SYSTEMS ANALYSIS OF METHODS

FOR MEASURING TRACE DISSOLVED

ORGANIC MATTER IN SEAWATER

George Francis Diehl

United States Naval Postgraduate School

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	SYSTEMS	ANALYSIS	OF METHODS
	FOR MEASURING TRA	CE DISSOLVED
	ORGANIC	MATTER IN SEA WATER
		by
	George	Francis	Diehl, Jr.
Thesis	Advisor :		E.D.	Tra£	anza

June 1971

Approved {^on. pub tic nzlca.bc; dlbtsiibiution witirnutcd.

Tio3;ii6

Systems Analysis of Methods for Measuring Trace Dissolved Organic Matter in Seawater

by

George Francis Diehl, Jr. Ensign, United States Navy B.E., Villanova University, 1970

Submitted in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE IN OCEANOGRAPHY

from the

NAVAL POSTGRADUATE SCHOOL June 1971

ABSTRACT

The diffuse field of marine analytical organic chemistry is systematically analyzed to select schemes for measuring the trace amounts of dissolved organic compounds which can be a- dopted as standard, rapid, routine tools to advance the ocean- ographic understanding of this important aspect of the ocean.

The many methods considered for measuring dissolved organ- ic compounds in seawater have been systematically reduced to three systems that are potentially routine and rapid for ship- board work. These include quantitative gas chromatographic analysis of all amino acids and qualitative results for histi- dine, cystine, tryptophan, and arginine; gas chromatographic analysis of lipids including light hydrocarbons, fatty acids, and sterols, and; autoanalysis with tetrazoleum blue after charcoal adsorption for soluble sugar compounds like glucose, sucrose, and fructose.

Pumping systems or glass samplers on a chemically inert hydrographic wire followed by pre-centrifugation and inverse multiple filtration in combination with a selected analytical scheme will provide a system for routine analysis of seawater. Synoptic data of these compounds can be compared with total organic carbon which is currently the only routine analysis for organics in seawater.

TABLE OF CONTENTS

y

I. INTRODUCTION : : 8

A. DISSOLVED ORGANIC COMPOUNDS PRESENT

IN SEAWATER 11

1. Soluble Sugars 12

2. Amino Acids 15

3. Lipids 15

II. SAMPLING PROCEDURES 17

. A. MATERIALS 23

B. SAMPLING DEVICES 24

C. QUALITY CONTROL 26

III. STORAGE AND CLEANING OF APPARATUS 27

IV. FILTRATION PROCEDURES 29

A. CELL RUPTURE 32

V. ISOLATION PROCEDURES 35

A. AMINO ACIDS 35

1. Evaporation Techniques for Concentration 48

B. LIPIDS '48

1. 'Liquid Extraction 49

2. Lipoid Substances Measured and Other Isolation Procedures 56

C. SOLUBLE SUGARS 59

1. Recent Improvements 61

2. Enzymatic Assays 61

3. Other Techniques 64

VI. IDENTIFICATION. OF INDIVIDUAL SPECIES 65

A. AMINO ACIDS 65

B. LIPIDS 68

C. SOLUBLE SUGARS 70

VII. DISCUSSION AND CONCLUSIONS 71

BIBLIOGRAPHY 81

INITIAL DISTRIBUTION LIST 95

FORM DD 1473 96

\LIST OF TABLES

I. Properties of seawater and. ace ano graphic processes that are influenced by dissolved

organic matter 9

II. Naturally occurring organic substances 13

III. Dissolved organic matter sampling devices 19

IV. . Methods of amino acid analysis 36

V. Methods of lipid analysis 50

VI. Properties of Amberlite adsorbents 58

VII. Methods of soluble sugar analysis 62

LIST OF FIGURES

1. Distribution of Free Organic Constitutent s in Seawater 1*1

2. Flow Diagrams for Current Methods of Amino Acid Analysis 43

3. "Ideal" Analytical Schemes for Measuring

Dissolved Organics in Seawater 79

\

ACKNOWLEDGEMENT

I thank Dr. E.D. Traganza for his helpful ideas and direction throughout this work. The encouragement and ex- periences of Dr. L.M. Jeffrey and her group at Texas A & M University; that of the Chemistry Department staff at Woods Hole Oceanographic Institute; and that of all the oceano- graphers who patiently answered my inquiries, were invaluable I am grateful also to Dr. E.C. Haderlie, Dr. C.P. Rowell, Miss J. Keene, and Miss Lyke at the Naval Postgraduate Schocl for helping to complete this paper.

I. INTRODUCTION

The presence of trace amounts of organic matter in sea- water is well documented, but their importance is not as well known [Vallentyne 1957; Collier 1953; Wangersky 1965; Kalle 1965; Wagner 1969]. Their minute concentrations have defied routine analysis and prevented an understanding of their role in oceanography. Only recently has it been recognized that these dissolved organic compounds account for important phy- sical, as well as biological and chemical differences between natural seawater, and salt solutions of the same mineral composition [Table I]. In order to determine the precise role these organics play, so that they may be used to predict and understand oceanic processes, reliable, routine systems to identify these dissolved species are essential. A main objec- tive of this thesis is to systematically select current tech- niques which will make it possible for the chemical oceanographer to place less emphasis on analytical chemistry and more on a study of the ocean.

Dissolved organic matter is operationally defined as that organic substance passing through a filter with a pore size of 0.^5 microns ,. under laboratory conditions. Because of this small size, and the fact that on the average, every gram of organic matter is dwarfed by 36,000 grams of salts in 900,000 grams of water, the essence of the problem is to re- move the relatively enormous amount of salts, while

TABLE I ■

PROPERTIES OF SEAWATER AND OCEANOGRAPHIC PROCESSES THAT ARE INFLUENCED BY DISSOLVED ORGANIC MATTER

BIOLOGICAL

1. Energy Sources [Johannes, et al . 1969]

2. Growth stimulators, e.g., vitamins [Johnston 1955]

3. Organic toxins [Hood, et al . I960]

k . Physiological processes of organisms [Provasoli 1963]

5. Distribution of bioacoustic properties [E.D. Traganza, personal communication]

6. Pollutants and contaminants [Goldberg 1970]

7. Photosynthesis, metabolism, mineralization [Wagner 1969]

CHEMICAL

1. Interaction in calcium carbonate system [Chave and Suess 1970]

2. Interaction in nutrient cycle [Menzel and Ryther 1970] 3- Fluorescence' [Traganza 1969]

M . Ion-exchange properties of clays [Duursma 1970]

5. Interaction in CO2 system

6. Synthesis of marine humus [Kalle 1966]

pwvqTraT.

1. Water mass characterization [Blumer 1970]

2. Sea slicks and surface viscosity [Jarvis, et al . 1967]

3. Surface tension [Lumby and Folkard 1956]

4. Color [Christman 1970]

5. Sound transmission [Hood 1966]

6. Heat balance at air-sea interface [Hill 1962]

7. Foaming properties [Garrett 1967]

8. Surface potential [Jarvis 1965]

GEOLOGICAL

1. Formation of petroleum in marine sediments [Slowey, et al 1962]

2. ProDerties and comDOsition of sediments [Degens, et al . 1964]

3. Radiocarbon dating [Emery i960]

4. Occurrence in ferromanganese minerals on sea floor [Graham 1959]

5. Dating by pigments [Vallentyne 1957]

6. Natural light and heavy hydrocarbon seepage from oil bearing submarine sediments

concentrating the minute amount, of organic matter to reason- ably measurable quantities.

Many concentration and desalting procedures have been proposed to separate classes of compounds from "sea salts." These techniques are scattered throughout the literature in oceanographic, chemical, engineering, and geological jour- nals. Many of these incorporate reactions or procedures which are specific for particular compounds, while others give positive results for distinct molecular groupings. Some methods have been completely worked out; in others, only preliminary steps have been attempted. Each of the pub- lished papers contributes only relatively small pieces of information to the organic puzzle. No real effort has been made to step back and examine what progress nas been made in this field, and into which areas to direct future work. Such an approach at this point will be very valuable.

The analysis of organic matter in seawater can be thought of as a system of interconnected procedures. As compound isolation and identification methods are improved, particular techniques stand out as superior in various ways. This be- comes evident by their successful use in field and laboratory work. At the same time, other methods seem to have no re- deeming value in the light of more sophisticated, accurate, and practical procedures. A careful examination of the ad- vantages and disadvantages of each technique, and of their precision, sensitivity, and selectivity will be valuable to

10

future investigators for optimizing systems for trace organic analysis in the sea.

In the sequence of organic compound identification one must start with accurate sampling, followed by nondestruc- tive filtration or centrifugatlon, concentration and/or desalting which must not alter the samples, and finally, quantitative determination of the individual species present. Laboratory procedures and handling often will cause degrada- tion or alteration of the dissolved species, which are indi- genous to the very dilute, natural marine environment. In addition to this inherent source of erroneous results, there is the obsequious contamination from such externals as samplers, sampler handlers, filters, reagents, and so forth.

This paper is directed to the search for routine organic analysis and to the need for a more consolidated effort to optimize the procedures for measuring dissolved organic mat- ter. The results of this work will be used at the Naval Postgraduate School to focus on the "ideal" compound, or compounds, which are relevant, relatively easy to measure, and are variable in the ocean. Data on this or these com- pounds' presence and concentration will be complemented with a total dissolved carbon method to study the marine environment .

A. DISSOLVED ORGANIC COMPOUNDS PRESENT IN SEAWATER

There are many types of dissolved organic compounds in the ocean. Stumm and Morgan (1970) have concisely tabulated

11

these naturally occurring sub-stances [Table II]. The distri- bution of the free const itutent.s has been instructively pre- sented by Degens (1970) [Figure 1].. This paper will show the results of a systems analysis for soluble monosaccharides, lipids and their derivatives, and free and combined amino acids. These three groups were chosen because they are sig- nificant in the ocean, and much has been published on methods to identify these compounds in seawater. 1 . Soluble Sugars

Among the life substances, monosaccharides or hexoses are principally derived from planktonic activity in the eu- photic zone. They are the hydrolysis products of higher carbohydrates. Knowledge of their presence and concentra- tion can therefore be a valuable measure of the past and present biological activity in an area.. Paradoxically, with all methods that have been developed to quantitatively measure individual organic compounds in the sea, the techniques used for monosaccharides have been, in the past, the least numer- ous, least sensitive, least accurate, and least reliable. This was due, in part, to the fact that monosaccharides are especially susceptible to bacterial digestion, decomposition, and rearrangement during concentration and desalting proce- dures. Also, monosaccharides are unstable, very reactive organic compounds which may affect the diagentic processes [Josefsson 1970]. However, there is now a good deal of op- timism for improving these methods to the point where development of a routine analysis is likely.

12

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INDOLES

PHENOLS

AMINO SUGARS

UREA

HYDROCARBONS

SUGARS

FATTY ACIDS

AMINO ACIDS

(FREE)

AMINO ACIDS

(COMBINED)

FIGURE 1 'Distribution of free constituents in sea water. The individual samples have been grouped into systematic classes of compounds and have been plotted in the form of cumulative frequency diagrams to summarize' the information in a comprehensive form. The diamond-shaped figures represent the 2 sigma range. The data are presented in jug C/liter to allow a direct comparison to the total dissolved organic matter which is generaiiy reported in mg C/liter. ( Degens, E.T., 1970 )

11

2. Amino Acids

Amino acids are another important water soluble hydro- lysis product, derived from proteins. They have been found to be biologically important to marine ecology in many ways. These include excretion by plankton and utilization .by algae, bacteria, and marine invertebrates. They are essential for growth in some species of phytoplankton by satisfying certain micro-nutrient or vitamin requirements [Shiraishi and Provasali 1959].

Amino acids have been found in all oceans, and are variable with depth. They are continuously being recycled in the marine environment. Flux data as well as concentra- tion levels are keys to understanding community energetics. A reliable method to measure this component is needed to correlate the many biological events and other properties in the sea.

3 . Lipids

The class lipids includes compounds that may be chemically unrelated, but are all characterized by their solubility in organic solvents like ethyl acetate or chlor- oform. They are commonly esters of fatty acids. Their deri- vatives include hydrocarbons, mono-, di-,and triglycerides, sterol esters, steroids, free fatty acids, and phospholipids.

Lipids have been more extensively examined because they appear to compose from 10-50% of the total dissolved organic carbon in seawater [Jeffrey 1970], and they are relatively easily separated from seawater by liquid

15

extraction. They have been found in natural surface slicks, deep anoxic waters, and surface waters, in both coastal and oceanic environments. They were found to be directly pro- portional to the total dissolved organic carbon which in turn is proportional to the distance from land and depth of water [Jeffrey 1970].

The high concentrations of lipids found in sea water result from the fact that they are more resistant to biolo- gical attack than amino acids or carbohydrates and because of their relative greater abundance. A knowledge of their concentrations would help determine the natural background of dissolved organics in the oceans, before human pollution becomes extensive.

Hydrocarbons, considered a lipid in this study, have been found in all depths of the seas [Swinnerton and Linnenbom 1967]. Garrett (1967) found that lipids are a pri- mary constitutent of natural sea slicks that alter the phy- sical characteristics of the air-sea interface. In addition, vertical and horizontal fatty acids profiles may be used to study mixing rates and current patterns in the ocean [Slowey, et al. 1962].

A more detailed study of the relationship of lipids to marine organisms can be found in a paper by Lovern (1964). In this valuable paper Lovern discusses the lipids of marine plants, invertebrates, vertebrates, mammals, and their metabolic roles, for example, in marine diets.

