(15) W. J. Sweeney, "Petroleum and Its Products," W. J. York, N.Y , 1950. (16) E. T. Commins, Afmos. Environ.,3, 565 (1969). (17) M. J. Lyons, Nat. Cancerlnst., Monog.,9, 193 (1962). (18) S. K. Ray, and R . Long, Combust. Flame, 8, 139 (1964).
Sweeney, New
RECEIVEDfor review September 13, 1974. Accepted NOvember 25, 1974. This work was supported by Research Grant No. GP-33751 from the National Science Foundation.
Analysis of Sulfur-Containing Gases by Gas-Solid Chromatography on a Specially Treated Porapak QS Column Packing T. L. C. de Souza, D. C. Lane, and S. P. Bhatia Pulp and Paper Research Insbtute of Canada, 570 St John's Boulevard, Pointe Claire, P Q , Canada H9R 3J9
The quantitative analysis of mixtures of sulfur-containing gases such as those emitted from the stack of a kraft pulp mill is best performed by the use of a gas chromatograph. The degree of success depends primarily upon the type of solid support, stationary phase, column material, and detector used. Up t o now, various packings with different stationary phases have been tried with little or partial success. This note describes how acceptable results can be obtained on a Teflon column by the use of a specially treated Porapak QS packing-a porous polymer composed of ethylvinyl benzene cross-linked with divinyl benzene t o form a uniform structure of a distinct pore size (Dow Chemical Co., Freeport, Texas). One of the many publications available on the analysis of sulfur gases by means of gas-solid chromatography, by W. L. Thornsberry, Jr. ( I ) , described the use of an acidwashed silica gel column packing "Deactigel" (Applied Science Laboratories, State College, Pa.) for the isothermal separation a t 122 "C of percentage quantities of COz, COS, HzS, CS2 and SO2, using a thermal conductivity detector. Thornsberry's claims have been partially substantiated by Mitchell and DeLew ( 2 ) for the separation of ppm levels of COS, H2S, CH3SH, and CH3CH2SH (no SO2 or CS2 were present in the mixtures tested) with temperature programming and a flame photometric detector (FPD). However, an attempt t o separate a mixture of H2S, CHsSH, (CH&S. COS, and SO2 in the parts per million range in our laboratory, following very faithfully the procedure described by them, was unsuccessful, because of problems arising from tailing after the SO2 peak. For the determination of sulfur compounds a t the ppm level in air, Bruner et al. (3) used a Teflon column packed with graphitized carbon black treated with 0.5% phosphoric acid and 0.3% Dexsil 300 (Analabs Inc., North Haven, Conn.). However, a t 40 "C, they found tailing problems with CH?SH a t concentrations below 100 ppm. At 80 "C, they obtained a good symmetrical peak of CH3SH, but a t the expense of losing separation of H2S and SO2 from the air peak. Adams e t al. ( 4 ) used an elaborate procedure. First, they adsorbed the sample a t -78.5 "C on activated silica gel. Then they desorbed by heating the silica gel in a vacuum, and trapped the evolved gas a t -195-8 "C. Finally, they transferred the gas t o a gas-liquid chromatographic column for conventional analysis under programmed temperature conditions. Since water vapor interfered in the subsequent analysis, it was removed in a two-step procedure: condensation a t 0 "C followed by adsorption on a solid desiccant
such as anhydrous calcium sulfate. No mention was made of any particular volume or weight of individual gas constituents recovered. For similar analysis, R. K. Stevens et al. ( 5 ) used a 36foot long, 0.085-inch i.d. Teflon column, packed with 40/60 mesh Teflon beads coated with a mixture of polyphenyl ether and orthophosphoric acid. The disadvantages of this column are t h a t it is very difficult to pack, develops a considerable back pressure, and costs around $200 if bought commercially; also, it seems unable t o separate COS and (CHdnS. We have now found t h a t all the above-mentioned problems can be overcome by the use of a specially treated Porapak QS column, described in this paper.
