Water Research Vol. 15, pp. 457 to 462, 1981 0043-1354/81/040457-06502.00/0 Printed in Great Britain Pergamon Press Lid

EXCLUSION CHROMATOGRAPHY WITH CARBON DETECTION. A TOOL FOR FURTHER

CHARACTERIZATION OF DISSOLVED ORGANIC CARBON

R. GLOOR, H. LEIDNER, K. WUHRMAh~q and TH. FLEISCHMANN

Federal Institute for Water Resources and Water Pollution Control, Ueberlandstr. 133, CH-8600 Diibendorf-Ztirich, Switzerland

(Received September 1980)

Ahstract--A method is introduced for determining quantitatively molecular weight distributions of organic compounds in natural waters on a routine basis. Sample preparation, exclusion chromatography with either Sephadex or TSK SW (Toyo Soda, Japan) and DOC detection are discussed. The application of the method is shown with examples such as waste water treatment, ozonation of lake water and ground water infiltration.

INTRODUCTION

Various techniques for determining organic water constituents have been developed. In general most research focussed on volatile organic pollutants since this fraction is more readily accessible to the analyst with sophisticated gas chromatography (GC) and mass spectroscopy (MS) techniques. It should be stressed, however, that volatile compounds constitute a very small portion of the dissolved organic carbon in natural waters (Giger & Roberts, 1978; Steelink, 1977). The bulk of organic compounds are not suffi- ciently volatile or of too high molecular weight for direct GC application. Derivatization techniques prior to GC analysis and high performance liquid chromatography (HPLC) (Armentrout et al., 1979; Pitt et al., 1976; Kasiske et al., 1978) permit more polar molecules to be detected. Carboxylic acids (Gloor & Leidner, 1976), phenols (Armentrout et al., 1979), amino acids (Kasiske et al., 1978) and carbo- hydrates (Eklund et al., 1977) are frequently deter- mined. However, a significant fraction of the organic matter still remains unidentified, including substances often referred to as aquatic humus (Gjessing, 1976), yellow acids (Ghassemi & Christman, 1968), fulvic acids (De Haan & De Boer, 1978) or by other general terms (Steelink, 1977; Swift & Posner, 1971; Davis, 1980). This last group of compounds is only operatio- nally defined. It is essential, therefore, to establish analytical methods for the further specification of dis- solved organic matter. As part of a continuing research program on this subject, we used exclusion chromatography in combination with DOC detection (Gloor & Leidner, 1979). In contrast to techniques where the analyst focuses directly on specific com- pounds or fractions, our approach consists in first fractionating all dissolved organic matter into mol- ecular weight (MW) groups and in further specifying

these fractions in a second step. This paper shows that the first step alone, the molecular weight fractionation of DOC, already opens new prospects on the behav- ior of organics in surface and ground waters.

MATERIALS AND METHODS

Sample preparation

A sample volume containing a minimum of 1.0 mg DOC was filtered through a 0.45-p.rn Millipore filter. The sample was then passed through a high capacity weak acid ion exchanger (Sephadex CM-50, Pharmacia, Sweden) in the sodium form (Baecini &Suter, 1979). The polyvalent cations in the solution were exchanged, and soluble sodium salts were formed. After evaporation to dryness (vacuum 12 mm Hg/40°C) in a rotary evaporator, the residue was redissolved in a few ml of distilled water (samples may be stored in dry form). The aqueous concentrate was adjusted to a pH between 2 and 3 with dilute hydrochloric acid and gently purged with nitrogen to remove dissolved carbonic acid. Upon addition of a few mg of phosphate buffer salt (Na2HPO4, KH2PO4), the pH was adjusted to about seven. The samples were stored in sealed vials under nitro- gen to prevent any access of CO2.

DOC measurement (Unor Majhak, Germany) before and after ion exchange proved that no organic carbon was lost within the limits of accuracy of the instrument (+0.1 mg1-1 DOC). This was also true for the concen- tration step. Loss of certain volatile compounds during evaporation is negligibly small.