16

II. SAMPLING PROCEDURES

On critical analysis of the methodology for measuring dissolved organic matter in seawater, it becomes apparent that despite the improved analytical techniques for isolating and identifying the trace organic constituents, methods for sampling the seawater have not progressed to the same level of development. In fact, in many papers dealing with the determination of these dissolved species the sampling method is often obscure or undefined. This seems to indicate that perhaps not enough consideration is given to this fundamen- tal step in the whole system. The results of current organic analysis are for the most part dependent on a large, uncontaminated sample that is representative of the seawater desired.

The primary reason for proper sampling procedures is to prevent probable contamination of the seawater sample from the device itself, or during transfer and storage of the sampler. This contamination is effected by a variety of degradation or condensation reactions catalyzed by container walls resulting in loss or modification of the compound sought, or its phase transfer. Furthermore, regarding or- ganic constituents, contamination of even a few orders of magnitude below total organic carbon (generally <1 milli- gram/liter) is critical relative to the concentrations of the individual component compounds present .

17

Many seawater sampling devices have been proposed or used Table III is a compilation of samplers that have either been designed specifically for dissolved organic matter, or sam- plers that have been modified for this use because of their large volume and potential low contamination. Table III was prepared using certain criterion for the "ideal dis- solved organic matter sampler." These criteria are based on those of Clark and his co-workers [1968] and include:

1. minimum contamination

2. large volume sample

3. ease of shipboard handling

H. ease of cleaning and short turnaround time

5. simplicity of design

6. reasonable cost

7. prevention of organic reactions, e.g., oxidation, and

8. ease of transfer and storage-

With respect to the large volume criterion, it must be re- membered that there are analyses that require small volumes that may have a better chance of becoming routine for oceanographic studies.

These devices in Table III collect large volume samples in two ways: either by continuous pumping on board, or by collecting descrete samples. They have been designed for surface collection, or at depth. Some samplers may include in situ filtration (see filtration section). There is very little critical comparison of various samplers [Gordon 1969]j and it is hoped that Table III will be helpful in list! .

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t/1 ►- z Ul z £ o	1. Deslqned for work at 4500 meters. 2. Allows for evacuation or filling with inert gas to prevent oxidation of sensitive organic compounds. 3. Easily dissembled and can be cleaned" ul trasonically. 4. Collected 1n situ thru a Milllpore filter. 5. Glass liner can be removed and stoppered.	1 > m 1/) • ^s C3 *J >< <o t- l/l ^ •*- 1 — IA 3 >, 9""— OI *Q i- c •a 01 - is> 0 33 XI L- • 10 r— O	1. Practical and compact. 2. Device can be stored and handled without removing samples.	1. Cheap and simple In construction. 2. Sampler Is hauled up 1n a horizon- tal position to reduce water pressure on container. 3. Tested to 2000 m. 4. Young (1969): "... light and cheap, but not rugged enough for routine work."	1. Satisfactory flushing. 2. Averaqe turn-a-round time on surface is 15 minutes. 3. Tested to 4000 m. 4. Can be cleaned ultrasonically.
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DIMENSIONS OF SAMPLER AND EASE OF HANDLING	137 x 16.5 (O.D.) cm; can be lowered on 0.425 cm. hydrographic wire	designed to be supported singly on the end of a hydrotiraphlc wire	the Nisken Rosette 1s 18" x 18" (O.D); remote control depth sensor; accomodates reverslnq thermometers on each bottlr:	can be used on 4 mm hydrotiraphlc wire; sampler 1s easily repaired	slmph' to operate and rugqetl; easy to repair; used from a low hydro - boon; lightweight and compact.
u. o a Ul Ul 2: _l =3 O- _l 31 O <t > co	1-15 1. adjusted by tie rods	0	1.7 1.; 5.0 1.; and 30.0 1.	0	0 CO
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0 Ul ee z 0 0 u. — CO CO Ul — 0 CO >- 3: _l <-> «t — z =: «£ 3	dissolved organic matter	dissolved organic carbon	large volume hydrographic casts	radioactive trace metals and particulate organic matter	14 C analysis
c_ or O ul Z CO	Rupture-disc triggered sampler	01	IA OI *J «J O .£> C OJ z	"Tantalus" bottle 1	IA OI a. E m iA en OJ ji L.

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22

alternatives to solving the contamination-sampling problem. In this regard, a standard sampling procedure is preferable and may be obtained by utilizing the desirable aspects of each of the samplers listed. An ideal sampler for universal usage may be a possible outcome of this study, similar to the results of Clark and his co-workers (1968).

A. MATERIALS

Most investigators in this field will insist that glass is the best material for collecting uncontaminated samples, but glass is expensive and a difficult material to use in constructing samplers. Metal containers are not often used, except for stainless steel, due to Impurities and also reactivity with organics . Some plastics emit soluble plas- ticizers into a sample and thus contaminate it. It has been shown that such commercially used plasticizers as the phthalite esters bleed out of container walls during storage or processing [Nishiwaki and Fukai 1970]. Jeffrey (1970) recommends polyvinyl chloride because it is less expensive, allowing a more universal standard usage, and that any contamination from this plastic by soluble plasticizers that is possible, is apparently at a very low and acceptable level. In most, cases, the proper procedure before using any material in a sampler is to examine its specific interference with the organic compound or compounds under study.

23

B. SAMPLING DEVICES

If synoptic profiles are desired, large multiple quanti- ties of water must be collected. This implies the Nansen bottle type casts , but more commonly a single sampler fasten- ed to the end of a hydrographic wire. The resulting collec- tion and shipboard filtration becomes a slow and extremely cumbersome task. Several investigators have overcome this problem by pumping seawater samples from depth and, in some cases, subsequently through a filter, and adsorption or ex- traction unit, in one continuous operation. This technique provides large volume samples up to 9000 liters providing three milligrams of organics [Jeffrey 1969]. This procedure also eliminates the extra handling involved in filtering, transfer, and storage. However, attention must be given to contamination by gain or loss of dissolved compounds in the tubing, and the rupture of biological cells by the pumping technique. Also, it should be noted that with deep water pumps, submersible pumps have the advantage that they take in samples at the desired inlet at the end of 'the tubing, and are bubble-free; whereas deck-operated pumps may take in "samples" from any opening or leak along the entire length of the pump's tubing and are unsuitable for gas-free sampling. Nevertheless, recently, more and more investigators are using submersible pumping (and filtration) units in their study of dissolved organics [Jeffrey 1969; G.W. Harvey, personal com- munication]. It is not clear whether this technique is wholly satisfactory with respect to contamination, or how much mixing

24

takes place in the line, but it-is potentially very valuable mainly because it automates the analysis. Woods Hole Oceano- graphic Institute [Dr. Blumer and Dr. Edhardt , personal com- munication] is pumping from 4 depths with 4 pumps at each depth, to obtain synoptic replicate profiles.

The sea surface contains higher concentrations of dis- solved organic matter than other positions in the water col- umn [Garrett 1970]. It is an area of increased microbial activity [Williams 1967] and a possible significant site of photochemical reactions [E.D. Traganza, personal communica- tion]. Collection of seav/ater samples in this microenviron- ment is vital. Organic matter, especially polar and water insoluble species, show sharp concentration gradients in these layers. The collection problem here is to obtain representative samples from a thickness from one centimeter to several Angstroms. Garrett (1965) and Harvey (1966) and others not listed in Table III [Goering and Menzel 1965, 1967] have proposed samplers to do this . Although much of the early work on sea slicks [Jarvis 1967, and Williams 1967] have been done using Garrett's sampler, low efficiency with respect to total collection of materials present, due to adsorption on the Monel metal collecting screen, vertical mixing from below, and floating surface contaminates, all contribute to give only qualitative data, at best.

Harvey's rotating cylinder apparatus seems to avoid these problems and also makes it possible to sample a much larger

25

sea-surface area more rapidly and to collect layers as thin as 60 microns. James Reed (1969) has successfully used a modified Harvey "skimmer" to collect lipoid substances from the surface film of "windrows" in Monterey Bay, California.

C. QUALITY CONTROL

Quality control is an important aspect of accurate sam- pling that must be borne in mind no matter which sampling technique or device is used. Bowen [unpublished] and Hood (1968) have established quality criterion analysis for sam- ples which are collected individually or continuously, re- spectively. Generally, measurement of any chemical parameter that has established or easily measured concentration grad- ients should complement any hydrocast to assure that the sample is properly associated in the synoptic picture of the ocean. Actual sample depth, and recorded sample depth as indicated by the depth at which the tripping mechanism was activated are frequently not the same. This is especially important in the case of the very large volume samples [Schink and Anderson 1969]. Such chemical properties as salinity^ dissolved oxygen, silicate, and phosphate have been used to verify the organic concentrations. This is normally done by comparing consecutive organic subsamples for agree- ment with the known or established concentrations profiles of a chemical parameter at each hydrostation. This procedure has not been used in the majority of the papers reporting organic matter concentrations in seawater and is possibly a major

26

reason for the lack of agreement on many organic profiles published by various authors .

III. STORAGE AND CLEANING OF APPARATUS

Once a large water sample is collected for subsequent dissolved organic analysis, it must either be processed im- mediately, or preserved and stored for analysis in the insti- tutional laboratory ashore. If sampled properly, the task of immediately processing the water collected follows direct- ly, assuming that all glassware and materials that come in contact with seawater are scrupulously clean. If it is necessary to store the sample for future work, several pre- cautions must be taken. During storage, the quality of the sample may change due to adsorption of organics or detritus onto container walls, and to microbial degradation or utili- zation. The former problem is solved by following the same criteria for choosing the type of material which is suitable for sampler construction. The latter problem has been " resolved to varying degrees by the use of acidification [Webb and Wood 196'7], chloroform [Chau and Riley 1966], HgCl2 [Josefsson 1970], thymol [Palmork 1963b] , and deep- freezing of the sample [Degens , et al . 1964]. These proce- dures, although used to prevent bacterial activity, may also kill organisms, releasing their cellular components [Webb and Wood 1967; Gilmartin 1967]. Parker (1967a) suggests that if immediate analysis is not practical, then freezing is

27

permitted. However, while stored at low temperatures, ad- sorption of volatile organics , and CO2 fixation by contami- nating micro-organisms may be possible sources of additional organic matter. In general, any method of attenuating microbiological activity should be examined carefully to avoid creating additional sources of experimental error.

In all micro-organic analysis, ultra clean chemical apparatus is vital. This is especially true when working in the parts per billion organic concentrations found in sea- water. If possible, work should be done in a "clean" room to prevent contamination from such unsuspected sources as room air vents [Blumer, personal communication]. Such substances as stopcock grease and impure solvents should never be used. Glass ana plastic equipment must be thorough- ly cleansed of all possible contimination. Dr. Jeffrey [personal communication] has recommended a scheme which she uses in all trace organic work. In it, polyvinyl chloride is cleaned initially with soap and water, then rinsed with "clean" ethyl alcohol and again with acetone. As for cleaning glassware, a good scrubbing with soap and water is done initially, followed by distilled water rinses. The glass is then soaked in good cleaning solution (chromic acid and HpSOij), then rinsed with distilled water. To remove any traces of insoluble grease that the cleaning solution may not have oxidized, rinses with acetone, chloroform, and alcohol' are adequate. After drying, the glassware should be covered with aluminum foil. If samples of less than a liter

28

are processed, it is worth the time to finally rinse with

•v

organic-free distilled water. This can be obtained by putting 5 grams/liter of potassium persulfate in distilled water and leaving it overnight, or by ultraviolet radiation of water containing 1 ml/liter of ^Op [Jeffrey, personal communication]

When glassware is not in use, it should be covered, and then cleaned just before use. Another alternative is to store it in distilled water containing 5 gm/liter of persulfate solution .

IV. FILTRATION PROCEDURES

It is evident from Tables IV, V, and VII and the results of Parker (1967a) that many investigators fail to describe the details of their filtering procedures. Since many artifacts may result from improper filtering techniques, meaningful com- parison of results of various authors is not possible unless either a standard procedure is adopted, or the filtering step is described completely. That is, the type of filtration should be specified including the type of membrane used, pre- treatment , pore size, amount of positive or negative pressure, filter surface area and volume filtered. Parker (1967a) has presented a valuable comparative study of various methods of filtering natural waters .

The principal reason for filtering seawater samples is to obtain the dissolved organic fraction free of plankton, par- ticulate matter, and bacteria. The filtrate o-f seawater,

29

passed through a filter of 0.45 microns pore size is commonly considered to contain only the dissolved organic fraction. However, Ogura (1970) has found that dissolved organic parti- cles, less than 0.45 microns are sometimes retained on a 0.45 micron filter by adsorption onto the surfaces of inorganic and organic particulate matter that are held back by the fil- ter. This indicates that perhaps a group of dissolved com- pounds that may be susceptible to adsorption may never have been detected. Garrett (1967) realized this phenomena and did not filter his sample before analyzing for organics at the sea-surface interface. Ogura (1970) also indicates that further fractionation of the dissolved species is possible. This may be a useful tool to isolate the dissolved compounds by molecular size. For example, he found that in a Scenedesmus suspension, filtration through filters of de- creasing pore size resulted in the following breakdown:

SIZE RANGE % CONCENTRATION OF

(MICRONS) DISSOLVED ORGANIC CARBON

0.45-0.22 8

0.22 - 0.10 4

0.10 88

Parker (1967a) found that although a 0.45 micron pore filter does not remove all bacteria, a small pore size (viz. 0.22 micron) may trap hydrated macrcmolecules that are part of the dissolved fraction. Johannes (1968) noted that the E

30

bacteria that are allowed to pass a 0.45 micron pore filter are not enough to cause problems for the first couple of hours .

