Marine Chemistry, 6(1978) 27--40 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

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T H E D E T E R M I N A T I O N O F c~-AMINO A C I D S I N S E A W A T E R U S I N G A F L U O R I M E T R I C A N A L Y S E R

RODGER DAWSON and RICHARD GARY PRITCHARD*

Institut fi~r Meereskunde, Kiel (F.R.G.)

(Received March 3, 1977 ; revision accepted June 8, 1977)

ABSTRACT

Dawson, R. and Pritchard, R. G., 1977. The determination of a-amino acids in seawater using a fluorimetric analyser. Mar. Chem; 6: 27--40.

A method is described for the detection of free a-amino acids in the picomole range in seawater samples of less than 100 ml volume. The modifications made to a standard amino acid analyser to incorporate a fluorimetric detect ion system are described in detail, together with the methods for desalting the seawater samples on cation exchange resin and the concentrat ion procedures prior to analysis. A complete analysis of up to 30 amino acids requires around 3 h with a detection limit of around 0.05 pg of an individual acid per litre. Twenty samples of seawater from different depths in the open Baltic have been analysed for their FAA contents together with 3 samples taken from the Kie' Fjord. The total FAA content of the samples ranges from 4.5 to 84 pg/1 with a mean of around 25 pg/1. Five samples were hydrolysed prior to analysis in order to estimate the CAA content of the seawater. The values lay in the range 438--805 #g/1.

INTRODUCTION

T h e p a s t d e t e r m i n a t i o n s o f F A A in s e a w a t e r have b e e n r e v i e w e d in t h e f o r m o f T a b l e I. T h e a l p h a b e t i c n o t a t i o n s in t h e l a s t c o l u m n r e f e r to t h e fo l - l o w i n g c o m m e n t s r e g a r d i n g t h e m e t h o d o l o g i e s e m p l o y e d :

(a) R e f e r s t o t h e f a c t t h a t o r n i t h i n e was n o t d e t e c t e d in t h e s a m p l e s . T h i s a m i n o a c i d has b e e n s h o w n t o b e p r e s e n t in q u a n t i t i e s r e p r e s e n t i n g as m u c h as 10% o f t h e t o t a l F A A p o o l .

(b) P a p e r o r t h i n - l a y e r c h r o m a t o g r a p h y l a c k s t h e s e n s i t i v i t y r e q u i r e d f o r a n a l y s e s a t n a t u r a l s u b s t r a t e c o n c e n t r a t i o n s .

(c) Gas c h r o m a t o g r a p h i c d e t e c t i o n has b e e n e m p l o y e d a f t e r a d e r i v i t i s a t i o n p r o c e d u r e .

(d) T h e s a m p l e a p p e a r s t o h a v e b e e n e v a p o r a t e d o r l y o p h i l i s e d t o d r y n e s s in o n e o r m o r e s t e p s . T h i s m a y p r o d u c e r a n d o m losses o r c o n d e n s a t i o n s be- t w e e n c o m p o n e n t s ( see D a w s o n a n d M o p p e r , 1 9 7 7 ) .

* Present address: Dept. of Geochemistry, School of Environmental Sciences, University of East Anglia, Norwich, England

28

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31

(e) Ligand-exchange chromatography on Cu-Chelex 100 was employed for desalting, and in the opinion of the authors, after recovery test experi- ments, the method produces erratic or reproducible yields of FAA with heavy contamination of the samples with glycine.

(f) Key amino acids are not reported in the spectrum (particularly fi-alanine and 7-amino butyric acid and the methionine sulphoxides).

(g) The time required for analysis and the sample volumes involved detracts from the usefulness of the method for any sort of survey work.

(h) Ninhydrin reagent, often used for the determination of the total amino acid content of a water sample is not specific enough since ammonia, urea and peptides react with the reagent.

