

procedure, but other columns are also suitable, such as

1 to 2 foot columns packed with 1 to 3 percent SE-30 on

various supports.)
Column temperature. 200 to 230° C, depending on column packing,

etc. Other settings will vary from instrument to instrument

and must be determined individually.
Detector. A flame ionization detector is recommended because its

response is usually linear over a wide range. Use of

88 DETERMINATION OF SPECIFIC ELEMENTS

another kind of detector will probably necessitate prepar-
ing a correction curve for the specific detector.

Determination of total serum cholesterol

1. Measure 0.2ml serum into test tube.

2. Add 4.7ml 95 percent ethanol.

3. Add 0.3ml 33 percent aqueous potassium hydroxide solution.

4. Stopper tube and mix well.

5. Place in water bath at 55 °C for 15 minutes.

6. Remove tube and cool.

7. Add 5ml distilled water.

8. Add 10ml hexane and allow to stand till two phases appear.

9. Remove a 5ml aliquot of the upper (hexane) phase and trans-
fer to a 3-dram screw-cap vial.

10. Use a stream of nitrogen and a water bath to evaporate the
hexane aliquot to dryness.

11. Add 0.2ml of the internal standard solution to the dry residue.

12. Cap vial and mix well; inject about 3/xl of mixture into gas
chromatograph (exact size of sample is not important when internal
standardization is used — see p. 74) .

Analysis on GLC will take 7 to 15 minutes, depending on the
column.

Determination of free cholesterol

1. Measure 0.2ml serum into 15ml screw cap centrifuge tube.

2. Add 9ml 95 percent ethanol.

3. Add 3ml diethyl ether.

4. Shake and allow to stand for 5 minutes.

5. Centrifuge (600 to 700 X g).

6. Pour supernatant into 20ml vial.

7. Evaporate residue to dryness as previously described.

8. Add 0.2ml of internal standard solution.

9. Inject about 3/xl into gas chromatograph; GLC analysis time will
be the same as for total cholesterol.

Interpretation of chromatograms. First obtain a chromatogram by
injecting Zjx\ of the cholestane-cholesterol standard solution and calcu-
late the ratio either by using peak height or peak area, thus:

cholestane peak height (area) _ „
cholesterol peak height (area)

To calculate total cholesterol in the sample, measure peak heights
(or areas), multiply cholesterol peak height by the ratio, and divide by
the cholestane peak height. Since in the original procedure 5ml of
hexane was withdrawn from the mixture, the value must be multiplied
by 2. Thus, the calculation for total cholesterol in a sample becomes:

FUNCTIONAL GROUPS AND LIPID CLASSES 89

cholesterol peak height x R x 2 = ^ cholesterol (mg/lQOml)
cholestane peak height

To determine free cholesterol, follow the same calculation procedure
as for total cholesterol. Since the whole sample is used in the analysis
for free cholesterol, the calculation will be:

cholesterol peak height x R = frge cho,estero, (mg/100ml)
cholestane peak height

The ratio R should be determined from standards daily, as slight
changes in column conditions may affect it. It must also be remembered
that if changes in sensitivity factors have been made during the run,
these must be taken into account in the calculations.

THE DETERMINATION OF GLYCOLIPID SUGARS

By Using Anthrone

The method described is that of Radin et al. (4, 5).

Reagents: anthrone stock solution — 2 percent anthrone (re-
crystallized) in sulfuric acid; age 4 hours at room
temperature (stable for 2 weeks in refrigerator),
anthrone working solution — stock solution diluted 14
times with sulfuric acid- water 9:5 v/v; make up just
before use.

galactose standards — dry galactose at 100°C over
phosphorus pentoxide and dissolve in water to give a
concentration of 20mg per ml ; store this solution in
polyethylene bottle in refrigerator; prepare standards
by evaporating appropriate volumes to dryness in
colorimeter tubes.

hydrolytic solvent — ethanol-concentrated hydrochloric
acid 74:63 v/v.