Cellulose ester membranes [Millipore, Gelman, Schleicher and Schuell Companies] are the most commonly used filters in the field despite some of their inherent pitfalls. Glass fiber filters and metal fiber filters, such as silver, are being used more often. Membrane filters can be washed, are readily available, and allow a reasonable flow rate. How- ever, they contain 2 to 3% of their dry weight as detergents such as Triton X-10 0 [Chan 1967]. In addition to the con- tamination, this causes a foam in the filtrate if not complete- ly washed, and can cause cell rupture. Such interference may confuse the investigator, as reported by Wallace ana Wilson (1969) and result in erroneous results. Furthermore, Guillard and Wangersky (1958) found that unless membrane filters are washed before use, they will elute soluble car- bohydrates. In fact, they caution that significant errors will result if samples less than one liter in volume are passed through these filters. This soluble carbon can be removed by passing 60 ml. of 0 . IN HC1 through the filter [Parker 1967a]. Glass fiber filters owe their increased usage to the fact that they can be efficiently cleaned in an autoclave or by ignition. Parker (1967a) found that, out- side of filtration time, there was no significant difference in total carbon produced using a pre-washed 0.45 micron membrane [Millipore] filter, and a 0.45 micron silver fiber

31

filter [Silas]. The^ total filtration time in this study was much longer for metal fiber filters , than for membrane filters Jeffrey claims that Gelman 0.3 micron glass filters are as good as Millipore. They are easily sterilized, and they fil- ter faster because they are thicker and have more surface area [Hood 1968]. Menzel and Vaccaro (1964) recommend filtra- tion with pre-combusted glass fiber filters that have been rinsed with a small amount of sample prior to use. Blumer (1970) claims that clean extracted filter paper, although not as retentive as membranes, eliminates cell injury and allows gravity filtration. In a comparative study, he found that paper filtration and consecutive filtration through paper and clean membrane filters allowed the same materials to pass. He concludes that paper filtration is adequate for .qualitative identification of dissolved organics; whereas gravity filtration followed by membrane filtration is desirable for quantitative work.

A. CELL RUPTURE

As more precise methods of analysis are introduced, re- sults are blurred by cellular material rupturing on the fil- ter and falling into the filtrate, increasing apparent concentrations -of some organic species. It has been verified that plankton, especially naked flagellates, are subject to cell rupture above a certain, but unknown, pressure drop across the filter [Nishiwaki and Fukvi 1970]. Gentle posi- tive or negative pressures may be used to increase the flow,

32

but non-destruction of cells must be assured. Many reported values are probably inaccurate because of cell fragments adding to the dissolved content of a filtrate.

One of the reasons for using pressure to promote filter flow, is to overcome the resulting clogging of the pore af- ter passage of water over a period of time. This also, in effect, decreases the pore size allowing only progressively smaller and smaller particles to pass through. A system that would obviate applied pressure, and minimize the clog- ging limitation is a "cascade" filtering unit. Such a unit, in which the sample is passed through several membrane fil- ters of decreasing pore size, has been used by Lysyj and his •co-workers at the U.S. Interior Department's Department of Saline Water (1968). They used a two stage cascade unit. and are planning a multistage assembly. . Lewis and Traganza (1971) in their work have used a system of decreasing pore sized fritted glass filters with the added feature of filter inversion to allow gravity to work against the organisms. which might clog the filters, i.e., they fall away. Traganza (1969) and Pomeroy . and Johannes (1966) used systems based on this principle reported originally by Dodson and Thomas (196*0. Since the above problems are eliminated, this will improve the efficiency of each filter for fractionation.

Centrifugation has been used as a pre-filtration step to remove cells that might either rupture on the filter, or clog it. Parker (1967a) found that centrifugation at 6?00 x g followed by either membrane or metal fiber filtration

33

was very efficient in removing seston from seawater samples. Lewis and Traganza (1971) reached the same conclusion and have suggested constructing a large volume, 10-20 liter, centrifuge as a pre-filtration step.

In situ filtration is another solution to the cell rupture problem. Such devices as the "biodialystat " [Parker 1967b], the "rupture disc triggered" sampler [Clark, et al. 1966] and the submerged membrane filter apparatus [Williams 1969 ] may significantly minimize cell rupture as well as minimize sample handling. This technique has also been applied to submerged sampling-filtering pumps [Spencer and Sachs 1969]. Filtering at ambient pressure should provide a very repre- sentative sample of the dissolved organics as they exist in the marine environment. The biodialystat, whicb is not a filter in the strict sense, operating on the principle of sol- ute diffusion, has shown much potential. According to Parker (1967a), the "biodialystat" is more efficient than filtration. It not only keeps cell injury to a minimum by filtering in situ, but it is designed to prevent filter clogging and also to preserve the sample for 96 hours after collection by sealing and storing in the unit. Although work with the "biodialystat" has been limited primarily to fresh- water sampling, Parker [personal communication] believes there is no reason it can not be used in the marine environ- ment. This may be an optimistic view for salts usually kill good ideas conceived for freshwater.

3^

V. ISOLATION PROCEDURES

A. AMINO ACIDS

There has been a relatively large number of papers pub- lished on the isolation and measurement of dissolved, free and combined, amino acids. Table IV presents the major contributions for detection, isolation, and analysis of these important compounds in the marine environment . Amino acids have been found in most oceans, bays, estuaries, and gulfs at the surface and at depth. Typical concentration levels that must be detected range from <0 . 5 z^g/liter to 15/jg/liter (ppb). From the data in Table IV, all investigators have filtered their samples to remove particulate matter and bacteria. Most have used 0.45 micron pore-size membranes , while a few, in more recent papers, have used filters with a pore size of both 0.45 micron and 0.30 micron, with no apparent conflicting results with respect to concentration magnitudes. Degens , et al. (1964) filtered with vacuum. High pressure drops have been shown to be a cause of cell rupture which may partly explain his higher concentrations of amino acids. Bacterial decomposition, especially a problem with amino acids, has been inhibited either by freezing, or the addi- tion of chloroform, thymol, or HgCl , or a combination of a chemical preservative plus freezing.

When all the methods for detecting amino acids are viewed, four fundamental schemes are apparent. After- filtrat ion is

35

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41

complete, these are generalized by the flow diagram in Figure 2 .

42

00

CO-PRECIPITATION TO CONCENTRATE

ION-EXCHANGE DESALTING

IDENTIFICATION

(B)

DERIVATIVES OF AMINO ACIDS ARE PRODUCED

SOLVENT EXTRACTION OF DERIVATIVES

CONCENTRATE BY EVAPORATION

IDENTI- FICATION

(C)

CONCENTRATION BY EVAPORATION

ION-EXCHANGE

DESALTING

CONCENTRATE BY EVAPORATION		IDENTI- FICATION

(D)
ION-EXCHANGE DESALTING		CONCENTRATE BY EVAPORATION		IDENTIFICATION

Figure 2 . Flow Diagrams for Current

Methods of Amino Acid Analysis

43

The- details of each of these steps will follow later in this section.

The co-precipitation scheme A, developed at Texas A & M University, represents the first successful attempt to qual- itatively determine the presence of amino acids in seawater. However, poor recovery of <50% under most conditions, and the long and laborious work involved to concentrate a sub- stantial amount of organics makes this method less desirable than others [Chau and Riley 1966]. Commercial ferric chloride used to coprecipitate the organic matter introduces extraneous organic carbon which is difficult to avoid. And finally, especially for large water samples, it is difficult to remove the iron and other co-precipitated cations

Scheme B, which processes the chemical derivatives of amino acids, has much potential. One nice advantage of this technique is that the derivatives can be manufactured in

the field and preserved for analysis ashore, analogous to

4.0

the Mm addition step in the Winkler dissolved oxygen meth- od. The 2, 4-dinitro-l-f luoro-benzene method [Palmork 1963a] seems to work better in natural seawater than does the recent dansylation method ( l-dimethylaminonaphthalene-5-sulphonyl chloride) developed by Litchfield and Prescott (1970), [Webb and Jeffrey, personal communication]. However, the dinitro- phenyl derivatives are unstable and light sensitive. Palmork reported only the recovery of neutral and aromatic acids, and Chau and Riley (1966) found poor recoveries for other

Hk

amino acids by this method. Suitable automation of the chromatography, after the dinitrophenyl derivate manufacture and its extraction, may develop this into a useful method. Dansylation is the reaction of amino-terminal residues of proteins, peptides, or free amino acids with 1-dimethyl- aminonaphthalene-5-sulphonyl chloride (DSN-C1) to form highly fluorescent amino acid derivatives.

CH, CH

\ / 3

N

h,/\//

on p-i

R 0

I I

+ N - C - C

H H

OH

CH-j CH...

N

^

^/W

+ HC1

R C

qo _ m _ p _ r _ nil ~~2 N ° L J"

H

DNS-C1

a-amino Compound

"Fluorosphoric amino derivative"

Although Dr. Webb found that dansylation did not work well in seawater, his examinations were not exhaustive. Be- cause this process yields stable derivatives that are amen- able to highly sensitive fluorescence spectrophotometric analysis it should be more extensively investigated in the future [E.D. Traganza, personal communication].

Scheme C, first proposed by Palmork (1963b) was employed by Chau and Riley (1966), who 3 after a careful and complete study of procedures proposed up to that time, recommended it

^5

as "the. most reliable method to- determine amino acids." The salient feature of this scheme is that the sample is alternatively evaporated, while periodically removing salts from the mother liquor, and desalted with cation exchange resins. This method seems to work favorably and has been recently modified by Riley and Segar (1970) and is now claimed to be more precise, twice as sensitive, and less time consum- ing. The changes introduced by the latter investigators were the use of Dowex 50V/ exchange resins in place of Amberlite CG 120 resins in the desalting step, and a Chromascan [Joyce Loeble Co.] to identify the TLC spots.

Scheme D seems to be the method that most workers re- commend in current work [Webb, Jeffrey, Harvey, Blumer, per- sonal communication; Sicgel and Degens 1966; Kobbie and Crawford 1988; Andrews and Williams 1971]- Amino acids are concentrated and isolated from seawater by ligand ex- • change on copper-Chelex 100 resin. Combined amino acids are determined by hydrolysis of the column eluate, which is then run through the ligand exchange procedure again to obtain them as dissolved free amino acids.

Chelex 100 resin (Bio-Rad Laboratories, Richmond, Calif- ornia) has extraordinary select ivities for transition metals. Such metals as copper will not bleed from the column when seawater is passed. The resin structure is

.^CHoCOOH — ___

R - CU0 - N MLm 0

d "^-CH2C00-

HS

where :

M is the metal ion

N is the coordination number of the metal

L denotes the added ligand (e.g., amino acid) which becomes bound to the metal [Siegel and Degens 1966] The active sites of the resin are the inodiacetlc acid groups The degree of success using this method is varied, which may be explained by variation in individual technique, and the varying quality of the resins. It is something of an art and a bit of luck in selecting a good batch of resin. Webb and Wood (1967) and Riley and Segar (1970) have examined SiegelTs lead (1967) and worked out this method to a high •degree of accuracy. Webb found that under his conditions, the method is least good for the acidic protein amino acids; it is usually poor for taurine, but is very good for the phosphonic acids that he has tried [Webb, personal communication] .

Riley and Segar (1970) have found, using radioactive tracers, that maximum removal of amino acids occurred be- tween pH 9.0 and 9.-5. Appreciable loss of amino acids were found to occur if more than 50 ml. of distilled water is used to wash the column after desalting. Also, to remove traces of salt before introduction into the amino acid ana- lyzer, the evaporated remains were passed through a cation exchanger.

47

The_ recovery efficiency of the Cu-Chelex column falls with continued use, and it must be regenerated. Webb and Wood, and Riley and Segar regenerate when the efficiency drops to 80% .

1 . Evaporation Techniques for Concentration

Evaporation is the most common procedure to reduce the volume of samples before or after the desalting step. The fundamental criterion is to evaporate as gently as pos- sible to prevent sample rearrangement or destruction. In or- der to minimize thermal decomposition, Palmork (1963b) eva- porated the acidified sample under vacuum to about 20% of original volume in a climbing film evaporator. This evapo- rator heats small portions of the sample at a time, at moderate temperatures (~ 50°c). The rotary film evaporator operates similarly and allows removal of salt crystals periodically to improve the efficiency [see Scheme C, in this section].

B. LIPIDS

There have been many investigations to isolate lipoid substances from seawater. This is essentially due to their higher concentrations relative to that of proteins and car- bohydrates, and. the fact that lipids are readily extracted by non-polar organic solvents.

Just, as with amino acids and soluble sugars, a descrip- tion of the sampler used is frequently absent in lipid stu- dies. L.M. Jeffrey, in her extensive studies "of lipoid

M8

substances 3 recommends a polyvinyl chloride sampler rinsed with the extracting solvent. If quantitative results are desired, polyethylene samplers and containers should be avoided [Jeffrey 1970].

From the data in Table V, filtering is achieved, for the most part, by using 0.45 micron pore-membrane filters. How- ever, no description of precautionary filter washing is evident. Garrett (1967), to avoid possible losses due to surface active material, did not filter his samplers at all.

1 . Liquid Extraction

Riley and Skirrow (1965) concluded that solvent extraction is probably the most efficient method available for recovering dissolved organic matter from seawater for Qualitative analysis . Since lipids lend themselves to tbis method, most investigators employ extraction. The most com- mon system for lipid analysis after pre-f iltering the sample includes solvent extraction with vacuum distillation, to remove the solvent, followed by either paper or gas chromatography.

There are several drawbacks to solvent extraction. These include contamination from impure commercial solvents, and compound degradation during multiple or prolonged ex- traction. Also, lipids tend to adsorb onto solid surfaces in acidic solutions and since acidic conditions are desirable during extraction, compounds may be lost. A pH level of about 2 was found to be optimum [Blumer 1970]. At higher pH, free acids are partly ionized and incompletely extracted.