It is clear that special at tention should be paid to the concentration of amino acids from a seawater sample prior to analysis to ensure that:

(1) the sample is uncontaminated during the sampling and filtration pro- cedures;

(2) the recoveries from the desalting process employed are consistent; (3) the final concentrat ion procedures produce no alteration of the com-

ponents as a result of condensation or surface activated reactions; (4) the number of steps involved during the sample concentration proce-

dure should be reduced to a minimum to reduce the risk of contamination. The final separation and detect ion of the FAAs is performed by an auto-

matic amino acid analyser working on the ion-exchange chromatography system. The detect ion system of such an analyser should be sensitive enough to be able to carry out determinations on a small sized sample of seawater (less than 100 ml) and should produce good separations in a reasonable length of time. The reduction in the sample size also serves to decrease the risk of contamination during the concentrat ion steps since smaller quantities of reagents are required, and smaller sample bottles are easier to clean effectively. It is also possible to collect water samples with sterile water samplers in con- junction with a microbiological sampling programme since the sample volume demands are low.

METHODS AND MATERIALS

Instrumentat ion

The final separation and determination of individual amino acids is carried out using an automatic amino acid analyser (Locarte Amino Acid Analyser, The Locarte Co., London) modified for use with a fluorimetric reagent. The components are eluted with a stepwise buffer system using 3 buffers of in- creasing pH and molarity together with a mid-run temperature change-over and associated automatic regeneration and equilibration of the ion-exchange resin.

A full analysis of an extended hydrolysate requires 165 min when using a 9-mm i.d. column and 100 min when using a 6-mm column. Using the 9-mm

32

column it is possible to inject 2.5 ml of sample directly on to the column without significant loss of resolution. This amount is reduced to around 1.5 ml for the 6-mm column. The use of 6 -+ 1 micron particle size resin (Locarte, London) allows shorter columns to be used without loss of separa- tion and excessive increase in back-pressures (normally lying around 25 atmos- pheres).

The analyser was redesigned to incorporate a fluorimetric detection system by making the following modifications:

(1) The exit of the column was connected to a low-volume mixing block. (2) The reagent pump was fit ted with a pulse damper and pumped the re-

agent at one half of the speed of the buffer pump into the mixing block. (3) The output of the fluorimeter was fed through a potential divider and

low-pass filter to the terminal of a compensation recorder. The amino acids separated are displayed on the recorder as peaks and are

identified by comparison of the retention times with those of known stan- dards. Quantification is carried out using the digital output of integrated peak area which is linear with amino acid concentration.

Reagent, buffers and running parameters

The reagent consists of a mixture of 0.8 g o-phtal-aldehyde (Merck) pre- dissolved in ethanol (8 g/100 ml) and 2 ml 2-mercaptoethanol (Merck) dis- solved in 1 1 of 0.1 M p.a. sodium borate buffer adjusted to pH 9.5 with NaOH. 1% Brij 35 solution is added to the solution. The water used to make up the reagent should be as far as possible ammonia free; freshly drawn deionised water is usually good enough. The publications of Roth (1971), Roth and Hampai (1973) and Benson and Hare (1975) were consulted before deciding on the reagent composition. Contrary to Roth (1971) the pH was found to be somewhat uncritical and the molarity of the borate buffer was, out of convenience, fixed at 0.1 M instead of 0.4 M. This was found to pro- duce no significant change in sensitivity or stability. Previous reports sugges- ted that the reagent was unstable and that increase of fluorimetric back- ground was observed over a period of time.As a precaution against the absorp- tion of ammonia from the air, the reagent was kept in a sealed reagent bottle under a blanket of N 2 delivered by the N2 ballast system of the analyser. Fol- lowing these precautions, the reagent has proved to be stable for long periods of time (up to a month). The reagent has the advantage over the conventional ninhydrin reagent that it is prepared in a much shorter time, is cheaper and is far more sensitive when combined with fluorimetric detection (Xex = 340 nm, Xf1455 nm).

The reagent reacts with all a-amino acids without the necessity of heating, thus producing sharp peaks because of the reduction in dead-volume. Proline and hydroxy-proline are not detected unless they are oxidised with chlora- mine-T or sodium hypochlorite in strongly alkaline media (St. John, 1975). The ultimate sensitivity of the method is not governed by either the detector

33

or the reagent, but rather the purity of the buffer solutions required for elution.

The eluting buffers employed in the method are Durrum Pico Buffers System II (Durrum Chemical Corp., Palo Alto, Calif.). The buffer concen- trates are diluted before use with freshly drawn deionised water and protec- ted from the atmosphere with sulphuric acid traps. The buffers prepared in this manner produce relatively quiet baselines at moderate sensitivities.

