Identification of nonvolatile neutral organic compounds in tap water

Enoirommm International, Vol.4, pp.31-37, 1980 Printed in the USA.All rightsreserved.

0160-4120/80/070031-075(R.00/0 Copyright©1980PergamonPress Ltd.

IDENTIFICATION OF NONVOLATILE NEUTRAL ORGANIC COMPOUNDS IN TAP WATER

R. Shinohara, A. Kido, S. Eto, T. Hori, K. Koga,* and T. Akiyama* Kitakyushu Municipal Instituteof Environmental Health Sciences, Shinike 1-2-1, Tobata-ku, Kitakyushu804, Japan (Reveioed 6 DEC 79" Revised 26 FEB 80)

Nonvolatileneutral organiccompoundsin tap waterweree.jraminedby high-pfeuure fiquidchromatography, infrared spectrometry, IH- and 13C-FT-NMR, gel permeation chromatogntphy,elementary analysis, and field dmorption mass spectrometry.Neutral organic compoundsin 10 m* of tap water were continuouslyextractedby the AmberfiteXAD-2 resin ¢olnmn~and ~ a t t e d into four fractiom by ailiea-gel column chromatography. Removal of volatile compounds in fraction 3 inzluding polar oxygenatedcompoundswas carried out with microdiati~tioncolumn under high vacuumby umnga diffusionpump. Nonvolatileorganic compoundsimlatedfrom tap water wa'e identifiedas poly(vinyl acetate) derivativeswith molecularweightsrangingfrom 300 to 1300.

Introduction

1976; Farrah et al., 1978; Gjessing and Lee, 1967; Hall and Lee, 1974; Manka et aL, 1974; Weber and Wilson, 1975). Except papers by Pitt et al. (1975) and Hajibrahim et al. (1978), there have been few reports on nonvolatile organic compounds which dissolve in solvents such as benzene, ether, and chloroform. In this paper, we describe identification of nonvolatile neutral organic compounds from tap water by adsorption on Amberlite XAD-2 resin.

The need for information regarding trace organic compounds in tap water has been required because of detrimental effects of potential hazardous organic compounds on human health. The majority of work on trace organic compounds in tap water has dealt with volatile compounds identified by gas chromatography (GC) a n d / o r gas chromatography-mass spectrometry (GC-MS). Recently, many kinds of compounds including hydrocarbons, pesticides, and phthalates have been identified in tap water by G C - M S (Keith, 1977). We have also analyzed tap water by G C - M S and reported the presence and concentration levels of trace organic compounds (Koga et aL, 1978; Shlnohara et aL, 1979). On the other hand, it is known that 80%-90% (by weight) of organic compounds in the environment cannot be analyzed by gas chromatographic techniques even after derivatization. Previous investigations on nonvolatile organic compounds in environmental samples almost exclusively dealt with humic and fluvic acids (Chian, 1977; Chian et al., 1977; Farrah et aL,

Experimental Materials

paper was presented at the joint meeting of the American Chemical Society and the Chemical Society of Japan, Honolulu, Hawaii, April 2, 1979. *Present address: University of Occupational and Environmental Health, School of Medicine, Iseigaoka 1-1, Yahatanishi-ku, Kitakyushu807, Japan. 31

All solvents were purified by fractional distillation. Amberlite XAD-2 resin (Rohm & Haas, Co., Philadelphia; 20-50 mesh) was washed according to the method of Junk et aL (1974). Silica gel (Wako Pure Chemical Ind., Tokyo; 100-200 mesh) was washed with benzene-ethanol (1 : 1) in a Soxhlet extractor for 24 h, and reactivated at 145 ° C for 4 h before being used. A gel permeation chromatographic column (500 × 8 ram) packed with Shodex A-802 was purchased from Showa Denko Co., Tokyo. LiChrosorb SI-60 silica gel (Merck CO., New York; 5 #m) for high-pressure liquid chromatography was packed into a stainless steel column (250 × 4 ram) as a balanced density slurry in tetrabromoethane-tetrachloroethylene (6 : 4), washed

32

Shinohara, Kido, Eto, Hori, Koga, and Akiyama

with 250 ml of chloroform, and conditioned by pumping normal propyl chloride.