^9

10

>>

ro

c

et

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■o

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sz +->

QJ

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to (— z o		1. The greater part of these adds are presumably breakdown products during extraction. 2. Chloroform 1s easier to handle 1n continuous extraction systems. (Wagner 1969)	1. The results Indicate that this method was adequate for both qualatatlve and quantitative analysis of C,0 and C-q acids. 2. Because the solvent 1s removed by distillation in a current of nitrogen, low molecular weiqht adds are lost. (Wagner 1969)	Solvent extracts of sea water were separated into 8 lipid classes by solvents of increasing polarity.	Measurements were made Investigating primary production in a plastic sphere	Most phenolic compounds were found 1n hydroly- zates of the sea sediments
a: o ■>. o z «I >- or UJ > o UJ at	i — > — SS ^j UO		recoveries of 95% to 100% attained after 3 weeks were con- firmed by proces- sing known con- centrations; 85% recovery after one week	90% extraction efficiency based on C-14 labelled stearic acid	o 4-> o-> >. u Ol > o o « QJ Cn i- en	limit of detectloi was estimated to be 100 mgC/mJ per water as glycollic add	reproducible to _£ 10% confirmed by standards
u_ o t/l z <4 UJ JE	c t— O LlJ □		partition chro- matography on a silica column, eluted with chloroform con- taining increas- ing amounts of tert-butyl alcohol	gas-1 iquid chro- matography of methyl esters (method only permitted iden- tification of C10 " C2o'	fractionation by silicic add column chroma- tography, qas chromatography, followed by infrared spec- troscopic tech- niques to analyse each fraction	photometric with 2.7 dihydroxy- naphthaTene reagent	two-dimensional ascending paper chromatography; diazotized para- ni troani line was used as the spra reagent for phenols
o o UJ i. t- 5! oo UJ o		continuous extr- action in chlor- oform or other at pH 3 for 3 to 5 weeks	extraction 1n ethyl acetate at pH 3	extractloi In petroleum ether for steroids , and in ethyl acetate for other lipids at pH 2-3	1	saline residue was first leached and finally de- salted by ion- exchangers i
o o 3C t— UJ E z o ►— «I or 1— z UJ t- z c		vacuum distillation, at 60DC. .after extraction	concentration of extract by distilla- tion under nitrogen at atmospheric pressure after extraction	extract evaporated ^to dryness in a rotary evaporator	c o +-» re u o o. re > QJ	filtrate was evapora- ted to dryness, acid- ified with HCl and evaporated again J_n vacuuo; phenols and indoles were extractei from dry salts with ethyl acetate
z o t— «* c or c 1— 3 -j t- — u U- 2	1	20 1 . sample was frozen on collection (-70C) thawed, and filtered through MilH- pore filter	5 gallon sam- ples through a 0.45/(pore- sized mem- brane Milli- pore filter; chloroform was added	8 to 50 1. through a 0.45, pored Milli- pore membrane filter	1 QJ T- C r— re r— t. T- XI m qj *j	2 1 . samples were frozen; ) after thaw, / filtered through 0.45/f filter (type not described] under vacuum
	C £ u (J c c c ; c i	i 9 3 3 i j	free acetic, formic lactic and glycolic acids with average offshore concentra- tions of 0.1 mg/1	fatty adds of C- length 10-20 Including mono- and dl-unsaturated species at 0.1 to 0.8 mg/1	hydrocarbons, gly- cerol esters of fatty acids, sterol esters, sterols, P- and N- compounds 3 .4 to .8 53.	-o u re <j i— •.- ^~ r— en O E u CnO	i -5^< • eS* o re ^_ f > crr~ O 1 QJ re Xj i/> SlUTJ KTT o en o l_ c re o- *
i	1 — « ■a i or o ■_r: t 3 <-	3 jj E =r .o	Koyma and Thompson (1959) and (1964) Northeast Pacific waters and inshore waters off Washington State	Slowey et. al . (1962) Gulf of Mexico approximately (26°N, 90°W) at 10 m. to 3000 m.	Jeffrey et. al . (1963), Jeffrey (1966) and (1968) Redfish Bay, Texas waters at 20 m. and Gulf of Mexico water;	Anita et. al . (1963, 1964)	1 c r- 3 S-CT re o o 3 l/J «^o C 1- CM 4_> re U- r— ro qj qj **— re u c o * w^ a z c W t- CO oj o et «> t_ ~- cr.cn c . 0j .- cj " J= <— | o — ■ -o * *> — - 1

50

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53

At lower pH, the extracted material increases with extrac- tion time due to non-extractable polymers or to the cell fragments that have passed through the filter [Blumer 1970].

The solvents used most extensively by researchers are ethyl acetate, chloroform, and petroleum ether. Pentane, hexane, and carbon tetrachloride have been used in some studies. Jeffrey (1970) has found that ethyl acetate and chloroform removed more dissolved organic material by weight, e.g., including substituted groups, than did petroleum ether. However, in most cases, petroleum ether was found to recover higher percentages of carbon. As a result petroleum ether is a desirable solvent for such species as hydrocarbons, sterols, fatty acids, and triglycerides - the less polar lipids. Cbio'^o'^O''0^ and <=t^""~i scet^t-.p n-np* recommended for compounds with amino, hydroxyl, phosphate, or carboxylic acid groups.

Jeffrey (1970) found that ethyl acetate is more convenient than chloroform for extraction of seawater. How- ever, as noted, acidic acid is formed when the seawater is acidified and must be removed before extraction and prior to further analysis. Also, besides having a higher blank than either petroleum ether or chloroform, ethyl acetate was found to dissolve more water and salt than both other solvents. This latter phenomena makes it difficult to dry the extracts and it also may yield erroneous concentration levels. Chloroform, on the other hand, is conveniently heavier than salt water, and does not form acetic acid nor dissolve appreciable salt. Dr. Jeffrey has also found I

54

for aerobic coastal ,and oceanic waters, chloroform Is as efficient as ethyl acetate and much more efficient than petroleum ether for extraction. •

In his systematic examination of all dissolved organic constituents in seawater, Blumer (1970) chose pentane as the extraction solvent for hydrocarbons and fatty acids. Dr. Blumer ' s choice Is based on pentane 's high volatility and ease of purification.

Decomposition is postulated to be a cause of errone- ous concentration value-s after prolonged extractions [Kama- tani and Matsudaira 1966]. If more than three extractions are required to recover a significant amount of lipids, another method of isolation should be used, since the compounds may be aooreciablv destroyed ["Jef^r^7, ri^p^Qripj portiniuni c^^ion I. Blumer (1970) recommends mechanical agitation (Vibromixer) for qualitative results, and the use of separating funnels for quantitative investigations. An internal standard is a valuable tool to determine the recovery efficiency, and. thus the usefulness of a particular solvent in recovering certain dissolved constituents. Williams (1961) and Slowey (1962) both used an isotopic tracer technique to do this. Slowey used C-14 labelled stearic acid which was assayed by a pro- portional counter to calculate the percent of recovery. Williams (1965) used a soluble tracer in the form of penta- deconic acid to examine the recovery of specific components. It should also be remembered that the efficiency of a

55

particular solvent will vary with the biological and chemical characteristics of the water sampled.

2 . Lipoid Substances Measured and Other Isolation

Procedures

Jeffrey (1963,1966,1968) has published the most comprehensive studies on lipids. In her studies, lipid extract was separated by silica gel chromatography into eight fractions, from the most polar, hydrocarbons, to the least polar, phospholipids. Most other researchers have focused on only one or two lipid species.

Swinnerton and Linnenbom (1965,1967) have developed a system to purge the volatile hydrocarbons from seawater. They have detected low molecular weight hydrocarbons up to n-octane. For higher molecular weight hydrocarbons, Bluraer's (1970) pentane extract method is applicable.

Patty acid detection has progressed through the efforts of Slowey, et al . (1962), Jeffrey (1970), Williams (1961,1965), Garrett (1967), and Ushakov, et al.(1966). These isolations were made, for the most part, by extraction although Garrett coprecipitated his sample with FeClo before extraction. All but Ushakov subjected the total organic matter to extraction and followed by methylation and gas chromatography. Ushakov fractionated the fatty acid compo- nent first by thin layer chromatography before applying methylation and gas chromatography.

Other organic acids have been detected in seawater. These include phenolic, acids [Degens , et al . 1964], 1

56

chain organic acids [Koyma and Thompson 1959,1964], and volatile organic acids [Kamatani and Matsudaira 1966].

Sterols have been observed in studies by Adams (1968), Jeffrey (1970), Matthews and Smith (1968), and Riley and Taylor (1969). The latter paper introduced a new tool for isolating the dissolved organic components from seawater. These researchers used Amberlite polymeric adsorbents manu- factured by Rohm and Haas Company, Philadelphia, Pennsyl- vania. These adsorbents are hard, insoluble polymeric spheres of variable surface area, porosities, and polarities. The non-polar adsorbents are particularly effective for adsorbing non-polar solutes from polar solvents. Conversely, the high- ly polar adsorbents are very effective for adsorbing polar solutes from non- polar solvents. The physical properties of Amberlite XAD adsorbents are summarized in Table VI. Using appropriate solvents, Riley and Taylor (1969) were successful in completely recovering a variety of compounds from seawater including fatty acids, sterols, vitamins, surfactants, dyes, insecticides, and humic acids. They used Amberlite XAD-1. Calder and Fritz (1970) at Ames Laboratory have used XAD--7 . to isolate various lipids. They successfully recovered organic bases, carboxylic acids and phenols by eluting the Amberlite column with dilute strong acid (0.05 M HC1), dilute weak base (0.05 M NaHCO ) and dilute strong base (0.05 M NaOH) respectively. Desorption was achieved by elution with ether, pentane, or methanol. After neutralization, they

are readsorbed onto another column, stripped off, and char- acterized. Using a gas-chromatograTih-mass spectrometer

57

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combination, they were able to detect k2 compounds. The acrylic ester adsorbents (XAD-7 and XAD-8) are presently being used by several investigators at Woods Hole Ocean- ographic Institute to detect insecticide levels in pelagic waters .

Foam separation columns is another unique isolation tool now being developed at the Naval Research Laboratory by Wallace and Wilson (1969). Satisfactory progress is re- ported on fractionating such surface active materials as fatty acids, steroids, phospholipids, and proteins. The advantages of this method are that large volumes (~7 liters) of seawater can be processed in minutes; degradation of com- pounds is minimal; and sample handling is reduced. The method is specific for surface — active cornpounds • and allows chemical and chromatographic identification of a significant group of dissolved organics in seawater. Jeffrey [personal communication] cautions against possible contamination from the foaming gas and recommends a larger foaming tower than the ones used by Wallace and Wilson (1969).

C. SOLUBLE SUGARS

Most methods to detect and identify soluble sugars in seawater have been developed for "total sugars." These tech- niques involve hydrolysis of the organic matter and spectro- photomet.ric analysis of the resulting colored compounds. These colored compounds are the result of a reaction between the sugars and such reagents as n-ethyl carbazole, 'one,

59

phenol-sulfuric acid, and orcinol-sulfuric acid. Such methods are commonly insensitive and non-specific.

Lewis and Rakestraw (1955) studied the total carbohy- drate content in seawater by the use of the anthrone and n-ethyl carbazole methods. They found that, based on ara- binose as a standard, both methods demonstrated comparable selectivity but observed that anthrone was the more sensitive of the two. Chloride-ion correction factors, and decompo- sition of the colored reaction product were problems. Guillard and Wangersky (1958) modified these methods by using glucose as the reference, and by placing a thin layer of mineral oil over the sample to prevent oxidation during the reaction.

The anthrone method was further evaluated for determina- tions of total hexose^ keto- and aldo-hexoses . hexurnnif1. .acid, and pentose [Anita and Lee 1963]. These workers con- cluded that this was the most sensitive and precise colori- metric method to estimate these groups of carbohydrates. However, probable interference with other chemical consti- tuents and a threshold sensitivity that bordered on the natural concentration levels of marine sugars were two sericus drawbacks .

Dubois and his co-workers (1956) developed a phenol- sulfuric acid method to determine the presence of carbohy- drates. In this method higher saccharides are hydrolyzed into monosaccharides and then cycled into derivatives of furfural. These derivatives are condensed with phenol to give a chromophore in solution which has an absorption

60

spectrum maximum in % the visible region. The maximum absorption depends on the higher saccharides considered. Handa (1966) made a comparative study of the phenol- sulfuric acid, anthrone, and n-ethyl carbazole methods. He concluded from his study that the phenol sulfuric acid method is the "most recommendable for determining total carbohydrate in seawater." Handa has used this method in extensive studies of the carbohydrate content of Japanese coastal and oceanic waters [Handa 1967a, 1967b ,1970 ] .

1 . Recent Improvements

Recently, efforts have been made to analyze and identify individual soluble sugars on a more continuous basis. Such tools as enzymatic assays, chromatographic columns ion— exchange membrane electrodial,rsis and automatic analysis systems have been employed in these more sophisticated systems. These methods are listed in Table VII.

2 . Enzymatic Assays

Glucose, due to its relative abundance in the ocean (up to 45 . 6 /^g/ liter) [Josefsson 1970] and to its role in photosynthesis [Vaccaro, et al . 1968] is an important bio- chemical parameter to measure. Hicks and Carey (1968) have developed an enzymatic assay to determine glucose in seawater After pre-f iltration, glucose is coupled to a series reaction that yields a reduced coenzyme. Catalyzed by diaphorase the coenzyme is then allowed to reduce a dye, resazurin, to a highly fluorescent product, resarufin. The amount of

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63

resaruf.in produced is proportional to the amount of glucose present in the original sample. A fluorometer was used to measure the excitation and emission peaks of resarufin, which after calibration yielded the amount of glucose present in their 3 milliliter water samples. By comparison with standards, they found the assay to be sensitive to within 3 /^g/liter. Their results were confirmed independently by Yaccaro and Jannasch (1966). Vaccaro, and his co-workers (1968) have successfully employed this assay to demonstrate the occurrence and role of glucose in the open ocean.