The run parameters

Buffer A pH 3.19 Buffer B pH 3.86 Buffer C pH 4.60 Regen. 0.2 M NaOH Temp. c/o 48°--63°C Equilibrate buffer A

are as follows:

6 mm column 9 mm column (minutes) (minutes)

1 20 40 50 45 55 15 15 20 35 60 60

Buffer flowrates lie around 70 ml/h for the 9 mm column and 35 ml/h for the 6 mm column. Reagent flowates are 35 ml/h and 17 ml/h respectively. All samples are injected onto the column in a loading buffer with a pH of 1.8.

Sampling and storage o f seawater samples

Samples of seawater are collected with a sterile sampler (Niskin Sterile Water Sampler 2 1 vol.) and immediately filtered through 0.2 #m filters (Sartorius) in a sterile, precleaned filtration apparatus (Sartorius). 120 ml of sample is transferred to a glass bottle with teflon lined cap and disinfected and adjusted to pH 3 by the addition of pentachlorophenol in acidified aqueous ethanol. The samples are then stored at -20°C whilst awaiting analysis. All glassware used for the sampling and work-up of the samples is cleaned by rinsing with 20% HF solution followed by rinses with bidistilled (Quartz glass) water. The glassware is then dried in a cabinet at 450°C.

Desalting of seawater samples

The samples of seawater are desalted on cation exchange resin (Dowex 50W-X8) in the H ÷ form after acidification to pH 3. A maximum of 100 ml needs to be desalted since the detection system is sensitive to less than 20 picomoles of an amino acid. For a sample size of 100 ml, approximately 20 ml bed volume of resin was employed for samples from the Baltic with salinities less than 20%o (according to Garrasi, personal comm., much smaller quantities of resin may be employed without reducing the recovery efficiency). The cation exchange resin should preferably be well aged or cleaned and re-

34

generated over a long period of time and packed into glass columns 20 × 2 cm with glass frits and teflon stopcocks. A dropping funnel with a ground glass joint serves to contain the sample during dropwise passage through the column. The seawater sample is passed through the column (regenerated and converted to H + and rinsed with bidistilled water) at a flowrate of about 5 ml/ min. The column is then rinsed with 2 or 3 bed volumes of distilled water and then eluted with 80 ml (4 bed vols.) of 3 M a.r. NH4 OH into a rotary evaporator flask acting as a receiver. 0.1 ml of a solution containing 50% glycerine in ethanol is added (Dawson and Mopper, 1977) and the sample evaporated under reduced pressure at 40°C. The sample is transferred with about 5 ml of distilled water to a 25 ml flask where it is concentrated twice to dryness with intermediate washings with distilled water to remove the ammonia. The sample is taken up in 0.5 ml pH 1.8 buffer and is ready ]!or injection on to the column of the analyser. Usually only 0.2 ml of sample need to be injected to give reasonably sized peaks.

Recovery tests on extraction procedure

Spiked solutions containing different mixtures of amino acids were pre- pared by the addition of 5 nmole of each acid to 100 ml of 0.3 M NaC1 solu- tion made up in bidistilled water acidified to pH 3.0. The standards were taken through the whole procedure as described above. The results are shown in Table II, and indicate that the procedure is reasonably reproducible. The basic amino acids are only poorly recovered by the procedure as are the more acidic components; cysteic acid, taurine and urea. Urea reacts erratically with the reagent and has a very low response (20 times less than other acids) and thus it was uncertain what the exact recovery figure was. The determina- tion of urea should in any case be performed using a separate method, since little confidence can be placed on the results of the amino acid analyser for this component. All concentrations of amino acids in seawater were sub- sequently corrected according to the mean recovery percentage.

Blanks

A series of procedural blanks were carried out by taking a sample of 100 ml of bidistilled water, acidified to pH 3.0 with HC1 through the whole procedure. The results of several tests showed peaks on the chromatograms corresponding to: asp, 0.12; ser, 0.28; glu, 0.05, ala, 0.08; gly, 0.18 and orn, 0.08 pg/1. Small traces of thr, iso, leu, lys and arg were detected.

