Water R[,search Vol. 15, pp. 507 to 509, 1981 0043-1354/81/'040507-03502.00/0 Printed in Great Britain. All rights reserved Copyright © 1981 Pergamon Press Ltd

TECHNICAL NOTE

T H E U S E O F R E V E R S E P H A S E L I Q U I D C H R O M A T O G R A P H Y F O R S T U D Y I N G T R A C E

M E T A L - O R G A N I C A S S O C I A T I O N S IN N A T U R A L W A T E R S

JULIAN LEE*

Applied Biochemistry Division, DSIR, Palmerston North, New Zealand

(Received August 1980)

Abstract--Reverse-phase liquid chromatography was employed for the separation of natural water organics into fractions of varying polarity. Concomitant metals were monitored by electrothermal atomization atomic absorption spectrophotometry.

INTRODUCTION

Studies of the nature of organic compounds in surface waters have received considerable attention in recent years, particularly a class of naturally occurring poly- meric polyelectrolytes, operationally defined as fulvic acids (Weber & Wilson, 1975; Steelink, 1977; Perdue, 1978). These organic acids have been strongly impli- cated in trace metal speciation (O'Shea & Mancy, 1976; Pagenkopf, 1978; Vuceta & Morgan, 1978). Their separation and characterization is consequently of considerable importance to the understanding of various facets in metal complexation.

Steric exclusion chromatography has proven useful for the evaluation of metal-organic interactions in waters (Mantoura & Riley, 1975; Means et al., 1977). However organic fractions have not been well- resolved, with the separation of fulvic materials, after concentration techniques, achieved on a broad mol- ecular weight basis only.

Reverse phase liquid chromatography (RPLC) has developed rapidly over the last few years and provides a powerful tool for the separation of organics from complex mixtures. Non-volatile, polar, thermally un- stable molecules or high molecule weight compounds can be separated. The method has recently been well reviewed by Karger & Giese (1978). The technique has been employed in the separation of, polynuclear aromatic hydrocarbons in waters (Hunt et al., 1977; Ogan et al., 1979), drinking water extracts (Thruston, 1978) and other organics in natural waters (Jolley et al., 1975; Derenbach et al., 1978). Soluble water or- ganics are sorbed onto a stationary phase (usually hydrocarbonaceous and less polar than the mobile phase) and then eluted in a general order of decreas- ing polarity. The stronger solvent e.g. acetonitrile, is

* Geological Survey of Canada.

W.R. I 5/4--H

increased as the polarity of adsorbed organics de- creases.

Transition metal cluster complexes have been separated by RPLC (Enos et al., 1977) and recently atomic absorption has been used as a liquid chroma- tographic detector for a number of transition metals (Jones et al., 1976; Botre et al., 1976; Cassidy et al., 1976). However conventional AA is restricted in trace metal organic speciation studies in natural waters because of its limited sensitivity. Koizumi et al. (1978) have demonstrated the use of Zeeman atomic absorp- tion with liquid chromatography in the analysis of mixtures of vitamin B12 and Co(NO3)2.

This note demonstrates the potential of RPLC for the separation of high molecular weight natural water organics with the detection of associated Co and Cu by electrothermal atomic absorption.

EXPERIMENTAL

Natural water samples (0.5-2.0 ml) were pumped across a Brownlee Labs LiChrosorb RP-18 (10 #m) reverse phase column (4 mm i.d. x 25 cm) using a Waters Associates high pressure liquid chromatograph equipped with model 6000 pumps, a 2.0 ml sample loop and a variable u.v. detector operated at 254 nm (0.04 a.u.f.s.). The flow rate was 1.9 ml min -1 which developed 1500p.s.i. with 100~o water and dropped to 1000 p.s.i, with the change to 100~ acetonitrile. The system was operated isocratically initially with H20 as the solvent and switched to mixtures of H20 and acetoni- trile for selective elution of the adsorbed organics. The acetonitrile was distilled in glass prior to use. Baker "Ana- lysed" fiquid chromatography grade H20 was degassed by vacuum filtration, and solvent mixtures by refluxing.