In a recent paper, Andrews and Williams (1971) measured the oxidation rate and concentrations of glucose in the English Channel. In their method the sugars were isolated on a carbon adsorption column, eluted, and then determined enzymat ically by the glucose-oxidase system. Based on C . - labelled glucose as a tracer, they claim a sensitivity of within 1 y" g/liter of glucose. With concentrations of carbo- hydrates commonly within the range of 1-10 /'g/liter , the precision associated with such enzymatic assays are not assuring. Because of the high selectivity characteristic of enzymatic assays, and their elimination of any isolation and fractionation steps, more sensitive assays would be attractive .

3. Other Techniques

Several investigators have been successful in iso- lating sugars from seawater by using charcoal absorption columns [Wangersky 195.2; Schaefer 1965 ; and Keiling

64

Desorption from the column, always a~ problem with carbon absorption, has been achieved by gradient elution with 10- 20$ ethanol. Improved results have been obtained by pre- treating the carbon columns with Celite (hyf lo-supercel) [Keiling 1968]. Schaefer (1965) has worked out a set of optimum conditions and details for the isolation of soluble sugars from seawater.

Electrodialysis using ion-exchange membranes has been used by Josefsson (1970) to isolate soluble sugars from sea waters. This method has great potential for neutral molecules like sugars, which are not greatly influenced by an electrical field. Because desalting is continuously carried out electrically, the membranes do not require any chemical regeneration. A 2.2 liter capacity electrodialysis unit has been used by Josefsson. The membranes themselves are manufactured by milling ion-exchanger beads onto a binder such as polythene. An anionic and a cationic selective membrane are used simultaneously. Although the desalting time is 2^-30 hours, Josefsson claims that this time may be significantly reduced without loss of efficiency.

VI. IDENTIFICATION OF INDIVIDUAL SPECIES

A. AMINO ACIDS

From Table IV, it can be seen that there are basically four techniques that are used to identify the individual amino acids or their derivatives in a desalted seawater

65

sample.. These are two dimensional paper chromatography, two dimensional thin-layer chromatography, gas chromatography, and an automatic amino acid analyzer.

In a comparative study, Chau and Riley (1966) found that thin-layer chromatography is more sensitive and rapid, and the resolution of the amino acids is better, and the tailing is less, than in paper chromatography. Jeffrey [personal communication] recommends spraying with 0.1$ ninhydrin in n-butanol or acetone, and heating at 110°C for 15-20 minutes. In addition, the spots can be easily removed and eluted for spectrophotometric analysis. However, this method can only give partially quantitative data at best. Degens , et al . (1964) made visual comparisons of TLC spots with knowns and could only be accurate within ± 1S%. Quan- titative results are increased by using an analytical scan- ning device to examine spots representing the ninhydrin complexes. Riley and Segar (1970) used the Joyce Loeble Chromoscan with a thin layer scanner attachment. They found this to reduce their analysis time by several hours, and produced maximum precisions from ±0.03/^/1 for alanine to • ±0.5//g/l for phenylanaline . The eluate from a Cu-Chelex 100 column was found to contain a ninhydrin-negative material chromatographing in the non-leucine region [Wainer and King 1965]. Webb and Wood (1967) have seemed to solve this pro- blem by passing the eluate from the Cu-Chelex column through a "micro-column" of Chelex 100 in the NH^ form. Their results indicate complete purification.

66

Gas- chromatographic procedures, for natural amino acids [Gehrke, et al . 1968] and Kunisaki, et al . 1969 ] have been developed recently. These researchers found that n-trifluro- acetyl n-butyl esters to be the amino acid derivative best suited for gas chromatographic analysis.

0

ii

CH3 - CH - C - 0 - C^Hq

HN - C - CF-. II 0

N-trif luoroacety L, N-butyl ester of alanine

The particular advantage of these esters lies in the fact that three fluorine atoms are introduced into the molecule, increasing sensitivity to electron capture detection. Kuni- saki and his co-workers (1969) found that except for histi- dine and cystine, tryptophan and arginine, the reproducibility was very good for all other amino acids.

The amino acid identification tool used more and mere, and recommended by many workers, is the automatic amino acid analyzer. This device allows quantitative determination of amino acids by automatically recording the ninhydrin color value from the effluent of icn-exchange columns. Ion- exchange resins are used as the separation medium. Tech- nicon and Beckman instruments have been used. With this analyzer, the hydrolyzate of a protein or peptide may be analyzed in less than 2 4 hours; with minimum attention. An advantage of such continuous autoanalyzers irrcludes less

67

manipulation of the sample during the automated process. However, the problems of collection, filtering, desalting, and concentration remain.

B. LIPIDS

Based on the many studies and observations by Jeffrey (1963jl966,1970), the seawater-solvent extracts can be characterized both qualitatively and quantitatively.

Complete quantitative results are time-consuming, requir- ing separation of the lipid extract by elution from a silicic acid column, thin-layer chromatography of the fractions obtained, and chromatography of the li.pid derivatives. In some cases, mass spectrometry is necessary after chemical separation for absolute identification. Jeffrey was able to separate the lipid component into 8 fractions according to their polarity. The silicic acid columns used by Jeffrey were eluted in the following order, with l%9k%3 and 25% ethyl ether in petroleum ether; pure ethyl ether; acetone; 20/£ and 50% methanol in dichloromethane ; and pure, methanol . This elution scheme separated the 8 fractions. Blumer (1970) describes another scheme used in his pentane extract study. He also used silica gel and notes that the adsorbent should be partially deactivated to minimize catalytic reactions such as dehydration. Functional group tests [Wallace and Wilson I.969], solubility tests [Jeffrey 1963] and photometric techniques [Anita 1963; Riley and Taylor 1969] are applicable

68

to such. isolated fractions. These auxiliary tests are primarily used to confirm thin-layer or gas chromatography analyses .

Qualitative results can be obtained by thin-layer chro- matography of the lipid extract of 10-20 liters of water. Jeffrey [personal communication] recommends chromatography with Silica Gel G on 8M x 8" plates in 1) Toluene-ethyl acetate (19:1 by volume) for hydrocarbons, sterols, fatty acids, etc. (non-polar compounds); 2) chloroform-methanol- water (14:1) for increasingly polar compounds (Phenols, simple substituted phenols); 3) Butanol-acetic acid-water (4:1:1) for' even more polar compounds and finally, 4) Pyri- dine-acetone-NH^OH(10:6:5) . Spraying with 0.2$ 2,7 dichlo- rcf lucres cein in methanol shows up .lipids very vividly under an ultraviolet light (long wave length). No heating is necessary for development of the spots.

Gas chromatography has been used to identify lipoid substances by various workers [Jeffrey 1963,1966,1970; Slowey 1962; Williams 19.61,1965; Swinnerton and Linnenbom -1967; Ushakov, et al . 1966; Garrett 1967 and Blumer 1970]. This analysis has been applied to either the raw solvent extracts or to the methyl derivatives of the constituents of the fractions eluted from chromatographic columns, and thin-layer chromatographic spots.

69

C. SOLUBLE SUGARS

For the most part, final identification of soluble sugars has been attempted by analysis of enzymatic reaction products, spectrophotometric techniques, and simple analytical micro- methods [Table IV] . These methods have either been too in- sensitive to lower concentrations, or subject to excessive manipulation of chemical reagents while attempting to effect a desired reaction. Non-reproducibility and low resolution of the total sugar content into its individual species are also shortcomings. However, a new procedure, worked out by Mopper and Degens (197D has minimized these problems,

and is developed for use in an auto-analyzer system [Technicon 1965] ■

Mopper and Dozens pass the sample continuously through

an anionic column in the sulfate form. .The sugars are separated in the column as it 'is eluted with 89% EtOH. The eluted sugars are treated continuously with a basic solution of tetrazoleum blue. Tetrazoleum blue is more sensitive and less corrosive than either orcinol-sulfuric acid, anthrone or phenol- sulfuric acid. The limit of sensitivity is 10~" to 10~9 moles. The major success of this method is due to the devel- opment of the applicability of tetrazoleum blue for use in capillary tubing. Monosaccharide mixtures are fully resolved in 3-4 hours. This procedure should be able to complement Josefsson's electrodialysis procedure to give quantitative results for soluble sugars.

70

VII. DISCUSSION AND CONCLUSIONS

The primary objective of examining the chemistry and techniques used in the study of trace organic matter in seawater was to sort out methods of rapid analysis that can be used routinely by chemical oceanographers on board ship. Other criteria considered in this analysis include:

1. Selectivity for specific type of molecules, with respect to size, functional groups, etc.;

2. Destructive or non-destructive analysis with respect to compound alteration;

3- Continuous, or batch in operation;

4. Quantitative or qualitative analysis;

5. Reliability based on usage by independent researchers;

6. Rigorous, or quick analysis for approximate results with respect to quality and accuracy of the desired data.

One finding that is apparent is that there, is.no procedure standardization. This is evident especially in sampling and filtration. Because of this variability from technique to technique, it was extremely difficult to compare the results of independent researchers. Much of the published works on dissolved organics is qualitative or unreliable due to the use of unsatisfactory or incompletely tested analytical methods. Chau and Riley (1966) arrived at' this same conclu- sion, but. their findings were not emphasized, "and re

71

efforts, based on questionable processes continue to ramify. This report demonstrates the diverse procedures used, and should help to direct future efforts in a direction that will achieve routine, rapid, reliable techniques which can be used as tools of chemical oceanography.

The first analytical consideration must be the collection of representative samples of seawater from the water column. Table III lists samplers that have been used or recommended for collection of seawater for dissolved organic analysis. Samplers collect seawater in discrete volumes in situ or by pumping from depth to a vessel at the surface. Present ana- lysis systems require both kinds of raw seawater input. They are each useful with appropriate systems.

An ideal in situ or batch tyoe samclincr device has been developed by Clark and his co-workers (1968). Their "rup- ture disc-triggered" sampler is specifically designed for dissolved organic work, and it ensures minimum contamination by using glass. Although others recommended polyvinyl chloride there is always some quesiion about bleeding plasticizers and the problem of keeping this material clean. Clark's device is worthy of consideration as a standard for obtaining synop- tic dissolved organic profiles. Similar "rupture disc" bottles should be designed which can be attached in multiples on a chemically inert hydrographic "wire." Uncontaminated , representative seawater samples must be collected if analysis of trace organics is to become a routine matter.

72

Pumping systems, constructed of noncontaminating materials, such as those developed by Zeitoun and his co-workers (1965) have the advantage of collecting continuous samples which have the potential for obtaining continuous profiles as well as large volumes. There is still some doubt as to possible mixing, compounds alteration, or biological cell rupture during passage through the tubing and the pump itself. A feature which may be developed for pumping seawater from various depths is on line filtration, preferably at depth. Some attempt has been made by Laird (1967) as well as Zeitoun (1965). In the past, continuous pumping systems have been used for delivering large volumes to collecting systems epitomized by the charcoal adsorption columns used by Jef- frey ^ 1 Q 6 Q ^ While Jeffrey's ob^ectiire ,,t a s t ° n b t •Ci i ^ "i ^ r> c <=> amounts of organic material for identification, systems like this may have a future when more sensitive systems which require less water are available.

The results and observations of many workers suggest that combusted glass fiber and washed metal fiber filters be used in conjunction with an inverted multiple filter system. Pre-centrifugation may be useful with the develop- ment of a large volume centrifugation unit. The main advan- tage of glass and metal fiber filters over cellulose ester membrane filters is more effective filter cleansing. Means should be developed to use glass and metal filters "in line" with continuous pumping systems, as well as in batch f tion. The inverted multiple filtration principle us

73

Lewis and Traganza (1.971) minimizes filter clogging and com- pound and cellular alteration. Parker (1967a) and Lewis and Traganza (1971) both concluded that use of large volume centrifugation as a pre-f iltration step to remove biological cells that might rupture on, or clog the filter, is the most efficient separation scheme. Ideally, a simple and effective filtering procedure should be agreed upon for widespread use. In this manner, future studies would have a more representative "dissolved organic fraction" upon which to begin analysis, and compare independent results.

After proper "filtration," the seawater filtrate is assumed to contain organic and inorganic matter no larger than 0.^5 microns in diameter. Isolation of the dissolved organic matter in this sub— sample from tine relatively vast amount of inorganic salts (desalting), and subsequent frac- tionation into individual organic compounds, are both essen- tial and formidable steps in the analytical scheme. It is not a simple task to sort out accurate and reliable methods that are applicable to routine use aboard ship.

The results of this systems analysis of the methods for analyzing the three groups of dissolved organic compounds selected for consideration - amino acids, certain lipids, and soluble sugars - demonstrate that current methods employ a variety of techniques. After analysis and systematic eval- uation of all these techniques, those which would serve as rapid and potentially routine tools for trace organic work were selected.

Ik

Based on the data In Table IV and the recommendations of many investigators , free and combined amino acid concen- trations can best be measured by the use of a Cu-Chelex 100 resin column to selectively adsorb and remove the amino acids •from the dissolved salts. This step is followed by elution, concentration by film evaporation and identification adapted to autoanalysis of the concentrate. Andrews and Williams (1971) have successfully worked out the latest details of this scheme. The weakest link in this system is in the in- accuracy during autoanalysis of individual amino acids caused by chromatographic interference. The use of an identifica- tion scheme developed by Gehrke (1968) and Kunisaki (1969) and their co-workers would obviate this shortcoming by forming an amino acid ester ohst is amenable uo sensitive gas chroma- tography. These authors report that n-trifluoroacety 1 n-butyl ester derivatives allow excellent precision with most free and bound amino acids. With appropriate automated gas chro- matography as an improvement over the autoanalyzer [Figure 3], this should be a reasonably rapid and routine technique that could be taken to sea.