Equipment: colorimeter

screw-cap tubes, 10 to 15ml
constant temperature bath set at 58° C

Procedure :

1. Evaporate a sample solution containing 0.2 to 1.7mg cerebroside
(or other galactolipid) to dryness in a screw-cap tube.

2. Add 3ml hydrolytic solvent and place in water bath at 58° C
for 3 hours.

3. Add 5ml toluene (as an anti-splashing agent) and evaporate to
dryness under vacuum.

4. Add 10ml water and 2ml chloroform, cap the tube, and centrifuge
at 600 to 700 X g.

90 DETERMINATION OF SPECIFIC ELEMENTS

5. Transfer duplicate 2ml aliquots to colorimeter tubes, add 1ml
toluene, and evaporate to dryness.

6. Add 5ml of the anthrone working solution and mix well.

7. Develop the color (green) by heating for 6 minutes at 100°C or
16 minutes at 90°C.

8. Read the color at 625m^ against a blank anthrone solution.

9. Prepare a standard curve from evaporated aliquots of standard
galactose solution (as described above) treated as in steps 5 through 8.

If the complete fatty acid composition of the glycolipids is known,
the average molecular weight can be determined and the galatose con-
tent can be used to calculate moles of glycolipid. If this information is
not available, conversion of sugar content to quantity of glycolipid can
be calculated on the basis of expected average sugar content for the
particular glycolipid.

By Gas-Liquid Chromatography

Carbohydrates liberated from glycolipids can readily be analyzed by
GLC. Preparation of the trimethylsilyl derivatives of sugars is quite
easily and rapidly achieved with the commonly used silylating agents
such as hexamethyldisilazane and trimethylchlorosilane in dry pyridine.
Quantitative yields of the polytrimethylsilyl derivatives can usually be
obtained within a few minutes at room temperature. The derivatives
can be separated on short columns packed with nonpolar phases such as
SE-30 (2 to 3 percent) at temperatures about 140°C to 160°C. Mix-
tures of sugars can be quantitatively determined by using the internal
normalization procedure, and a single sugar can be analyzed by using
an appropriate internal standard. However, while the preparation of
derivatives and their GLC analysis are relatively simple matters, the
quantitative liberation of the sugars from the glycolipids presents diffi-
culties. Under some hydrolytic conditions (such as aqueous acid),
some sugars may degrade. These difficulties have been discussed in de-
tail recently by Sweeley and Vance (6). The best method available at
present appears to be anhydrous methanolysis : the products liberated
from most glycolipids under these conditions are equilibrated anomeric
mixtures of relatively stable methyl glycosides that can be readily con-
verted to derivatives for GLC analysis.

DETERMINATION OF N-ACETYLNEURAMINIC ACID
(IN GANGLIOSIDES)

By Using Resorcinol

The method described is from Svennerholm (7,8).

Reagents: hydrochloric acid, density 1.19 (at least 36.4 percent),

Fe+++ less than 0.0001 percent.

0.1M solution of copper sulfate.

FUNCTIONAL GROUPS AND LIPID CLASSES 91

resorcinol stock solution — 2g resorcinol in 100ml de-
ionized water (stable for months in refrigerator),
resorcinol working solution — 10ml stock solution
added to 80ml concentrated hydrochloric acid that con-
tains 0.25ml of the 0.1M copper sulfate solution (stable
one week in refrigerator).

blank — same as working solution, without resorcinol.
Equipment: spectrophotometer
centrifuge
water bath
test tubes, 10 to 15ml

Procedure: Three 2ml samples containing 5 to 30/xg of N-acetyl-
neuraminic acid are pipetted into test tubes. Two of the samples are the
unknowns in duplicate, and the third is the blank. To the two unknowns
add 2ml of the working resorcinol reagent, and to the third tube add
2ml of blank sample reagent. Prepare a standard curve using N-acetyl-
neuraminic acid in the range 0 to 30^. Prepare as for samples using
the 0/xg tube as the blank. Heat the tubes for 15 minutes in a bath of
boiling water. Cool and place in ice bath. Extract the color with 4ml
of a solution of n-butylacetate-n-butyl alcohol, 85:15 v/v. Shake well
and allow to settle in the cold, or transfer to centrifuge tube and centri-
fuge at 300 to 400 X g in a cold room or a refrigerated centrifuge. Com-
plete this part of the procedure within 1 hour of the heating step. Read
absorbance at 580m//. in 1cm cells. Subtract absorbance of blank sample
from test samples and read /xg of N-acetylneuraminic acid from curve.
If greater sensitivity is desired (0.05 to 0.50/xM of N-acetylneuraminic
acid), the color may be read in 50mm microcells.