Apparatus A Nihon Denshi JGC-20 KP gas chromatograph equipped with a flame ionization detector was used for the analysis of volatile compounds in distilled matter. A Nihon Bunko A-3 infrared (IR) spectrometer was used to measure IR spectra taken in liquid film of samples. A Hitachi Model-635 high-pressure liquid chromatograph equipped with silica-gel column (stainless steel, 150x4 mm), a variable wavelength UV detector, and a solvent programmer for gradient techniques, was used. Nuclear magnetic resonance (NMR) of proton and carbon-13 was measured with a Nihon Denshi JNM-FX 60 Fourier transform N M R (FT-NMR). Field desorption (FD) mass spectra were obtained by using the JMS-01SG-2 mass spectrometer attached with an EI-FI-FD ion source. Extraction and preliminary separation Trace organic compounds in tap water was continuously adsorbed on XAD-2 resin by passing 10 m 3 of tap water at an approximately flow rate of 250 ml/min for 1 month through the column (30 × 4 cm) sampler previously described (Shinohara et al., 1979). The

TAP WATER Concentration

I

Extraction

XAD-2 resin with adsorbed compounds was extracted with freshly distilled ether in a Soxhlet extractor for 24 h. The extract was separated into acid, basic, and neutral fractions with 0.1 M NaOH and 0.1 M HC1. The neutral fraction was concentrated to less than 2 ml in a Kudema-Danish evaporator. Compounds in the concentrated ether were adsorbed on about 0.4 g of silica gel which was added on the top of a silica-gel column (20 × 1 cm), and separated into four fractions as shown in Fig. 1. After removal of solvent from each fraction by using nitrogen gas stream, the residues were weighted, neglecting the loss of high-volatile organic compounds. A trace amount of light-yellow liquid remained in fraction 1 which essentially consisted of aliphatic hydrocarbons. Fraction 2 yielded 9.9 mg of organic compounds including aromatic hydrocarbons. In the case of fractions 3 and 4, 217 and 99 mg of brownish-black oil were obtained, respectively, and these fractions included polar oxygenated compounds. Nonvolatile compounds in fraction 3 were analyzed in detail because of the large amount in this fraction. Volatile components in fraction 3 were eliminated by the use of the microdistillation apparatus (Fig. 2) at 60 ° C under high vacuum (diffusion pump, 80 liter/ sec) for 1 week. After distillation, inside of the apparatus was kept at approximately 8 x 10 -3 Torr. The

i0,000 liters Amberlite XAD-2 resin column (50 X 4 cmi.d.) Ether, Soxhlet, 12 hr

f

Removal of acid and basic organics

[ Bases

Acids

Separation

Silica-gel column (20 X i cm i.d.)

I

l IsoSctane

IsoSctane-benzene

Benzene-ethyl acetate

Benzene-methanol

Removal of volatileorganics

HPLC Fig. 1. Extraction and fractionation scheme for organic compounds in tap water.

Compounds in tapwater

33

microdistillation column was cut into six segments, and organic compounds in each stage were dissolved in 2 ml of dichloromethane. Gas chromatographic data (Fig. 3) of each stage showed that the amount of volatile compounds increased on going towards the upper stages. Absence of volatile compounds in the bottom (stage 1) of the microdistillation column was estimated from the absence of GC peaks on its gas chromatogram. The bottom fraction gave 125 mg of a residue consisting of nonvolatile compounds which had a high viscosity and odor like pyruvic acid.

each vial was evaporated with gentle heating under a nitrogen stream. Results and Discussion

In order to obtain the information of functional groups, IR and 13C-NMR spectra were measured. IR spectra of LC peaks 5, 6, 7, 10, and 11 shown in Fig. 5 indicate a fairly great similarity. Therefore, the nonvolatile organic compounds isolated from tap water was assumed to consist of homologous components. The stretching vibration of the polymeric OH group and CH (assigned to CH 3 and CH 2 groups) was observed at 3420 and 2945 cm- 1, respectively. Absorption of vc. 0 vibration at 1720 c m - i indicated the presence of ester groups. In LC peak 5, absorptions of C = C and C~___C groups were observed at 1600 and 2250 cm-i, respectively. 13C-NMR spectrum (Fig. 6) of LC peak 5 in CDC13 showed the presence of a methylene from paraffin structure, methylene substituted with the OH group, and ethylene, being essentially identical with IR spectral data. Similarly, 1 H - N M R spectrum supported IR and IaC-NMR spectral data, as shown in Fig. 7. According to the result of elementary analysis, nonvolatile compounds corresponding to LC peak 5 had the composition of (C5_6H7~801_2) n and no nitrogen. On the basis of the above results, the possible structure of isolated nonvolatile organic compounds was suggested to be poly(vinyl acetates).