Other methods of amino acid analysis include the derivative reaction techniques developed by Falmork (1963a) and Litchfield and Prescott (1969). The potential of these methods lies in the fact that they minimize compound alteration by pro- cessing amino acid derivatives that are manufactured in the raw seawater sample. The dansylation reaction, which yields a highly 'fluorescent product, is desirable due to the I

75

sensitivity of fluorescence analysis. However, until such problems as reaction rate, low selectivity and recovery are solved, these derivative techniques are not now applicable.

The majority of the research on lipid analysis [Table V] has been based on liquid extraction. Although this tech- nique is simple, it is not attractive due to the time consuming multiple extractions required and the cumbersome handling involved. A recently developed adsorbent technique is superior in this respect, and has successfully isolated fatty acids, sterols, vitamins, surfactants, dyes, insecticides, humic acids, phenols, and organic bases from sea salts [Riley and Taylor, 1969; and Calder and Fritz, 1970], Both research teams used Rohm and Haas Amberlite adsorbents. Since the

according to their polarity, after neutralization, the eluted sample is readsorbed onto a second Amberlite column, stripped off, and characterized by gas-chromatography [Figure 3]- Such an analytical arrangement could be set up in a ship- board laboratory to analyze these important organic consti- tuents at sea. Reagents, a few adsorption columns, and a gas chromatograph would be all the materials that are needed to routinely and selectively measure fatty acids, phenols, and sterols from this broad spectrum of compounds.

Light hydrocarbons have been successfully assayed by a method developed by Swinnerton and Linnenbom (1967). This method is extremely accurate due to highly developed gas chromatography. In addition, Swinnerton has refined hj

76

system to a relatively rapid, and routine analysis of hydro- carbons up to n-octane. For heavier hydrocarbons Blumer's pentane extraction method is reliable , but suffers from the shortcomings of other liquid extraction techniques.

Foam separatory columns are used to isolate surface active lipids and proteins. This technique which is currently being developed at the Naval Research Laboratory may become a useful tool in some analyses, but is not very practical for general shipboard use.

Because "total sugar" methods are not selective, and enzymatic assays are not sensitive enough, most investigators measure soluble sugar concentrations utilizing charcoal adsorp- tion columns. Continuous or batch sampling is applicable to carbon columns, but of course the adsorbed sugars must be eluted. Successful desorption of the sugars has been achieved by gradient elution -with 10-20^ ethanol [Keiling 1968]. Other advances include pre-treatment of the carbon with Celite, a hyf lo-supercel , to accelerate flow through the. column. Such columns, seven feet in length, have been success- fully used by Jeffrey (1969) to isolate three milligrams of organic matter from 9,000 liters of seawater pumped from depth. The unattractive feature is the time consuming desorp- tion. However, once the raw seawater is pumped through the columns, they may be sealed for later elution and fraction- ation. The newly developed method of Mopper and Degens (1971) allows identification of the desorbed sugars on an

77

autoanalyzer, after evaporation- in a climbing or rotary film evaporator [Figure 3].

Ion-exchange membrane electrodialysis first proposed by Jeffrey and Hood (1958) has recently been developed to a high degree of accuracy by Josefsson (1970). In this method, 2.2 liters at a time are electrodialyzed through an anionic and cationlc membrane simultaneously. This desalting step may be effected in several hours with sufficient voltage. However, this is not reasonably rapid for shipboard use. The Mopper and Degens autoanalyzer method (1971) is also applicable to the desalted seawater after film evaporation.

Figure 3 summarizes systems which are considered poten- tially the most rapid and accurate methods for measuring trace concentration levels of amino acids, lipids, and soluble sugars in seawater. Batch type samples collected by modified "rupture disc" bottles are proposed in a normal hydrocast mode. Large volume pre-f iltration centrifugation and/or reverse flov; multiple filtration are proposed as procedures for removal of the undissolved fraction. Soluble sugar analysis is patterned after the semicont inuous charcoal scheme developed by Jeffrey (1969).

Once these systems for measuring these organic compounds are a reality, they can be complemented with Edhardt's (1969) ultraviolet autoanalyzer technique for measuring total organic carbon. Further useful correlations may be obtained by measuring urea concentrations which are apparently already a routine matter with the use of the autoanalyzer [Newel]

78

BATCH SAMPLING AND FILTRATION

— f

CU-CHELEX RESIN COLUMN

FILM EVAPO- RATION

ESTERI- FI CATION

GAS

CHROMATO'

GRAPHY

AMINO ACID ANALYSIS SYSTEM

BATCH OR CONTINUOUS SAMPLING AND BATCH FILTRATION

AMBER- LITE ADSORP- TION COLUMN

SEQUEN- TIAL ELU- TION AND NEUTRAL- IZATION

AMBER- LITE AD- SORPTION COLUMN

GAS CHRO- MATO GRAPH OF METHYL DERIVA- TIVES

LIPID ANALYSIS SYSTEM

CONTIN- UOUS

PUMPING AND "IN LINE" FILTRA TION

charcoal column

ETHANOL

GRADIENT

ELUTION

FILM EVAPORATION

AUTOANALYSIS WITH TETRAZO- LEUM BLUE

SOLUBLE SUGAR ANALYSIS SYSTEM

Figure 3- "Ideal" Analytical Schemes for Measuring Dissolved Organics in Seawater

79

The results of this study have unquestionably supported gas chromatography and the "autoanalyzer" as powerful tools which are beginning to solve some of the difficult analytical problems of the organic chemical oceanographer . There are more refinements to be made, especially in autoanalysis of specific organic compounds. Perhaps some professional society should sponsor a conference on organic systems. This paper could be a useful basis for organizing this type of effort.

So many physical, biological, geological, and chemical processes and properties are influenced by dissolved organic matter that it is imperative that new analytical methods be developed and used. For example, glucose and glycine are virtually ubiquitous and should be able to indicate biological trends in the ocean. These two compounds are "ideal" in that they are readily measured, highly variable, and widely occurring. Rout ine -measurements of such compounds may reveal or predict many marine processes. We may not be able to discover the entire spectrum of dissolved organic matter before it has changed significantly, but wide analytical coverage of key compounds may provide an adequate index.

Future plans should be to develop and use automated ana- lytical systems similar to those described in this paper. If these systems can be developed and coupled with "auto- analysis" of total organic carbon, it would be possible to construct a complete synoptic picture of the importance of dissolved organic matter in oceanic processes and properties of seawat'er.

80

..BIBLIOGRAPHY

1. Adams, D. and A. Richards, 1968. "Dissolved Organic Matter in an Anoxic Fjord, with Special Reference to the Presence of Mercaptans." Deep Sea Res. 15: ^4 71 —

4 81. '

2. Armstrong, F.A.J . , P.M. Williams and J.D.H. Strickland,

1966. "Photooxidation of Organic Matter in Seawater by Ultraviolet Radiation, Analytical and Other Implica- tions." Nature. 211: 481-483.

3- Armstrong, F.A.J. , C.R. Stearns and J.D.H. Strickland,

1967. "The Measurement of Upwelling and Subsequent Biological Processes by Means of an Autoanalyser and Associated Equipment." Deep Sea Res. 14 ( 3 ) : 38I-389 .

4. Anita, A.L., and C.T. Lee, 1963. Studies on the Deter- mination and Differential Analysis of Dissolved Carbo- hydrate in Seawater. Fish. Res. Bd . Canada. (Manuscript rep . ser . 168 . )

5- Anita, A.L., and C.T. Lee, 1964. "The Determination of Free Aminosugars in Seawater." Limnol_. Oceanogr. 9:12.

6. Andrews, P., and P.J. Williams, 1971. "Heterotrophic Utilization of Dissolved Organic Compounds in the Sea. III. Measurement of the Oxidation Rates and Concen- trations of Glucose and Amino Acids in Seawater."

J. of Mar. Bio. Ass. U.K. Vol 51 (in press).

7. Bader, R.G., D.W. Hood and J.B. Smith, 19 60. "Recovery of Dissolved Organic Matter in Seawater and Organic Sorption by Particulate Material." Ceochim. . Cosmochim. Acta. 19: 236-243-

8. Baker, A- ,1966. "Volatile Fatty Acids in Aqueous Solu- tion by Gas-Liquid Chromatography." Jnl. Gas Chromatogr 4(11): 418-419.

9. Barnes, H., and D.M. Finlayson, 1963. "Estimation of Lactic Acid in Seawater Solutions and Homogenates . " Limnol. Oceanogr. 8: 292-294.

10. Baylor, E.R., andW.H. Sutcliffe, 1963. "Dissolved Organic Matter in Seawater as a Source of Particulate Food." Limnol. Oceanogr. 8: 369-371.

81

11. Bishop, A.D. and L.R. Louden, 1965. "Separation and Identification of Amino Acids in Galveston and Baffin Bays, Texas." Ocean Sci. and Ocean Eng. 1965. Vol.2 Mar. Tech. Soc, Am. Soc, Lira. & Oc. pp. 1104-1108.

12. Blumer, M. , 1970. "Dissolved Organic Compounds in Seawater. Saturated and Olifinic Hydrocarbons and Singly Branched Patty Acids." In: D.W. Hood ed. Symposium on Organic Matter in Natural V/aters, Institute of Marine Sciences, University of Alaska.

13. Bodman, R.H., et al . , 1961. "A Multi-Purpose Large Volume Water Sampler .". W.H.O. I. Contribution No. 1208.

14. Bohling, H., 1970. "Untersuchungen Uber Freie Geloste Aminosauren in Meerwasser." Mar. Bio. 6(3): 213-225.

15. Bursa, A.S., 1968. "Starch in the Oceans." Jnl.Fish. Res. Bd. Can. 25 :. 1269-1284 .

16. Bykora, E.L., 1.967. "Study of Organic Substances in Subsurface Water." Ore;. Veshchestvo Pidzemn. Vod. Ego.

Zncheie Neft. Geo. 189-198.

17. Cahn, R.D., 1967 . "Detergents in Membrane Filters." Science. 155: 195-196.

18. Calder, G.V. and J.S. Fritz, 1970. "New Method for Isolating Minute Amounts of Organic Compounds Found in Water." Ames Laboratory Newsletter, 11 Dec 1970.

19. Calkins, V.P., 1943. "Micro-determination of Glycolic and Oxalic Acids, "Ind. Eng. Chem. [Anal. Ed.] 15: 762-763.

20. Chapman, G. and A.C. Rae , 1967. "Isolation of Organic Solutes from Seav:ater by Co-Precioitation . " Nature . 214: 627-628.

21. Chau, Y.K. and J. P. Riley, 1966. "The Determination of Amino Acids in Seawater." Deep Sea Res. 13: 1115- 1124.

22. Chave, E.E. and E. Suess, 1970. "Calcium Carbonate Saturation in Seawater: Effects of Dissolved Organic Matter." Limnol. Oceanogr. 15(4): 633-637.

23. Chave, K.E., 1965. "Carbonates: Association with Organic Matter in Surface Seawater." Science , 148: 1723-1724.

82

24. Christman, R.F. , 1970. "Chemical Structures of Color Producing Organic Substances in Water." In: D.W. Hood [ed. ] Symposium on Organic Matter in Natural Waters , Institute of Marine Sciences, University of Alaska.

25. Clark, R.C., M. Blumer and S.O. Raymond, 1968. "Rup- ture Disc-Triggered Sampler." Deep Sea Res. 14 : 125- 128.

26. Collier, A., 1953- "The Significance of Organic Com- pounds in Seawater." Trans. N. Am. Wildl. Conf. 18: ^463-^70 .

27. Corv;in, J.R., 1969. "Volatile Oxygen-Containing Organic Compounds in Seawater: Determination," Bull, of Marine Sci. 19(3): 504-509.

28. Creach, P.V., 1955. "The Presence of Citric and Malic Acids in Littoral Marine Waters. C .R. Acad . Sci . , Paris, 240: 2551-2553.

29. Degens, E.T., J.H. Reuter and K.N. P. Shaw, 1964. "Bic- chemical Compounds in Offshore California Sediments and Seawaters." Geochim. Cosmochim. Acta. 28: 45-66.

30. Degens, E = T„, 1970. "Molecular Nature of Nitrogenous Compounds in Seawater and Recent Marine Sediments." In: D.W. Hood [ed.] Symposium on Organic Matter in Natural Waters, Institute of Marine Scinnces, Univer- sity of Alaska.

31. Dodson, A.N. and W.H. Thomas, 1964. "Concentrating Plankton in a Gentle Fashion," Limnol. Oceanogr. 9(4): 455-456.

32. Dubois, M. , K.A. C-illes , J.K. Hamilton, 'P.A'. Rebers, and P. Smith, 1956. "Colorimetric Method' for the Determination of Sugars and Related Substances." Analt. Chem. 28(3): 350-356.

33. Duursma, E.K., 1966. "Prediction of Dissolved Organic

Compounds in Natural Waters from Composition and Recom-

position Processes ." Proc . I. B. P. -Symposium, 0ct.l0-l6, 1966. pp. 285-287.

34. Duursma, 1967. "Van-Dorn-Type Sampler," Deep Sea Res. 14: 133-134.

35. Duursma, E.K., 1970. "Organic Chelation of 6oCo and 6^Zm by Leucine in Relation to Sorption by Sediments." In: D.W. Hood [ed.] Symposium on Organic Matter :' Natural Waters, Institute of Marine Sciences, Univer- sity of Alaska.