If this procedure is applied to gangliosides that have been sepa-
rated into mono-, di-, and trisialogangliosides (compounds containing
1, 2, and 3 moles of N-acetylneuraminic acid per mole of ganglioside),
then the N-acetylneuraminic acid content may readily be used to calcu-
late the weight of individual gangliosides. A typical preparation of total
gangliosides extracted from mammalian brain contains 24 to 27 percent
N-acetylneuraminic acid.

This procedure can be applied to glycolipids containing N-glycolyl-
neuraminic acid in place of N-acethylneuraminic acid; however, either
a standard curve using the N-glycolyl compound must be used or the
absorbancies must be corrected for the 30 percent greater molar absor-
bancy index of the glycolyl derivative. Values read from the N-acetyl-
neuraminic acid standard curve would have to be multiplied by 0.77.

By Gas-Liquid Chromatography

The hydroxyl groups on N-acetylneuraminic acid can readily be
silylated to yield a compound amenable to GLC analysis. Thus, the

92 DETERMINATION OF SPECIFIC ELEMENTS

methyl ester of N-acetylneuraminic acid, obtained on methanolysis of
gangliosides, can be converted to the trimethylsilyl derivative with
any of the available silylating reagents. This product can be subjected
to GLC on a column such as 2.5 to 3 percent SE-30. For quantitative
determinations it is necessary to use the internal standard technique
(p. 87), as described for the GLC determination of cholesterol. The
choice of a suitable internal standard for N-acetylneuraminic acid
determinations has not been extensively studied. It appears, however,
that a sugar such as mannitol or a suitable amino sugar should prove
satisfactory.

THE DETERMINATION OF PLASAAALOGENS

By Colorimetry

The method described is that of Gray and Macfarlane (9). It is a

two-stage reaction procedure in which the aldehyde is first split off the

plasmalogen and then condensed with fuchsin reagent to give a color.

Reagents: fuchsin reagent — dissolve lg rosaniline hydrochloride

in 700ml boiling water; filter, cool; add 5.0g sodium

metabisulfite and 100ml of IN HC1 and make up to

1 liter with water; decolorize for 48 hours and store at

2°C in a dark, glass-stoppered, narrow-necked bottle.

sulfite water — 0.5 percent sodium metabisulfite in

0.1N HC1.

octan-2-ol (capryl alcohol), low ketone grade,
dimethyl acetal of palmitaldehyde, prepared from com-
mercially available palmitaldehyde bisulfite (see p. 80).
acetic acid- water 90: 10 v/v.
Equipment : spectrophotometer

15ml centrifuge tubes, glass-stoppered

Procedure:

1. To determine total aldehyde in lipid samples, prepare a stock
standard solution by dissolving 48mg palmitaldehyde dimethyl acetal
in 20ml chloroform ; prepare a working standard by diluting to 100ftg
palmitaldehyde per ml. Prepare a standard curve of palmitaldehyde in
the range 5 to SOfig. Place suitable amounts of the working standard in
centrifuge tubes and remove solvent. Add 0.5ml 90 percent acetic acid
and store at 50°C for 45 minutes, prepare one blank). Add 2.0ml
fuchsin reagent, and after 20 minutes at room temperature add 2.0ml
sulfite water and 5.0ml capryl alcohol. Shake vigorously and centrifuge
for 4 minutes. Read the optical density of the colored layer against the
blank at 546mia. Now place an amount of lipid sample containing 5 to
45 fig of aldehyde in centrifuge tubes and procede as with the working
standard.