EXPERIMENTAL Analytical System. The system consisted of a F&M 5754 dual column gas chromatograph, a flame photometric detector operating at 750 volts, developed by Brody and Chaney ( 6 ) ,and an electrometer, both manufactured by MicroTek Instrumeiits Inc., Austin, Texas. The detector was operated with a 394-nm filter. An Infotronics CRS 11-HSB integrator was used for peak area measurements and was later replaced by a Hewlett-Packard Model 3373B. The column was an acetone-washed Teflon tube in. 0.d. and 18 inches long, packed with 80/100 mesh acetone-washed Porapak QS. A 2-cm3 stainless steel sampling valve, connected to a vacuum and a mercury manometer, was used to introduce the sample into the column at a constant absolute pressure of 20 cm Hg. Introduction of the sample gas at the same pressure allows repeat analysis from any sample source. Column Preparation and Conditioning. Two to four grams of 8O/lOO mesh Porapak QS column packing contained in a fine sintered glass funnel is washed with jets of chemically pure acetone by suction for 5-10 minutes only. Treatment with acetone for more than ten minutes leads to poor performance of the column. The material is air-dried by continuously pulling air through the suction funnel till the grains of the packing are loose and free-flowing. An 18-inch long, lk-inch 0.d. acetone-washed and dried Teflon tube is then lightly packed with the treated Porapak QS by using the vibration technique (glass wool plugs on either side of the packing) to an effective packed length of approximately 1 2 inches. The column thus prepared, and with a carrier helium gas flow of about 55 cm3/min, is conditioned overnight at 240 "C in an oven, the effluent gas being led away from the detector to waste. Preparation of Standard Gas Samples. High purity gases obtained commercially (Matheson of Canada) were used in making two levels of gas concentrations with gas-tight syringes in 500-ml glass cylinders having Teflon taps on either end. A 500-ml glass cylinder was evacuated and purged with pure iV2 at atmospheric pressure. Five milliliters of Nz were withdrawn by means of a syringe; then, in its place, a 5-ml sample of the pure sulfur-containing gas was introduced. From this 1% gas, further dilutions were made as described above, to obtain ppm concentrations of the gas. ANALYTICAL
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T a b l e I. Typical Conditions Used t o O b t a i n C h r o m a t o g r a m s S u c h as the O n e S h o w n i n F i g u r e 2 Column and Acetone-washed Teflon 18 in. X 1/8 in. packing 0.d. column packed with 80/100 mesh acetone-washed Porapak QS. Column Programmed, s t a r t 30 "C, post- injection delay of 1 min. to 210 "C by temperature 40 "/min increment. C a r r i e r gas Helium at 31 cm?/min. Sample 2 cm3 at 20 cm Hg absolute. volume Detector Flame photometric with 394-nm filter. Detector 115 "C temperature Gases to detector Air 50 cm3/min. H2 155 cm3/min. 0 2 15 cm3/min. Chart speed 1 inch/min.
Figure 1. Typical chromatogram obtained with untreated Porapak QS (R = CH3)
The glass cylinders were later replaced by exponential dilution flasks made of Plexiglas as described by Williams and Winefordner ( 7 ) and coated with a dilute solution of Siliclad (Clay Adams, Parsippany, N.J.) and dried. This treatment is necessary to prevent the adsorption of the reactive gases on t o the walls of the flasks. At other times, use was made, very successfully, of the spinning-syringe calibrator ( 8 ) of 50 cm3 volume which delivers a constant flow of the air-diluted gas of interest and which is further diluted by a diluent gas such as N2 before the mixture is led to the gas chromatograph. Calibration curves obtained by using these techniques were satisfactory in the range of 0.5 to 70 pprn, using conditions shown in Table I.