83

36. Ehrhardt, M., 1969. "A New Method for the Automatic Measurement of- Dissolved Organic Carbon in Seawater." Deep Sea Res. 6: 393-397.

37- Emery, K.O., i960. The Sea off Southern California > John V/iley & Sons, N.Y.

38. Fell, J.W., 1967. "Distribution of Yeasts in the Indian Ocean." Bull. Mar. Sci. 17: 454-470.

39- Fiadeiro, M. , L. Solorzano and J.D.H. Strickland, 1967. "Hydroxvamine in Seawater," Limnol. Oceanogrr. 12: 555-556.

40. Garrett, W.D., 1965. "Collection of Slick-Forming Materials from the Sea Surface," Limnol. Oceanogr. 10: 602-605.

41. Garrett, W.D., 1967a. "The Organic Chemical Composition of the Ocean Surface." Deep Sea Res. 14(2): 221-227.

42. Garrett, W.D., 1967b. "Stabilization of Air Bubbles at the Air-Sea Interface by Surface Active Material." Deep Sea Res. 14(6): 661-672.

43. Garrett, W.D., 1970. "Organic Chemistry of Natural Sea-Surface Films." In: D.W. Hood [ea. ] Symposium on Organic Matter in Natural Waters, Institute of Marine Sciences, University of Alaska.

44. Garside, C. and J. P. Riley, 1969. "Thin-Layer Chro- matographic Method for the Determination of Plant Pigments of Seawater and Cultures," Analytica Chemica Acta. 46(2): 179-191.

45. Gehrke, C.V/., et al., 1968. "Quantitative Gas-Liquid Chromatography of Amino Acids in Proteins and Biological Substances," Analytical Biochemical Laboratories, Inc., Columbia, Mo.

46. Ghassemi, M. and R.F. Christman, 1968. "Properties

of the Yellow Organic Acids of Natural Waters," Limnol . Oceanogr. 13: 583-597-

47. Gerard, R. and M. Ewing, 1961. "A Large Volume Water Sampler," Deep Sea Res. 8: 298-301.

48. Gilmartin, M., 1967. "Changes in Inorganic Phosphate Concentration Occurring During Seawater Sample Storage," Limnol. Oceanogr. 12: 325-328.

84

l\9. Gjessing, E.T., 1965. "Use of Sephadex Gel for the

Estimation of 'Molecular Weight of Humic Substances in Natural Water." Nature. 208: 1091-1092.

50. Goering, J. J. and D. Wallen., 1967. "The Vertical Distri- bution of Phosphate and Nitrite in the Upper One-Half Meter of the Southeast Pacific Ocean," Deep Sea Res. 14: 29-33.

51. Goering, J.J. and D.W. Menzel, 1965. "The Nutrient

Chemistry of the Sea Surface," Deep Sea Res. 12: 839-843.

52. Goldberg, E.D., 1970. "Air Transport of Organic Con- taminants to the Marine Environment," In: D.W. Hood [ed.] Symposium on Organic Matter in Natural Waters, Institute of Marine Sciences, University of Alaska.

53. Gordon, D.C., Jr., 1969. "Examination of Methods of Particulate Organic Carbon Analysis," Deep Sea Res . , 1969, 16: 661-665.

54. Graham, J., 1959- "Metabolically Induced Precipitation of Elements from Seawater," Science, 129: 1428-1429.

55. Grasshoff, K., 1965. "Automatic Determination of Fluoride, Phosphate, and Silicate in Seawater." m : Automation in Analytical Chemistry, Technicon Sympo- sium, 1965, pp. 304-307. "

56. Guillard, R. and P. Wangersky, 1958. "The Production of Extracellular Carbohydrates by Some Marine Flagel- lates," Limnol. Oceanogr. 3: 449-456.

57- Hall, K.J., W.C. Weimer and G.C. Lee, 197 0. "Amino Acids in Estaurine Environment," Limnol. Oceanogr. 15(1): 162-164.

58. Handa, N., 1966. "Examination of the Applicability

of the Fhenol Sulfuric Acid Method to the Determination of Dissolved Carbohydrate in Seawater," Jnl . Oceanogr. Soc. Japan 22(3) : 79-86.

59. Handa, N., 1967a. "The Distribution of the Dissolved and the Particulate Carbohydrates in the Kuroshio and Its Adjacent Area," J. Oceanogr. Soc. Japan: 23(3): 1-9.

60. Handa, N., 1967b. "Identification of Carbohydrates in Marine Particulate Matters and Their Vertical Dis-

tribution." Rec. Oceanogr. Works Japan, 9(1): 6

85

61. Handa, N., 1970. "Dissolved and Particulate Carbohy- drates." In: \D.W. Hood [ed.J Symposium on Organic Matter in Natural Waters, Institute of Marine Sciences, University of Alaska.

62. Harvey, 1966. "Microlayer Collection from the Sea Surface." Limnol. Oceanogr. 11(4): 608-613.

63. Hicks, S.E. and P.G. Carey, 1968. "Glucose Determina- tion in Natural Waters," Limnol. Oceanogr. 13: 36O-363.

64. Hill, H.j 1969. "Retention of Marine Particles by Screens and Filters," Limnol. Oceanogr. 15(3): 451- 454.

65. Hobbie, J.E., C.C. Crawford and K.L. Webb, 1968. "Amino Acid Flux in an Estuary." Science, 159: 1463-1464.

66. Holm-Hansen, 0., et al . , 1965 . "Fluorometric Deter- mination of Chlorophyll." Journal du Conseil, 30(1).

67. Holm-Hansen, 0., J.D.H. Strickland and P.M. Williams, 1966. "A Detailed Analysis of Biologically Important Substances in a Profile Off Southern California," Limnol. Oceanogr. 11(4): 548-561.

68. Holm-Hansen, 0., 1968. "Measurement of Deoxyribonu- cleic Acid in the Ocean and Its Ecological Significance,' Limnol. Oceanogr. 13(3): 507-514.

69. Hood, D.W., et al . , i960. "Measurement of Toxicity of Organic Wastes to Marine Organisms," Jnl. of Water Poll. Cont. Fed., Sept. i960, pp. 982-993-

70. Hood, D.W., G.K. Park and J.M. Prescott, i960. "Organic Matter in Seawater; Amino Acids, Fatty Acids; and Monosaccharides from Hydrolysates ( Abstr ) , "' Bull . Geo. Soc. Am. 71: 1890.

71. Hood, D.W., 1963. "Chemical Characteristics of the Marine Environment," Publ. No. 10, Great Lakes Res. Div . , The University of Michigan, pp. 91-111.

72. Hood, D.W. , 1966. "Occurrence and Concentration of Organic Matter in Seawater." Presented at the ONR Workshop on June 1966, Washington, D.C. 20360.

73- Hood, D.W. 3 ed. 1970. Symposium on Organic Matter in

Natural Waters. Institute of Marine Science Occasional Publication No. 1., University of Alaska.

86

74. Home, R.A., 1969 . Marine Chemistry. Wiley Inter- science, New York.

75. Jarvis, N.L., 1965. "Adsorption of Surface-Active Material at the Air-Sea Interface." U.S. Naval Research Laboratory, Washington, D.C. (NRL Report 6220).

76. Jarvis, N.L., W.D. Garrett, M.A. Scheiman and CO.. Timmons, 1967. "Surface Chemical Characterization

of Surface-Active Material in Seav/ater." Limnol. Oceanogr 19: 88-96.

77- Jeffrey, L.M. and D.W. Hood, 1958. "Organic Matter in Seav/ater: An Evaluation of Various Methods for Isola- tion." Jnl. Mar. Res. 17: 247-271.

78. Jeffrey, I.M., B.F. Pasby, B. Stevenson and D.W. Hood, 1964. "Lipids of Ocean Water," pp. 175-198. In:

U. Colombo and G.D. Hob son [ed.] Advances in Organic Geochemistry . Macmillan Co., N.Y.

79. Jeffrey, L.M., 1966. "Lipids in Seawater." Jnl. Am. Oil Chem. Soc. 43: 211-214.

80. Jeffrey, L.M., 1969. "A Method for Isolation of Dis- solved Organic Matter from Seav/ater and Some Chemical Characteristics of tne Isolated Material." Disser- tation, Texas A & M University, Doctor of Philosophy, Department of Oceanography.

81. Jeffrey, L.M., 1970. "Lipids of Marine Waters." In: D.W. Hood [ed.] Symposium on Organic Matter in Natural Waters , Institute of Marine Sciences, University of Alaska.

82. Johannes, R.E., et al . , 1969 . "Are Dissolved Amino Acids an Energy Source for Marine Invertebrates?" Come. Biochem. Physiol. 29: 283-288.

83. Johnston, R. , 1955. "Biologically Active Compounds in the Sea." J. Marine Biol. Assoc. U.K. 34: 185-195.

84. Johnston, R. , 1964. "Seav/ater, the Natural Medium of Phytoplankton, Trace Metals, and Chelation, and General Discussion," J. Marine Biol. Assoc. U.K. 44: 87-91.

85. Josefsson, B.O., 1970. "Determination of Soluble Carbohydrates in Seav/ater by Partition Chromatography after Desalting by Ion-Exchange Membrane Electrodia- lysis, Analytica Chem. Acta., 52(1): 65-73.

87

86. Kalle, K., 1966. "The Problem of Gelbstoff in the Sea," Oceanogr, Mar. Biol. Am. Rev. 4: 91-104.

87. Kamatani, A. and C. Matsudaira, 1966. "Extraction and Determination of Organic Acids in Seawater and Marine Sediments." Jnl . Oceanogr. Soc . Japan. 22(3): 87-92.

88. Keiling, R. , 1968. Methodes de Separation et de Dosage des Glucides a 1'Etat Part iculaire , Colloidal et Disscres dans ie Milieu Marin Lequide, Genoa Mussec Civico de Sroria Maturale Giacomo Doria Annoli , 77: b 5 - 7 9 •

89. Khailov, K.M., 1968. "Organic Macromolecules Dissolved in Seawater." Geokhimiya . 5: 595-603-

90. Kihara, H.K. and H. Kuno, 1968. "Microassay of Protein with Nitrocellulose Membrane Filters," Anal. Biochem. 24: 96-105.

91. Koyama, T. and T.G. Thompson, 1959. "Organic Acids of Seawater." A.A.A.S. Preprints of Int. Oceanogr. Congr. 1959: 925-926.

92. Koyama, T. and T.G. Thompson, 1964. "Identification and Determination of Organic Acids in Seawater by Partition Chromatography." Jnl. Oceanogr. Soc. Japan 20(5) ; 7-18.

93. Kunisaki, N., T. Yoneda, Y. Ishiha.ra, 1969. "Quanti- tative Analysis of Amino Acids by Gas-Liquid Chroma- tography and Its Application." Bull, of Fac. of Fish., Hokkaido University, 20(3): 193-201.

94. Laird, J.C., D.P. Jones and C.S. Yentsch, 1967. "A Submersible Batch Filtering Unit," Deep Sea Res . 1.4(2): 251-252.

95- Larimers, W.T., 1965- "Natural Water Fractionation:

Theory and Practice." Verh. Int. Verein. Theor. Angew. Limnol. 16: 452-458.

96. Leavandowsky , M. and E.S. Hodgson, 1965. "Amino Acid and Amine Receptors in Lobsters." Comp. Biochem. Physiol. 16: 159-15.1.

97. Lewis, G.J. and N.W. Rakestraw, 1955- "Carbohydrates in Seawater," Jnl. Mar. Res. 14 : 253-258.

98. Lewis, L.W. and E.D. Traganza. 1971. "Characteristic Chemical Patterns of Dissolved Organics in V.or.'\ M.S. Thesis, U.S. Nava] s t graduate School, Ju

88

ti

5

99- Linnenbom, V.J. and J.W. Swinnerton, 1970. "Low Mole- cular Weight Hydrocarbons and Carbon Monoxide in Sea- water." In: D.W. Hood [ed.] Symposium on Organic Matter in Natural Waters, Institute of Marine Sciences, University of Alaska.

100. Litchfield, CD. and J M. Presoott, 1970. "Analysis by Dansylation of Amino Acids Dissolved in Marine and Preshwaters , " Limnol. Oceanogr. 15(2): 250-256.

101. Lovern, J. A., 1964. "The Lipids of Marine Organisms," Oceanogr. Mar. Biol. Ann.' Rev. 2: 169-191.

102. Lurnby, J.R. and A.R. Folkard, 1956. "Variation in the Surface Tension of Seawater in situ, " Bull. Inst. Oceanogr., Monaco, 53(1080): i-19.

103- Lysyj, I., et al . , 1968. "Development of Analytical Techniques for the Determination of Trace Organic Material in Waters," Office of Saline Water, U.S. Dept . of Interior, Res. and Dev. Prog. Rep. No. 327-

104. Lysyj, I., et al . , 1967. "Methods for the Determination of Trace Organic Materials in Water." Office of Saiins Waters, U.S. Dept. of Interior, Res. and Dev. Prog.

Rep. , No. 239.

105. Matthews, W.S. and L.L. Smith, 1968. "Sterol Meta- bolism. Ill: Sterols of Marine Waters, Lipids." 3(3): 239-246.

106. Menzel, D.W. and J.H. Ryther, 1968. "Organic Carbon and Oxygen Minimum in the South Atlantic Ocean," Deep Sea. Res. 15: 327-337-

107. Momzikoff, A., 1969. "Studies on the Fluorescent Com- pounds of Seawater." Cahiers de Biologie Marine 10(3)'- 221-230.

108. Mopper, K. and E.T. Degens, 1971. "A New Chromato- graphic Sugar Auto-Analyzer with a Sensitivity of 10~10 to 10-H Moles." [in press]

109. Nakajima, T. and B.E. Volcani, 1969. "3 , 4 ,-dihydro- xyproline: A new Amino Acid in Diatom Cell Walls." Science. 164: 1400-1401.