2. Determine the phosphorus content of the samples (see p. 84).

FUNCTIONAL GROUPS AND LIPID CLASSES 93

The ratio of moles of phosphorus to moles of aldehyde in a plasma-
logen is unity. The ratio of moles of aldehyde in the sample (from step
1) to the moles of phosphorus in the sample (from step 2), multiplied
by 100, gives the "Plasmalogen Value," or the percent of phosphorus
that is present in the sample as plasmalogen.

By Two-Dimensional Thin-Layer Chromatography (10)

This method is based on the specific hydrolysis of plasmalogens to
the 2-acyl lysophosphoglyceride in the presence of a mercuric chloride
spray reagent.

Reagents: chloroform-methanol-water 60:35:8 v/v/v

chloroform-methanol- water-acetic acid 65:43:3:1 v/v

petroleum ether (bp. 40 to 60° C) -diethyl ether-acetic

acid 80:20:1 v/v/v

aqueous ammonia (sp. gr. 0.880)

5mM mercuric chloride in deionized water

18N sulfuric acid
Equipment: 20cm X 20cm TLC plates

TLC spreader and board

Silica Gel H (E. Merck A.-G., Darmstadt, Germany)

10ml stoppered tubes

oven at 180°C, situated under extractor fan

Procedure: Prepare TLC plates (about 500m//, thick) using a Silica
Gel H slurry prepared in ion-free water. Before activating plates at
110°C, wash the adsorbent by allowing chloroform-methanol-water
(60:35:8) to ascend to the top of the plate. This removes material which
interferes with charring by sulfuric acid. Silica gel washed in bulk may
be used, but remember that separations will vary and the variations
must be ascertained in advance for each silica gel used.

Duplicate lipid samples (containing 0.2 to 0.5/xg atom of phos-
phorus) are plated as 1cm bands, one 2cm in from the left and the
other 3cm in from the right-hand edge. Develop the plate in the freshly
prepared chloroform-methanol- water-acetic acid 65:43:3:1 v/v to 8cm
from the top of the plate. After removing the solvent, redevelop the
plate to the top using petroleum ether (b.p. 40 to 60°C) -diethyl ether-
acetic acid 80:20:1 v/v/v. This solvent mixture causes the lipids, other
than phospholipids and glycolipids, to migrate to a position above the
first solvent front. The residual acetic acid left on the plate after de-
velopment is neutralized by supporting the chromatogram above an
aqueous ammonia solution (sp. gr. 0.880) in a sealed dish for 5 minutes.
The excess ammonia is then drawn off under vacuum (0.5mm mercury
or less) for 30 minutes. The left-hand lipid track is then sprayed with
5mM mercuric chloride while the remainder of the chromatogram is
screened. The plate is turned through an angle of 90° and the second
solvent front is marked at 5cm from the top of the plate. The

94 DETERMINATION OF SPECIFIC ELEMENTS

plate is then reactivated by evacuating at 0.5mm mercury or less
over dark blue self-indicating silica gel for one hour. The mercuric
chloride-treated lipids are developed in the second dimension with
chloroform-methanol-water 60:35:8 v/v/v. The chromatogram is then
dried, sprayed with 18N sulfuric acid, and charred at 180°C for 1
hour in an oven situated beneath an extractor fan to ensure removal
of volatilized sulfuric acid and mercuric chloride.

The charred areas are removed and placed in 10ml stoppered
tubes. Appropriate blank areas (corresponding to large, medium, and
small lipid spots) are also taken and are added to tubes containing
evaporated aliquots (0.1ml) of a standard phosphate solution equivalent
to 0.05/xM of phosphorus. Total phosphorus is then determined on all
samples and on blank plus standard samples. The phosphorus deter-
mination procedure used by the originator of this method was that of
Sloane-Stanley and Eldin (11), but other procedures, such as the one
previously described (pp. 84-85), can be applied.