RESULTS AND DISCUSSIONS T h e acetone-washed Porapak QS column gave clean separations, with a base line stable throughout t h e entire 7 minute temperature program from 30 to 210 "C for a mixture of H2S, COS, S02, CHsSH, (CH3)2S, and (CHdzSz in the parts per million range. Some holdup of t h e sulfur compounds occurred on the acetone-washed Porapak QS. Conditioning of the column with two or three injections of concentrated gas mixtures, followed by temperature programming to 240 "C, helped to stabilize t h e column. Figures 1 and 2 illustrate typical chromatograms obtained before and after the acetone-wash treatment of Porapak QS, respectively, in the separation of six gas components usually found i n kraft recovery furnace flue gases. Table I1 gives t h e results obtained on a standard gas sample prepared manually by syringe injections in glass gas cylinders. This method is liable to give greater discrepancies between the quantity of the pollutant present and t h e amount found, due t o human errors involved in such a sample preparation. 544
Table 11. Separation a n d Recovery of Sulfur Compounds in a S t a n d a r d Sample, Using T r e a t e d P o r a p a k QS Packing Retention
Compound
time, sec.
Amount present, Amount found, ppm
v/va
ppm, V / V b
H2S
77 5.1 5.3 108 5.1 5.2 SO2 182 5.1 4.7 CH$H 213 5.1 5.0 (CH3)zS 270 8.4 9.9 (CH,)$$ 361 7.1 9.2 a F o r method of preparation of standard samples, see text. Average readings of three tests, with a spread of *5%.
cos
Figure 2. Typical chromatogram obtained on an acetone-washed Porapak QS column (R = CH3)
However, in future work, we intend to use permeation tubes, which should provide closer concordance between the standards and the values obtained. In our experience, acetone-washed Porapak QS is the best column for the separation of these six sulfur compounds in the parts per million range, compared t o others we tried and rejected. Different batches of Porapak QS were tried and, without t h e acetone-wash step, they always led to tailing after the elution of SO2 with subsequent interference with the resolutions of CHsSH, (CH&S, and (CH3)2S2 peaks. Acetone-washed Porapak Q gave separations inferior to those obtained with acetone-washed Porapak QS, with tailing and resolution problems. T h e most important advantages of this column, compared t o those of others, and particularly to t h a t of Stevens', are t h a t it is very short, easy to pack, inexpensive, with a power of high resolution, free of bleed even at a temperature of 240 "C, because of the porous bead packing which serves the function of both the liquid phase and solid support. Retention data are very constant since there is no liquid phase t o be lost because of continual bleeding. Yet another advantage is t h e remarkable property of this column in allowing rapid elution of water (present in flue gases) and other highly polar molecules with little or no tailing.
ACKNOWLEDGMENT We are indebted to M. E. J. MacMillan who has previously investigated porous polymers such as conventional Porapak columns for gas chromatographic analyses of sul-
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fur compounds, and thus contributed to the development of this technique. Our thanks are also due to S. Prahacs and D. W. Clayton for their guidance in preparing this paper, and to J. R. Chauvette for his technical assistance.
(5)R. K. Stevens, J. D. Mulik. A. E. O'Keeffe, and K. J. Krost, Anal. Chern., 43,827-831 (1971). (6) S. S.Brody and J. E. Chaney, J. Gas Chrornatogr. 4, 42 (1966). (7) H. P. Williams and J. D. Winefordner,J. Gas Chrornatogr., 4, 271-272 (1966). (8) Robert R. Austin Co., P.O. Box 3655, Industry, Calif., Bulletin No. 100470.
LITERATURE CITED (1) W. L. Thornsberry, Jr.. Anal. Chern., 43, 452-453 (1971). (2) E. Mitchell and R. DeLew. "Applications Tips," Aug. 20, 1971,
Varian Aerograph. Walnut Creek, Calif. Bruner, A. Liberti. M. Possanzini, and I. Allegrini, Anal. Chern., 44,
(3) F.
2070-2074 (1972). (4) D. Adams, R. K. Koppe, and D. M.
Jungroth, Tappi, 43,
6 (1960).
RECEIVEDfor review July 17, 1974. Accepted October 31, 1974. Work partially supported by the National Research Council, Industrial Research Assistance Programme, and the Ministry of the Environment, Air Management Branch, Government of Ontario.