The water sample components were separated by RPLC and eluting with 70~o H20/30~o acetonitrile followed by a 20~ H20/80~o acetonitrile mixture and finally with pure acetonitrile. Fractions (1-2 ml) were collected and Cu and Co analysed by electrothermal atomization AA. The natural waters had been previously fraetionated on Ami- con PM-I0 and UM-2 ultrafilters having nominal molecu- lar weight cut-offs of 10,000 and 1000 a.m.u, respectively.

507

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RESULTS AND DISCUSSION

The liquid chromatography RP-18 reverse phase column with water and acetonitrile as eluting solvents separates compounds into several polarity ranges. Preliminary experiments using fluorescent TLC plates had been carried out to determine the best solvent system for the separation of natural water organics. The RPLC profile of organics from a swamp water (12.5 mg 1-1 TOC) which had previously been frac- tionated on PM-10 and UM-2 Amicon membranes is shown in Fig. 1. On the basis of the membrane ultra- filtrations the organics have a nominal molecular weight range of between 1000 and 10,000 a.m.u. One of the features of the recorder tracing is the substan- tial amount of very highly polar compounds eluted with the solvent front (H20 only) which are not adsorbed on the column. Two further broad groups of compounds were then eluted with increasing non- polar character as the acetonitrile concentration in the solvent was increased. It is postulated that the more polar fulvic acids were eluted in fractions I and II with group III peaks being represented by such essentially non-polar types as polynuclear aromatic hydrocarbons and hydrocarbons with high CH ratios. Considering the complexity and polydispersity of ful- vic materials in natural waters, the RPLC shows reasonable resolution and peak sharpness. The sample consists primarily of natural organics, with man-made pollutants such as pesticides and PCB's not expected to be present in any significant quantity due to the isolation of the collection site.

Super-imposed on the chromatogram are Co and Cu profiles obtained by analysis of the eluant. As was expected the metal had eluted with the more polar

fractions (I and 11). Most of the Co appeared in frac- tion I, while the more tightly bound Cu eluted with a broad, somewhat bimodal, distribution across frac- tion It. It is noteworthy that the Cu peak did not coincide with the Co maxima. No metal was found in the later non-polar fractions. It should be noted that the metal in the injected sample represents that which did not pass through a UM-2 membrane (nominal MW cut-off 1000) after x5 diafiltration with de- ionized water. It is likely that the non-dialysable frac- tion of the total metal is "bound" either by covalent inner-sphere binding or by strong electrostatic attrac- tion to carboxylic or phenolic groups.

Natural water organics from other sources showed similar elution profiles.

C O N C L U S I O N S

One of the advantages of this technique for natural water organic separations is that it allows pre-concen- tration of the analyte at the beginning of the column, as water is a very weak solvent. Large volumes of sample may be delivered to the column for improved sensitivity. Little & Fallick (1975) have pumped 200ml of river water across a #-Bondapak Cla column and obtained a similar elution profile to that shown in Fig. 1. The use of the RP mode also pro- vides interesting information on the degree of hydro- phobicity of water organics and on the distribution of "bound" metal fractions. The effects of pH and counter ions on metal equilibrium has not been con- sidered in this preliminary study.

In future studies it will be necessary to collect indi- vidual peaks or groups of peaks for further analysis by other LC columns and GLC-MS techniques.

5 0

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T 30

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0

a/-..-a.

2 4 6 ~ tO 12 14 16 18 r ~ - - | i r , i

22 24 26 28

Time, rain

Fig 1. RPLC record of natural water organics and the distribution of Cu and Co. Conditions: Brownlee Labs 10/ms RP-18, column 25cm x4mm i.d., flow rate 1.9mlmin -1. detector u.v. 254nm, 0.04a.u.f.s. Sample: Silver Centre, Northern Ontario, 0.5 ml injected; UM-2 <MW<PM-I0, ( 0 0 0 )

Cu, ( ) Co.

Reverse phase liquid chromatography 509

Specific groups of compounds from RPLC separ- ations should also prove useful in metal binding ex- periments and provide valuable information on the nature of transport agents in natural waters.

Acknowledgements--The author wishes to thank Dr S. Behrandt of the Instrument Laboratory, Carleton Univer- sity, Ottawa for his assistance in the use of the Waters Associated Liquid Chromatograph, and Professor C. H. Langford, Department of Chemistry, Carleton University, Ottawa for his valuable comments.

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