The total lipid phosphorus content of the original diacyl and mono-
vinylether-monoacyl phospholipid mixture plus the 2-acyl lysophos-
pholipid left after hydrolysis with mercuric chloride is determined.
These values for percent phosphorus can be converted to amounts of
phospholipid by multiplying by an appropriate factor. The factors can
be determined from the actual fatty acid composition of each phos-
pholipid as determined by GLC. However, for many purposes the
factors given by Williams et at. (10) for human serum phospholipids
may be sufficiently accurate:

phosphatidyl choline 26.2

phosphatidyl ethanolamine 25.0

phosphatidyl serine 25.7

lysophosphatidyl choline 17.5

lysophosphatidyl ethanolamine 16.0

If the fatty acid composition of the lipids differs markedly from that
of serum phospholipids and would mean fairly large differences in
molecular weight, then factors for the lipids being analyzed must be
determined.

When the percentages of the intact and the 2-acyl lysophospho-
lipids are known, the percent plasmalogen can be determined by the
difference between the two.

DETERMINATION OF THE AMOUNT OF TRANS DOUBLE BOND

By Infrared Spectrophotometry

Compounds with isolated trans double bonds exhibit an absorption
band in the infrared due to a C-H deformation about the trans double
bond. This absorption band has its maximum at about 10.3ju and can
be used to determine quantitatively the isolated trans double bond

FUNCTIONAL GROUPS AND LIPID CLASSES 95

content of compounds. The procedure outlined below is the one de-
scribed in The American Oil Chemists' Society Handbook of Official
and Tentative Methods, vol. 1. The method is not applicable to lipids
that contain large quantities of conjugated unsaturation, to compounds
that contain functional groups that modify the intensity of the C-H
deformation about the trans bond, nor to compounds which give rise
to absorption bands near 10.3//,. For example, long-chain fatty acids
with less than 15 percent isolated trans isomers must be converted to
their methyl esters for analysis, because efficient correction for an
absorption band of the carboxyl group at 10.6//, is not possible at that
concentration of trans.

Equipment: any infrared spectrophotometer covering the region 9
to 11//, (900 to 1150cm-1) with a wavelength scale read-
able to 0.01^ and fitted with a cell compartment for
holding 0.2 to 2.0cm cells.

fixed thickness absorption cells with NaCl or KBr
windows from 0.2 to 2.0cm.

Reagents: carbon disulfide (dry, ACS)

standards of elaidic acid, methyl elaidate, and tri-
elaidin

Procedure: Weigh 200mg of standard; place standard and sample
into a 10ml volumetric flask. Dilute to volume with CS2 and mix.
Transmittance at the trans absorption maximum should be 20 to 70
percent; if not, use a different sample weight or cell thickness.

Using matched absorption cells, fill one cell with carbon disulfide
and the other with sample or standard solution. Place the cells in the
reference and sample beam holders in the spectrophotometer. Measure
the transmittance or absorbance over the range 9 to 1 1/x. Different in-
struments will require different programs in order to obtain a satis-
factory curve. Once a satisfactory program is obtained, all subsequent
measurements must be made using the identical program conditions.

From the charts obtained with the standards and samples, read the
transmittance at 10.36//,. Convert to absorbance and calculate absorp-
tivities. Draw a base line on the charts from 10.10/x to 10.65//, for acids,
from 10.20^1 to 10.59/x for methyl esters, and from 10.05/x to 10.67//.
for triglycerides (see Fig. 14). Measure the distance from the zero
line of the recorder chart to the absorption peak (distance ab), cal-
culate the fractional transmission (be) as the distance to the absorp-
tion peak (ab) divided by the distance to the base line (ac), convert
to absorbance, and calculate the "background corrected absorptivity."
If the chart paper is calibrated in absorbance, subtract the absorbance
at the base line (c) from the absorbance at the 10.3//, maximum (b) to
get the absorbance of the sample.

Calculate percent trans isomer as elaidic acid, methyl elaidate, or
trielaidin as follows:

DETERMINATION OF SPECIFIC ELEMENTS

100

0 9 10

wavelength (microns)
Infrared absorption of rrans unsaturation in esters.