Exclusion chromatoc2raphy For a considerable time, we used Sephadex as a separ-

ation medium (Pharmacia, Sweden). Unfortunately, these gels require low flow velocities because they are not com- patible with pressure. In the course of our study, a new silica-based packing TSK SW (Toyo Soda, Japan) became available which overearne some difficulties met with Sephadex gels (Fukano et aL, 1978; Wehr & Abbott, 1979; Saito & Hayano, 1979). This material offers an advantage over the Sephadex packing in terms of separation speed as well as resolution, although it is more limited in capacity. Eiution patterns for our samples were found to be depen- dent on the ionic strength and pH of the eluent. This

457

458 R. GLOOR et al.

Sephodex G25 (900xl5mm)

o

(~ ' Z'O ' 4'0 ' 6'0 ' 8'0 ' K )O" ' 2 0 ' , 4 0 m i n , m l

,5oo~ 3goo o~o ~o ,2'oo Molecular weight

Fig. I. Calibration of Sepbadcx G-25 column, l ml stan- dard containing 1 mg of dextran blue and fructose. Eluent 10-2M phosphate buffer (Na2HPO4/KH2PO4), pH 7.0. Flow rate 1 ml rain -1. Detection: DOC detector; carrier

gas 9 lh- 1 attenuation 1, gain 2. Recorder: 6 cm h- 1.

TSK 2000 SW (500x7 5 mm)

,,xoo A ruc,o.

i i i i 2 0 0 0 0 5000 500 200

Moleculor weight

Fig. 2. Calibration of TSK 2000 SW column. 200 #1 stan- dard containing 200#g of dextran blue and fructose. Eluent 10 -2 M phosphate buffer (Na2HPOJKH2PO4), pH 7.0. Flow rate 1 mlmin -t. Detection: DOC detector; carrier gas 501h -1, attenuation 1, gain 2. Recorder:

0.5 cm min- 1.

TSK 2 0 0 0 SW

(500 x Z 5 m m )

Summer

8 1 2 7 1 7 9

Im

i ) 2 0 0 0 0 t I '000 ( 2 0 0

5'000

molecular weNht

Winter

12 / 1 7 / 7 9

Im

o

I . , I I >20 (3130 [ IOOO ~200

5"000

molecular weight

20m

0

I I ,20oo01 ,~oo ~ooo

Molecular weight

20m

t ,2oooo I ,&o ,'280 5 0 0 0

Fig. 3. Molecular weight distribution of DOC in Lake Grvffenst~ water (Zurich, Switzerland) at ! and 20 m depth, collected in summer and at winter time. 200 #1 of each sample injected (concentration factor

200 times). Conditions as in Fig. 2.

Exclusion chromatography with carbon detection 459

regarded both Sephadex and TSK SW, in agreement with previously published observations (Swift & Posner, 1971; Determann, 1967; Saito & Hayano, 1979; Gjessing, 1976). The two parameters were controlled by using a 10-Z-M phosphate buffer (pH 7.0) as eluent (1.39 g i-1 Na2HPO4, 0.73 g 1-1 KH2PO4). The buffer reservoir was kept under nitrogen to prevent access of CO2 from the air. The Sephadex was either G-25 fine (separation range 200-5000 MW) or G-10 fine (separation range 50-700 MW). TSK-2000 SW was supplied in a packed column (7.5 x 500 ram) by Varian (Walnut Creek, CA, USA). The solvent was delivered with an Altex 110 pump (Altex, Berkeley, CA, USA). A loop injector (Rheodyne, 710, Berkeley, CA, USA) was used for introducing the samples with either a 1-ml loop (Sephadex) or a 200-#1 loop (TSK SW).

Detection and quantification The column effluent was split and an aliquot

(0:4 ml min - 1) was monitored on-line for organic carbon in a DOC detector (Gloor & Leidner, 1979). The chromato- gram represents the molecular weight distribution of all organic carbon in the sample, and the total area of the record is equal to the total DOC. If necessary, quantifi- cation of specific fractions was done by the cut and weight method of the recordings.

R E S U L T S A N D D I S C U S S I O N

Calibration of the separation columns

Calibration was performed by determination of the pore volume in the columns by using Dextran blue (Pharmacia, Sweden) for excluded and fructose (meth- anol in case of Sephadex G-10) for permeated species (Figs 1 and 2). Assuming a linear relationship between log of molecular weight and elution volume, the frac- tion range can be calibrated from the gel specifica- tions supplied by the manufacturer (Sephadex, Phar- macia, Uppsala, Sweden; TSK-Gel SW type, Toyo Soda, Japan). Throughout this paper, we use the data from the dextran calibration. This method is simple and proved to be sufficiently precise for our purposes since we were interested in monitoring changes in the molecular weight distribution rather than in establish- ing the exact molecular weight of a specific species.