110. Newell, B.G., B. Morgan and J. Cundy, 1967. "The Determination of Urea in Seawater." Jnl . Mar. Res. 25: 201-202.

89

111. Nishiwaki, Y. and R. Fukai, [Eds], 1970. Reference Methods for Marine Radioactivity Studies, Tech. Report #118, 1970, IAEA, Vienna.

112. Niskin, S.J., 1968. "A Deck Command Multiple Water Sampler," Marine Science Instruments , Vol. 4, Plenum Press, N.Y., 1968.

113. Ognar, G. and M. Schnitzer, 1970. "Humic Substances: Fulvic Acid-Dialkyl Phtlalote Complexes and Their Role in Pollution," Science, 3.70: 317-318.

114. Ogura, N., 1965. "Method of Concentration of Dissolved Organic Substances from Seawater by Dialysis," Jnl . Oceanogr. Soc. Japan 21(5): 14 .

115- Ogura, N. , 1970. "On the presence of 0.1 - 0.5 Micron

Dissolved Organic Matter in Seawater." Limnol. Oceanogr. 15(3): 459-461.

116. Palmcrk, K.H., 1963a. "The Use of 2 :4-Dinitro-l- Flurobenzene in the Separation and Identification of Amino Acids in Seawater." Acta Chem. Scand. 17: 14 56- 1457.

117. Palmork, K.H., 1963b. "Studies on the Dissolved Organic Compounds in the Sea." Norw. Fishery Mar. Invest. 13: 120-125.

118. Park, K., W.T. Williams, J.M. Prescott and D.W. Hood, 1962. "Amino Acids in Deep Seawater." Science . 1 3 8 : 531-532.

119. Park, K., et al . , 1963. "Amino Acids in Red Fish Bay, Texas." Inst, of Mar. Sci. Vol. 9, pp. 59-63-

120. Parker, B.C., 1967a. "Influence of Method for Removal of Seston on the Dissolved Organic Matter." Jnl. Phycol. 3: I66-I73.

121. Parker, B.C., 1967b. "'Biodialystat : New Sampler for Dissolved Organic Matter," Limnol. Oceanogr. 12: 722- 723.

122. Pomeroy, L.R. and R.E. Johannes, 1966. "Total Plankton Respiration," Deep Sea Res. 13: 971-973-

123. Pomeroy, L.R. and R.E. Johannes, 1968. "Occurrence and Respiration of Ultraplankton in the Upper -500 M. of the Ocean," Deep Sea Res". 15: 381-391.

124. Plunkett, M.A. and N.W. Rakestraw, 1955- "Dissolved

•ganic Matter in the Sea." Deep Sea Res . 3 ( 12-14.

90

125- Provasoli, L., 1963- "Organic Regulation of Phyto-

p'lankton Fertility. "In : The Sea, N.M. Hill, ed. Inter- science Publishers, Inc., New York, Vol. 2, pp. 165- 219.

126. Reed, J.R., 1969. "Collection and Characterization of the Surface Film of Windrows in Monterey Bay." Thesis, U.S. Naval Postgraduate School (R269).

127. Richards, F.A. and R.A. Kletsch, 1964. "The Spectro- photometry Determination of Ammonia and Labile Amino Compounds in Fresh and Seawater by Oxidation to Nitrate." Recent Res. Fields Hydrosphere, Atmos Nucl . Geochem. 1964: 65-81.

128. Riley, J. P. and G. Skirrow, 19 6 5. Chemical Oceano- graphy , Academic Press, New York, Volumes I and II.

129. Riley, J. P. and D, Taylor, 1969. "The Analytical Concentration of Traces of Dissolved Organic Materials from Seawater with Amberlite XAD-1 Resin," Analytica Chemica Acta. 46(2): 307-309.

130. Riley, J. P. and D.A. Segar, 1970. "The Seasonal Varia- tion of the Free and Combined Amino Acids in the Irish Sea." Jnl. of Mar. Biol. Ass. U.K. 50: 713-720.

131. Rohm and Haas Company, 5000 Richmond Avenue, Philadel- phia, Pennsylvania 19124.

132. Saz, A.K., S. Watson, S.R. Brown and D.L. Lowery, 196} . "Antimicrobial Activity of Marine V/aters," Limnol . Oceanogr . 8 : 63-67.

133. Schaefer, H., 1965. Isolierung von Gelosten Kohlen- hydraten aus dem Meerwasser . Hegolander wiss Meer- esunters . Bd . 12 : 253-260 .

134. Schink & Anderson, 1969. "Bag Sampler for Large Volumes," Jnl. Mar. Tech. 3(5): 49-58.

135. Shapiro, J., 1961. "Freezing-Out, a Safe Technique for Concentration of Dilute Solutions." Science . 133: 2063-2064,

136. Shiraishi, K. and L. Provasoli, 1959- "Growth Factors as Supplements to Inadequate Agal Foods for Tigriopus Japoniais . " Tohoku J. Agric. Res. 10: 89-96.

137. Siegel, A. and E.T.' Degens , 1966. "Concentration of Dissolved Amino Acid."- from Saline Water by Lij Exchange Chromatography." Science . 151 : 1-098-117".

91

138. Siegel, A., 1967. "A New.' Approach to the Concentra- tion of Trace Organics in Seawater," pp. 235-256. In: T.A. Olson and F.J. Burgess [ed. ] Pollution and Marine Ecology , Interscience, N.Y.

139. Sieburth, J. McN. and A. Jensen, 1968. "Studies on Algal Substances in the Sea. ""I Gelbstoff (Humic Material) in Terrestrial and Marine Waters." Jnl . Exp. Mar. Biol. Ecol. 2: 174-189.

140. Slowey, J.F., L.M. Jeffrey and D.W. Hood, 1962. "Fatty Acid Content of Ocean Water." Geochim. Cosmochim. Acta. 25: 607-616.

141. Spackman, D.H.3 W.H. Stein and S. Moore, 1958. "Auto- matic Recording Apparatus for Use in the Chromatography of Amino Acids." Anal. Chern. 30: 1190-1206.

142. Stephens, G.C. and R.A. Schinske, 1961. "Uptake of Amino Acids by Marine Invertebrates." Lirnnol. Oceanogr. 6: 175.

1^3. Strickland, J.D.H. and T.R. Parsons, 1965. "Determina- tion of Carbohydrate and Crude Fibre." Bull. Fish. Res . Board Canada #125.

144. SutcliiTe, W.H., E.R. Baylor and D.W. Menzel. I963. "Sea Surface Chemistry and Langmuir Circulation." Deep Sea Res. 10: 233-243.

145- Stumm, W. and J.J. Morgan, 1970. Aquatic Chemistry, Wiley Interscience, New York.

146. Swinnerton, J.W. and V.J. Linnenbom, 1965. "Gaseous Hydrocarbons in Seawater: Determination, Science .

5: 570.

147. Swinnerton, J.W. and V.J. Linnenbom, 1967. "Deter- mination of the C]_ to Cij Hydrocarbons in Seawater by Gas Chromatography." Jnl. Gas Chromatogr. 5-' 570-573-

148. Tatsumoto, M., W.T. Williams, J.M. Prescott and D.W. Hood, 196l. "Amino Acids in Samples of Surface Sea- water." Jnl. Mar. Res. 19: 89-96.

149. Traganza, E.D., 1969. "Fluorescence Excitation and Emission Spectra of Dissolved Organic Matter in Sea- water," Bull. Mar. Sci. 19(4): 897-904.

150.

Ushakov, A.N., D.M. Vityuk, V.A. Vaber and L.D. Bergel ' son, 1966. "Fatty Acids in Waters of- the Black Sea." Okeanolc ' .6: 891-89 •

92

151. Vaccaro, R.F. and H.W. Jarinasch, 1967- "Variations in Uptake Kenetics for Glucose by Natural Populations in Seawater," Limnol . Oceanogr. 12: 540-542.

152. Vaccaro, R.P. , S.E. Hicks, H.W. Jannasch and F.G. Carey 3 1968. "Occurrence and Role of Glucose in Seawater," Limnol. Oceanogr. 13: 356-360.

153. Vallentyne, J.R., 1957- "The Molecular Nature of Organic Matter in Lakes and Oceans, with Lesser Refer- ence to Sewage and Terrestrial Soils . " Jnl . Fish.

Res. Bd. Can. Ik: 33-82.

154. Van Beneden, G. , 1969. "V/ater Color. New Technique for Determining Humic Acids," Trib . Cebedeau 22(313): 626-631.

155. Van Dorn, W.C., 1956. "Large Volume V/ater Samplers," Trans. Am. Geophy . Union. 37: 682-684.

156. Vityuk, D.M., 1967. "Fatty Acid Fractions of the Dissolved Organics of the Black Sea. Din. Vod. Vop . Gidrokhim. Chern. Morya. 1967: 143-148.

157. Wainer, A. and J.S. King, Jr., 1965. "The Use of Cysteic Acid as an Internal Standard in Amino Acid Analysis." Jour. Chromatog. 20: 14 3.

158. Wallace, G.T. and D.F. Wilson , 1969. "Foam Separa- tion as a Tool in Chemical Oceanography," Naval Res. Lab. , NRL Report 6950.

159. Wagner, F.S., 3.969. "Composition of the Dissolved Organic Compounds in Seawater: A Review. Marine Science Institute, Texas University, Contribution 14: 115-153.

160. Wangersky, P.J., 1952. "Isolation of Ascorbic Acid and Rhamnosides from Seawater." Science . 115: 685.

161. Wangersky, P.J., 1965. "The Organic ' Chemistry of Sea Water." Am. Sci. 53': 358-374.

162. Webb, K.L. and L. Wood, 1967. "Improved Techniques for Analysis of Free Amino Acids in Seawater", In: Automation in Analytical Chemistry. Technicon Sym- posium.

163. Whaley, R.C., 1958. "A Submersible Sampling Pump," Limnol. Oceanogr. 3(4): 476-477.

164. illiams, E.D.F., 1969. "A Submerged Membrane r

.aratus for Microbiological Sampling," Mar' :.ol ■ 3": '78-80.

93

165. Williams , P.J. LeB . , 1966,' "The Wet Oxidation of Organic Matter', in Seawater," Limnol. Oceanogr. 12: 292-296.

166. Williams 3 P.J. and C. Askew, 1968. "A Method of Measuring the Mineralization by Microorganisms of Organic Compounds in Seawater," Deep Sea Res. 15(3): 365-375.

167. Williams, P.M., 1961. "Organic Acids in Pacific Ocean Waters," Nature. 189 : 219-220.

168. Williams, P.M. and A. Zirino, 1964. "Scavenging of 'Dissolved' Organic Matter from Seawater with Hydrated Metal Oxides." Nature. 204: 462-464.

169. Williams, P.M., 1965. "Fatty Acids Derived from Lipids of Marine Origin." Jnl . Fish. Res. Bd. Can. 22": 1107-1122.

17 0. Williams, P.M., 1967. "Sea Surface Chemistry: Organic Carbon and Organic and Inorganic Nitrogen and Phos- phorous in Surface Films and Subsurface Waters." Deep Sea Res. 14: 791-800.

171. Wood, L., 1966. "Determination of Free Amino Acids in Seawater, " In: Automation in Analytical Chemistry. Technicon Symposium, 1965~i New York, liediad, 196* 6" .

p. 652.

172. Young, 1969. "Beer-Keg Sampler," Limnol. Oceanogr. 14(4): 634-637.

173. Zeitoun, M. , Jeffrey and D.W. Rood, 1965. "Continuous Pumping-Extraction System for Lipids in Seawater,", Texas A & M University (College Station, Texas).

94

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95

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Za. REPORT SECURITY CLASSIFICATION

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3 REPORT TITLE

Systems Analysis of Methods for Measuring Trace Dissolved Organic Matter in Seawater

DESCRIPTIVE NOTES (Type ol report and, inclusive dates)

Master's Thesis; June 1971

fy au tmORIS) (First name, middle initial, last name)

George P. Diehl, Jr

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June 1971

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97

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ABSTRACT

The diffuse field of marine analytical organic chemistry is sys- tematically analyzed to select schemes for measuring the trace amounts of dissolved organic compounds which can be adopted as stan- dard, rapid, routine tools to advance the oceanographic understanding of this important aspect of the ocean.

The many methods considered for measuring dissolved organic com- pounds in seawater have been systematically reduced to three systems that are potentially routine and rapid for shipboard work. These include quantitative gas chromatographic analysis of all amino acids and qualitative results for histidine, cystine, tryptophan, and arginine; gas chromatographic analysis of lipids including light hydrocarbons, fatty acids, and sterols, and; autoanalysis with tetrazoleum blue after charcoal adsorption for soluble sugar compounds like glucose, sucrose, and fructose.

Pumping systems or glass samplers on a chemically inert hydro- graphic, wire followed by pre-centrifugation and inverse multiple fil- tration in combination with a selected analytical scheme will provide a system for routine analysis of seawater. Synoptic data of these compounds can be compared with total organic carbon which is currently the only routine analysis for organics in seawater.

DD.FrU473

S/N 0101 -807-681 1

(PAGE 1)

96

Security Classific

SecuriU' Classification

KEY WORDS

Systems Analysis

Methods

Dissolved Organic Matter

Measurement

) .ZWA473 <bw)

BOLE H T

97

Security Classification

Thesis D56713 c.l

128447 ■

Diehl

Systems analysis of methods for measuring trace dissolved organic matter in seawater.

128447

Diehl

Systems analysis of methods for measuring trace dissolved organic matter in seawater.

thesD56713

Systems analysis of methods for measunn

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