(Fig. 14)

FUNCTIONAL GROUPS AND LIPID CLASSES 97

percent trans = absorptivity (A/bc) of sample X 100

absorptivity of elaidic acid, methyl elaidate, or trielaidin

Where A = absorbance = log 100/7
b = internal cell length in cm
c = concentration of sample in g/liter
T = percent transmission

By Gas Chromatography

Equipment: Any gas chromatograph equipped to use capillary
columns. Stainless steel capillary columns (length 50m, i.d. 0.254mm)
coated with an 8 percent w/v solution of EGSS-X (Applied Science
Laboratories, Inc.) in methylene chloride. A polar wetting agent such
as Alkaterge T (an amine surfactant) is added to the packing solution
to give a concentration of 0.2 percent. Suitable prepacked columns also
can be purchased from Perkin Elmer Company.

Conditions for analysis: These are the conditions used by Lavoue
and Bezard (13), who employed a Barber Colman Model 10 chro-
matograph equipped with a Sr90 ionization detector. Suitable operating
conditions for other instruments and detectors must be determined
by individual investigators.

Column temperature: 186° to 187°C

Flow rate at column outlet: 0.2 to 0.3 ml/min.

Injector temperature: 300° C

Carrier gas pressure at inlet to injector: 8 to 10 psi

"Split" ratio: about 1:100 (The injected sample is split into two

streams so that only a portion of the sample enters the

column.)
Amount of solution injected: about 2jA
Rate of back flush (scavenger) : 60 ml/min.

The authors found that in addition they had to employ a shunt
(flow rate 8 ml/min.) to reduce the dead volume between the column
outlet and the inlet to the detector. This seems to be necessary when
using capillary columns with an argon ionization detector that has a
large internal volume relative to the volume eluted at the column outlet.
This problem is not encountered with a flame ionization detector.

The detector temperature was 220° C and the carrier gas was argon.

Using this set of conditions, the authors were able to determine
efficiently the composition of mixtures of methyl esters of oleic, elaidic,
9-trans-\2-trans, and 9-cis-l2-cis linoleic acids.

The one disadvantage of this procedure is the use of the polar
column packing, which has under these conditions a very short life
(5 to 10 days). Separation of many cis and trans isomers can be
achieved using a nonpolar column packing such as an Apiezon grease.
If suitable conditions for separation can be found using a nonpolar
column then it is clearly wiser to use nonpolar packings.

98 DETERMINATION OF SPECIFIC ELEMENTS

REFERENCES

1. Bartlett, G. R. (1959) /. Biol. Chem. 234: 466.

2. Zak, B., D. A. Luz, and M. Fisher (1957) Am. J. of Med.-Technol. Sept.-Oct.,
p. 283.

3. F & M Scientific (Div. of Hewlett-Packard) Bull. No. 116, "A rapid method
for serum cholesterol analysis by gas chromatography."

4. Radin, N.S., J. R. Brown, and F. B. Lavin (1956) /. Biol. Chem. 219: 972.

5. Radin, N. S. (1958) in Methods of Biochemical Analysis, vol. 6, p. 162.
Interscience Publishers, New York.

6. Sweeley, C. C, and D. E. Vance (1967) in Lipid Chromatographic Analysis,
ed. G. V. Marinetti; vol. 1, p. 465. Marcel Dekker, Inc., New York.

7. Svennerholm, L. (1957) Biochim. Biophys. Acta 24: 604.

8. (1963) /. Nenrochem. 10: 613.

9. Gray, G. M., and M. G. Macfarlane (1958) Bio chem. J. 70 : 409.

10. Owens, K. (1966) Biochem. J. 100: 354.

11. Sloane-Stanley, G. H., and A. K. Eldin (1962) Biochem. J. 85: 40.

12. Williams, J. H., M. Kuchmak, and R. F. Witter (1966) Lipids 1: 89.

13. Lavoue, G., and J. Bezard (1969) /. Chromatogr. Sci. 7 : 375.

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