Examples

Five examples which are part of various projects in this Institute and represent different areas of interest shall illustrate the potentials of the method.

Example I, Lake water. Samples were collected from the highly eutrophic Lake Greifensee (Ziirich, Switzerland) at the surface and at 20 m depth (max. lake depth: 30m during times of stratification (27 August, 1979, thermocline at 7 m) and complete mixing (17 December, 1979). The molecular weight distributions (Fi& 3) of the winter samples appear very similar, demonstrating good mixing in the lake. The summer sample from 20 m depth had a molecular weight distribution similar to the two winter samples. The surface sample exhibited increased amounts of organic carbon (1) in the excluded fraction (MW > 20,000) and (2) in the molecular weight range of 500 to a few 1000. This indicates that phototrophic

Sephodex G25 ( 9 0 0 x l 6rrcn)

0

[ ]Before ozonation

[ ~ After ozonation

i i I I ~5000 5000 100(3 500 (200

M o l e c u l a r weioht

Fig. 4. Molecular weight distribution of DOC in Lake Greifensee water (Zurich, Switzerland) before and after ozonation on Sephadex G-25. 1 ml of concentrated lake water (concentration factor 200 times) injected. Ozonated

chromagotram superimposed. Conditions as in Fig. 1.

production predominantly adds dissolved, high-mole- cular weight organic matter. Low MW compounds are either not released or are immediately eliminated by e.g. heterotrophic microbial metabolism.

Example 2, Reaction of organic matter with ozone. Ozone, usually applied as a disinfectant, reacts with dissolved organic compounds in aqueous solutions. The mechanisms of these oxidations are the subject of investigations by Hoign~ & Bader (1978, 1979), and the following results are part of a common project with this group. Filtered surface water from Lake Greifensee (13.11.78) with a dissolved organic carbon content of 4.3 mg I-1 C was ozonated for 5 min. The initial concentration was 13 mg 1-10a . Figure 4 illus- trates the molecular weight distribution of the lake water before and after ozonation as determined with Sephadex G-25. The amount of organic carbon com- pounds with a molecular weight > 1500 is reduced in the sample treated with ozone, and an increase in low-molecular weight compounds is observed. A new organic carbon peak appears in the region of molecu- lar weight 500. Figure 5 shows the same two samples fractionated on Sephadex G-10 (separation range between MW 700 and 50). The decrease in high MW compounds caused by ozonation is illustrated even more clearly since all compounds with molecular weights larger than 700 are appearing in the excluded front peak. The effect of ozonation upon MW distri- bution of organic water constituents was studied earlier by Lienhard & Sontheimer (1979) by using

460 R. GLOOR et al.

Sephodex GIO (800 xlGmm)

~ Before ozonation

i [ ] After ozorK}tion

100<50 Molecular weight

Fig. 5. Molecular weight distribution of D e c in Lake Greifensee water (Zurich, Switzerland) before and after ozonation on Sephadex G-10. Ozonated chromatogram

superimposed. Conditions as in Fig. I.

Sephadex G25 (900xlGmm]

River Lirnmat

A Groundwoter 0

I t I I I

)5000 2000 I000 500 ~ 200

Molecule weight

Fig. 7. Molecular weight distribution of D e c in river Limmat water (Zurich, Switzerland) and corresponding ground water formed by river water. 1 ml of 200-times con-

centrated sample injected each. Conditions as in Fig. 1.

ultrafiltration techniques. Their results also suggest a shift towards low MW compounds during ozonation, However, these authors only examined a fraction of "aquatic humus". Our results show the effect of ozo- nation on all dissolved organic material present in the lake water. The reaction mechanism and identity of the products of ozonation are presently under investi- gation.