Separation by high-pressure liquid chromatography (HPLC) For other instrumental analyses, separation of the nonvolatile compounds was attempted by HPLC. A LiChrosorb SI-60 silica-gel column was conditioned with propyl chloride for more than 20 min at a flow rate of 2 ml/min. A 20/~l aliquot of nonvolatile extract was injected into the SI-60 column and separated by gradient elution. A linear gradient of methanol in propyl chloride (0% to 4% over 14 min) was used after holding 100% propyl chloride for 4 min. The ratio of methanol in propyl chloride was increased to 80% for 3 min, and finally 80% methanol was held for 7 min. By this solvent programming, 11 LC peaks were obtained as shown in Fig. 4. The LC effluent peaks were trapped in glass vials; collection was started and stopped at approximately 10% of the peak height. The solvent in

To Diffusion pump

Thermocouple wire

Glass fiber tape X~

2-4 V

Heater wire

0

1

2

3

I

I

I

I

Fig. 2. MicrodistiUationapparatus.

4 cm I

34

Shinohara, Kido, Eto, Hod, Koga, and Akiyama

80Z MeOH

0

,/

5o

•

linear gradient0-~

,

\

~'o

5o

oR/ J

lOG

%

i00

~

I

I

25

2o

Micro distillation

, column

3

I

lk

3

7/ ;~

i

i 5

0

z 0

5

10

15

20

100'C

i0

ii0

;

I 20 Rt (rain)

5

Fig. 4. Liquid chromatogram of nonvolatile organic compounds.

25 Rt (rain)

300"C

Fig. 3. Gas chromatogram of each distillate by high vacuum system.

s---~

\ -oH.c,-C-:C-

~

16oo ,

L455

i~79

\

k

910 1280

7-A 730

i0

ii

V

V

2945

-CH21720 (polymeric)

3420-0H I

4000

I

3200

I

2400

I 25

-CO-

I

1900

[

1700

I

1500

I

1300

]

I

ii00

900

Wave

number

I

700 (Cm -I)

Fig. 5. IR spectra of nonvolatile components separated by HPLC (liquid film).

I

500

Compounds in tap water

35 Solvent

-CH=CH -

-CH2-OH

12B

-CH2-

67.9

I

I

I

I

120

I

i00

I

29.6

I

I

80

I

I

60

25.6

I

I

40

20 6 ppm

Fig. 6. 13C-NMR spectrum of LC peak 5 by FT-NMR. Solvent: CDCI3; Reference: Tetramethylsilane; Accumulation time: 33,000.

2.19

-CH2-C0

H -C-OI

CHCL 3

-CH2i. B2

~ -C-0-

5, 6

,

/

I

I

I

I

I

I

I

I

i0

9

8

7

6

5

4

3

I 2

1

0

6 ppm

Fig. 7. IH-NMR spectrum of LC peak 5 by FT-NMR. Solvent: CDCI3; Re~erence: Tetramethyl~ilAne;Accumulation lime: 30.

36

Shinohara, Kido, Eto, Hod, Koga, and Akiyama The range of molecular weight of nonvolatile organic compounds was measured by high-pressure gel permeation chromatography, and its ~hromatogram is shown in Fig. 8. Most of them were present in the range of 300-1500 calibrated with polystyrene. Measurement of exact molecular weight of LC peak 5 was attempted by FD-MS, providing the prominent ion peak for molecular ion of nonvolatile and/or unstable organic compounds. The FD mass spectrum of LC peak 5 gave many peaks in the range of 300-1300 as shown in Fig. 9, and this observation agreed with the result from gel chromatography. However, several mass peaks may be due to pyrolysis on an emitter of FD-MS. We may conclude that most of nonvolatile organic compounds isolated from tap water are hydrolyzed and oxidized poly(vinyl acetate) derivatives. Poly(vinyl acetate) is widely used as a raw material for poly(vinyl alcohols) and for synthetic paints in Japan.