The size of the blanks was considered acceptable at the sensitivities em- ployed (same as those employed for the seawater samples) and all results were subsequently corrected according to the mean reagent blank. Reagent blanks were found to be considerably higher if new cation exchange resin was used. The cation exchange resin employed for the desalting was therefore continuously reused in the same column for a long period without removal

TABLE II

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Recoveries of amino acids from spiked solutions

Recovery %

Amino acid 1 2 3 4 5 mean

CysSO2H 49 53 51 Taurine 52 58 55 Urea 30 30 Aspartic 108 86 103 100 104 100 Threonine 98 89 97 95 97 95 Serine 113 120 108 110 115 113 Glutamic 96 89 97 93 95 94 Glycine 115 113 101 105 102 107 Alanine 102 87 99 99 97 97 Cystine 90 85 92 90 91 90 Valine 90 85 93 91 90 90 Me thionine 80 73 86 84 78 80 Allo iso leu 82 86 84 Iso-leucine 84 79 91 89 85 86 Leucine 84 78 91 90 85 86 Tyrosine 73 69 70 70 71 70 Phenylalanine 81 74 71 72 72 74 ~-alanine 81 75 73 74 76 76 Ornithine 82 76 82 84 83 81 Lysine 83 78 78 86 87 82 Histidine 71 60 68 70 70 68 Arginine 55 53 52 56 54 54

Others : citrulline ; 90, cistathionine ; 81, ~/-aminobutyric ; 78, METO ; 108, METS; 98, aminosugars <20%.

for r e g e n e r a t i o n in larger ba tches . Each c o l u m n was r e g e n e r a t e d s e pa r a t e l y b y the passage of 2 N NaOH s o l u t i o n f o l l o w e d by convers ion to the H + fo rm wi th 3 N HC1 and f inal r ins ing to pH 3--4 wi th b id i s t i l l ed water .

RESULTS AND DISCUSSION

The resul t s o f a survey of t he free a m i n o ac id c o n t e n t s of w a t e r samples f rom d i f f e r e n t d e p t h s at 3 s t a t ions in t he Balt ic and one sampl ing si te in the Kiel F j o r d , us ing the m e t h o d s de sc r ibed above are p r e s e n t e d in t e rms of t o t a l f ree a m i n o acids in Tab le III . An e x a m p l e of a c h r o m a t o g r a m o b t a i n e d f rom a s eawa te r sample and the m o l e pe r cen t age of each a m i n o acid in t he sample is d e p i c t e d in F ig .1 . Al l the resul ts have been c o r r e c t e d for e x t r a c t i o n effi- c iency and r eagen t b lanks . A ful ler t r e a t m e n t o f the d a t a in c o m p a r i s o n to the bac t e r i a l m e a s u r e m e n t s ca r r i ed o u t on the same wa te r samples is s c h e d u l e d to a p p e a r in a s epa ra t e p u b l i c a t i o n (Dawson and Gocke , 1977) . F ive of the samples were h y d r o l y s e d wi th 6 M HC1 for 22 h a t 110°C p r io r to t he desa l t ing p rocedu re , in o r d e r to e s t ima te the c o n c e n t r a t i o n of c o m b i n e d a m i n o acids in s eawa te r (see also Table I I I ) .

36

TABLE III

Concentration of FAA in Baltic Seawater (May 1976)

Sample Conc. Total FAA Conc. CAA (#g/l) (~rn.w. 100) (#g/l)

Gothland Deep 2 m 30.2 438

20 m 4.5 50 m 22.8

100 m 27.5 150 m 19.6 200 m 22.1 240 m 13.0

Bornholm Basin 2 m 23.7

10 m 28.6 20 m 29.9 40 m 34.2 50 m 31.6 60 m 26.7 87 m 23.4

Danziger Bucht 2 m 38.6

10 m 27.9 20 m 26.3 40 m 36.8 60 m 21.3

100 m 28.2 526.6

Kiel Fjord 2 m 84.5 805 4 m 54.7 472 6 m 50.5 521

An average mole % of the individual amino acids includes the fol lowing amino acids in the indicated p ropor t i ons : cysSO2H; 3.4, tau; tr, me to ; 1.3, asp; 5.3, mets ; tr, thr; 4.2, set; 18.2, glu; 6.0, cit; tr, gly; 21.1, ala; 9.6, cys; tr, me t ; tr, iso; 1.0, leu; 1.8, tyr ; 0.9, phe; 1.9, t3-ala; 3.6, orn; 5.8, 3,-a.b.a.; tr, lys; 7.9, hist; 2.2, arg; 2.3., val; 2.0. Others include amino sugars and me thy l histidines in trace amoun t s .