Example 3, Adsorption of organics on suspended min- eral matter. Adsorption on suspended particulate matter must be considered as an elimination process for dissolved organic compounds in natural waters. A suspension of colloidal alumina (~,-A120 3, 1.0 g 1-1 in

Sephadex G25 [900x 16 ram]

r - ] Before odsorption

After odsorption pH83

I I i i 5 I ~5OOO 3000 tOO0 OO <200

Molecular weight

Fig. 6. Molecular weight distribution of D e c in Lake Griefensee water (Zurich, Switzerland)before and after adsorption on y-A1203. 1 ml of each sample injected (con- centration factor 200 times). Adsorbed chromatograms

superimposed. Conditions as in Fig. 1.

filtered lake water; Greifensee, 15 October, 1979; D e c 4.1 mgl -z) was stirred at two pH values (5.9 and 8.3) for 10 h. The alumina particles were then removed by centrifugation. Dissolved organic carbon in the lake water was reduced by 50~ in the sample with pH 5.9 and by 40Yo at pH 8.3. The molecular weight chromatograms of the organic compounds not adsorbed are shown in Fig. 6 in comparison with the original lake water organic mattter. It appears that molecules with a molecular weight larger than about 500 were adsorbed and that the degree of adsorption increased with an increase in molecular weight. Thus, adsorption may play a role in the removal of high- molecular organic compounds from natural waters, especially in systems with an excess of available sur- face area, e.g. lakes with a high input of inorganic particulate matter or in ground water carriers. A detailed study of the adsorption behavior of various molecular weight fractions is currently in progress by J. A. Davis at our Institute.

Example 4, Organic matter in ground water. Figure 7 and 8 illustrate molecular weight distribution of D e c in two ground waters formed by river water infiltra- tion. In both cases, the molecular weight pattern in the ground waters is quantitatively and qualitatively different from the corresponding river water samples. High-molecular weight organic compounds (MW > 50001 are completely absent from both ground water samples while lower molecular weight fractions are partly or not reduced at all by compari- son with the river water (~g. low MW compounds in the Limmat sample). The significant elimination of high-molecular weight compounds in the course of infiltration might be due to adsorption or microbial

Exclusion chromatography with carbon detection 461

Sephadex G25 ( 900 x 16ram)

J

.u_

Groundwater

50100 3~ * i i > O 1000500 (200

Molecular weight

Fig. 8. Molecular weight distribution of DOC in river Rhine water (Rheinau, Switzerland) and corresponding ground water formed by river water. 1 ml of 200-times con-

centrated sample injected each. Conditions as in Fig. 1.

• Before treatment DOC:~ rrlg I-' After treatment DOC:I6 rag1-'

TSK 2 0 0 0 SW

(7.5 x 500mm)

~20000 [ I 000 ';200 5000 500

Molecular weight

Fig. 9. Molecular weight distribution of DOC in sewage before and after (superimposed chromatograms) sludge treatment. 1 ml of 10-times concentrated sample injected

each. Conditions as in Fig 2.

degradation. A more detailed study on the fate of organic compounds during infiltration into ground water is presently under investigation in this institute (Schwarzenbach et al., 1980).

Example 5, Sewage purification. The effect of bio- logical sewage treatment (activated sludge) on the dis- solved organic matter is illustrated in Fig. 9. The main part of the influent DOC to the biological reac- tor comprises compounds of fairly low-molecular weight. This fraction was largely eliminated during treatment whereas the higher MW compounds seem to reappear in the effluent. This example might not be representative for all treatment plants. However, it demonstrates that the molecular weight distribution of organic compounds is an important parameter for the understanding of elimination mechanisms in bio- logical treatment systems.

CONCLUSION

The molecular weight distribution of DOC is a sig- nificant parameter for studying the alterations occur- ring to organic compounds submitted to natural tech- nical or water treatment processes. Exclusion chroma- tography with carbon detection proved to be a sensi- tive tool for measuring this molecular weight distribu- tion on a routine basis. The examples illustrate that the MW fractionation of the total DOC is highly in- formative. It leaves unresolved the problem of specifi- cation and quantification of nonvolatile dissolved or- ganics. We feel, however, that this technique is a use- ful primary step towards this final aim because it pro- vides for an efficient prefractionation. Applied on a preparative basis, fractions can be accumulated which are large enough for separation and identification of individual compounds by adequate specification methods.

Acknowledgements--The authors would like to thank P. Baccini, R. Schwarzenbach, J. Schneider, J. Hoign6 for valuable discussions and supplying samples, and J. Davis for reviewing the manuscript.

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