Molecular weight* 104 /

103

i~ 2

CH---~ C

I I

o L

i

I

0

5

i0

I

OH

O-C-CH 3 II O

I

15 20 Elution volume (ml)

Conclusion

Fig. 8. Gel chromatogram of nonvolatile organic compounds by HPLC. Exclusion limit: 5 x 103; Eluent: Tetrahydrofuran; Flow rate:

1.0 ml/min; Detector: UV-254rim; Molecular weight*: Calibrated by polystyrene.

At present, HPLC, F T - N M R (IH and 13C), and F D - M S are very useful analytical methods for chemical investigation of nonvolatile organic compounds. By

I00 323

v

.~ 50 a9

1205 7/,3

1073

451

1321

~4

aoo

400

500

600

700

800

900

I000

1100

1200

13b0

1~.bo

ro/z Fig. 9. FD-MS specLrumof nonvolatile organic compounds. Accelerating voltage: + 7 / - 5 kV; Emitter current: 20-24 mA.

Compounds in tap water

these methods, the presence of poly(vinyl acetates) in the environment was confirmed. These hydrophobic organic compounds may be accumulated in the human body by absorption over a long time. In order to find detrimental effect of these organic compounds on human health, many other biological studies such as mutagenic or carcinogenic tests are necessary. Acknowledgement--The authors wish to thank Dr. M. Sonoda, Research Director of this Institute, for his assistance and encouragement. They are also deeply indebted to Professor Y. Kato of Kyushu Institute of Technology for measurement of F T - N M R and to Prolessor S. Furukawa of Faculty of Phamaceutical Sciences, Nagasaki University, for elementary analysis.

References Chian, E. S. K. (1977) Stability of organic matter in landfill leachatcs, Water Res. 11, 225-232. Chian, E. S. K. and DcWalle, F. B. (1977) Characterization of soluble organic matter in leachate, Environ. Sci. Technol. 11, 158-163. Farrah, S. R., Goyal, S. M., Gerba, C. P., Wallis, C., and Shaffer, P. T. B. (1976) Characteristics of humic acid and organic compounds conceatratexi from tap water using the aquella virus concentrator, Water Res. 10, 897-901. Farrah, S. R., Goyal, S. M., Gerba, C. P., Mahajan, V. K., Wallis, C., and Melnick, J. L. (1978) Concentration of humic acid from tap water, Water Res. 12, 303-306.

37 Gjessing, E. T. and Ice, G. F. (1967) Fraction of organic matter in natural waters on Sephadex columns, Envirott Sci. Technol. 1, 631-638. Hajibrahim~ S. K., Tibbetts, P. J. C., Watts, C. D, Maxwell, J. R., and Eglinton, G. (1978) Analysis of carotenoid and porphyrin pigments of geochemical interest by high-performance liquid chromatography, Anal. Chert 50, 549-553. Hall, K. J. and Lee, G. F. (1974) Molecular size and spectral characterization of organic matter in a meromictic lake, Water Res. 8, 239-251. Junk, G. A., Richard, J. J., Grieser, M. D., Witiak, D., Witiak, J. L., ArgueUo, M. D., Vick, R., Svec, H. J., Fritz, J. S., and Calder, G. V. (1974) Use of macroreticular resins in the analysis of water for trace organic contaminants, J. Chromatogr. 99, 745-762. Keith, L. H. (editor) (1977) Identification and anaysis of organic pollutants in water. Ann Arbor Science Publisher, Inc., Ann Arbor. Koga, M., Shinohara, R., Kido, A., Eto, S., Hori, T., and Akiyama, T. (1978) Investigation of microorganics in tap water and river water by gas chromatography-mass spectrometry, J. Water Pollut. Res. (Tokyo) 1, 23-32 (in Japanese with English abstract). Manka, J., Rebhum, M., Mandelbaum, A., and Bortinger, A. (1974) Characterization of organics in secondary effluents, Environ. Sci. Technol. 8, 1017-1020. Pitt, W. W., Jr., Jolley, R. L., and Scott, C. D. (1975) Determination of trace organics in municipal sewage effluents and natural waters by high-resolution ion-exchange chromatography, EnvirorL $ci. Technol. 9, 1068-1073. Shinohara, R., Kido, A., Eto, S., Hori, T., Koga, M., and Akiyama T. (1979) Identification and determination of trace organic substances in tap water by computerized gas chromatography-mass spectrometry and mass fragementography, Water Res. (submired). Weber, J. H. and Wilson, S. A. (1975) The isolation and characterization of fluvic acid and humic acid from river water, Water Res. 9, 1079-1084.