Simple Electron Capture Gas Chromatographic Method for the Determination of Oral Hypoglycemic Biguanides in Biological Fluids Shaikh B. Matin, John H. Karam, and Peter H. Forsham Schools of Pharmacy and Medicine, University of California, San Francisco. Calif. 94 143
T h e biguanide compounds, phenformin, buformin, and metformin (Scheme I), are used clinically as oral hypoglycemic agents in the treatment of diabetes mellitus ( I ). T h e development of a simple, sensitive, and specific method for the measurement of these drugs in human bioligical fluids is necessary to investigate the mechanism of action, clinical pharmacology, and pharmacokinetics of these compounds
(2). Few chemical procedures for the identification and quantitation of phenformin have been reported ( 3 ) .These methods utilized color development ( 4 ), fluorescence ( 5 ), fluorescence quenching ( 6 ) ,pyrolytic gas chromatography ( 7 ) , and ion-pair extraction (8) techniques. However, all these reported methods lack sensitivity and/or specificity for the measurement of biguanide concentration in plasma after the administration of therapeutic doses to humans. In this report, a general method for the quantitation of biguanides using gas chromatography with electron capture detection is described. T h e method is based on the cyclization of biguanides to the corresponding 2-substituted 4monochlorodifluoromethyl-2,6-diamino-1,3,5-Striazine when acylated with monochlorodifluoroacetic anhydride. Using the developed method, plasma concentration of phenformin could readily be measured up to 24 hours following the administration of a single therapeutic dose to a diabetic patient.
I
H$c-N4\,/cb4H2 H3C
A
1 .I-O!PtTWYL3IC"IIN!Ot
Scheme 1. The oral hypoglycemic biguanides
and was used without further purification. The 1-benzylbiguanide and the 1-propylbiguanide which were used as the internal standard were synthesized according to a reported general procedure (9).
EXPERIMENTAL Apparatus. A Varian Model 1200 gas chromatograph equipped
with a "Ni electron capture detector was used. Chromatograms were obtained using a 2.12-m (6-ft) X 0.3-cm (0.125-in.) glass column packed with 3% OV-17 on Chromosorb W-AW DMCS H P (100-200 mesh), and conditioned for 48 hr at 280 "C with the first 24 hr without gas flow. The flow rate of carrier gas (5% CH4 in Ar) was 30 ml/min. The GCIMS for the representative samples were obtained on an AEI/MS-12 gas chromatograph-mass spectrometer. A 2.12-m X 0.31-cm glass column packed with 3% OV-1 was used for sample introduction. The operating temperature of the ion source was 250 "C. The trap current was 500 mA, and the electron energy was 70 eV. A PDP-computer was used to acquire and process the data. Materials. Phenformin, buformin, and metformin were supplied by the Ciba-Geigy Corporation, Ardsley, N.Y. Monochlorodifluro acetic anhydride was obtained from Peninsular Corporation
All urine was collected at specified time intervals from the diabetic patient who was being treated with phenformin and stored at -20 "C until analyzed. Blood samples were drawn into heparinized tubes, and the plasma samples obtained after centrifugation were kept frozen till analyzed. Method. An outline of the procedure for the determination of the compounds from plasma is presented in Scheme 11. Briefly, the method involves the addition of the appropriate internal standards, benzylbiguanide for phenformin and propylbiguanide for buformin and metformin and precipitation of plasma proteins by TCA in 1N HCI. The supernatant is separated, made alkaline with 10N NaOH and the compounds are extracted into 10 ml CH2C12. In the case of metformin, the basified layer is saturated with sodium chloride 0.5 g before extraction with CH2C12. Monochloro difluoracetic anhydride is added to the CH2C12 fraction and tubes are placed in a waterbath at 50 "C. When the evaporation of CH2C12 is complete, the excess anhydride is destroyed by adding 1N NaOH and the cyclized product is extracted into pentyl acetate (50 PI) and 1 to 2 ~1 are injected onto the gas chromatograph which was equipped with "Ni detector. Injector port, column, and detector temperatures were 250, 210, and 310 'C, respectively. The column temperature was maintained at 165 "C and 180 'C for the gas chromatography of metformin and buformin, respectively.
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