An unident i f ied peak is of ten found in the region of arginine with a concen t r a t i on a m o u n t i n g to up to 2--3% of the total (me thy l arginine is a possibil i ty). F r o m the samples analysed, it would appear tha t the concent ra- t ion o f c o m b i n e d amino acids exceeds tha t of the F A A by a fac tor o f a round 10. With the excep t ion o f the sample taken at 20 m in the G o t h l a n d Deep (4.5 pg/1), the mean concen t r a t i on o f F A A in the open seawater o f the Baltic at tha t t ime of year , is a round 25 gg/1. The concen t r a t i on o f the free and

i s t

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gly

37

"thr:

glu.

asp

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Fig.1. Chromatogram of a seawater ext rac t (20 ml sample) for F A A col lected at 6 m in the Kiel Fjord. The concen t ra t ions of the individual acids were quant i f ied as follows; in nmoles/ l : meto , 11; asp, 34.4; thr, 23.2; ser, 88; glu, 36; gly, 100; ala, 56; val, 16; iso, 9.6; leu, 12; galact, 4; tyr, 6.8; phe, 7.2;~-ala, 20.8; ~'-amino b.a., 14.4; orn, 44; lys, 12; hist, 7.2 ; arg, 9.6; cysS 02 H, 4 ; eit, t r ; tau, tr; cys, tr; glucos, tr; met , tr; urea, tr i phosph oser., tr; OH-lys, tr. The total concen t ra t ion o f amino acid in the sample lies around 51 pg/1, assuming a mean molecular weight of 100. tr denotes traces detected.

combined amino acids lies within the ranges reported by several authors for open sea samples (see Table I). The combined amino acids appear to be higher in concentration than those reported by Bohling (1972) for the North Sea but within the range reported by Garrasi and Degens (1976). The values for the FAAs are considerably higher than the values reported by Williams et al. (1976) and by Lee and Bada (1975, 1977). The proportions of the indi- vidual amino acids in the samples appear comparable with the data produced by Garrasi and Degens (1976) who employed a similar method together with an analyser with good resolution and sensitivity.

38

Future research objectives

All procedures used for concentrating organic components from seawater, however mild and uncontaminating, are open to criticism, simply because we are to a large extent ignorant as to the nature of these components in seawater It is for instance feasible that during the process of desalting on ion-exchange resins under weakly acidic conditions, labile peptide linkages are disrupted or metal chelates dissociated, and thereby larger quantities of " f ree" com- ponents are released and analysed.

The marine analytical chemist is therefore faced with the dilemma of having to change his concept of " f ree" amino acids in seawater since measure- ments made after preconcentrat ion or desalting produce alterations. The analyser system described is capable of detecting amino acids at their natural concentrations by direct injection of 2 or 3 ml of acidified seawater. The baselines produced by multiple buffer systems are at present somewhat un- acceptable but single buffer runs for the acidic and neutral amino acids with relatively quiet baselines at the lower picomole range are possible and may produce clearer information on substrate levels. Calculations based on micro- biological turnover rates for amino acids suggest that only a small part of the amino acids (also glucose) measured by existing methods involving precon- centration or desalting, is available to the heterotrophs as "free" compounds (Dawson and Gocke, 1977).

The problem of separating metal organic chelates from "free" components may be insoluble by the application of cation exchange columns with acidic eluting buffers and alternative techniques may be required. Recent investiga- tions (Garrasi and Mopper, personal communication) using a direct injection technique have already shown that distinct differences exist between samples desalted and those injected wi thout t reatment (quite apart from the reduced risk of contamination). For example, the presence of ~-alanine in several samples after desalting on cation exchange resin appears to be a product of the preconcentrat ion procedure either arising from contamination from the resin or as a result of hydrolysis of some higher molecular weight component on the columns.

ACKNOWLEDGEMENTS

We wish to thank Prof. Degens, Drs. Mopper, Garrasi and Gocke for the invaluable discussions leading to the product ion of this report, Mr. L. Lobel, the Locarte Company, London, for discussion leading to the modified machine and Mrs. B. Lohmann for the efficient preparation of samples. The project was supported by the Sonderforschungsbereich 95 of the German Research Society.

39

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