sisting of a U-tube made from borosilicate glass 4 mm. in outside diameter, and packed with 0.2 gram of Burrell high-activity charcoal held in place with wads of glass wool and bent wire, is purged and sealed as in preparation of the sample tubes. T h e tube is unstoppered and cooled in ice water, and 20 liters of the air to be tested are aspirated through it at the rate of 0.5 liter per minute. It is then restoppered and kept a t reduced temperature until time of analysis. Samples may be stored for 2 to 3 weeks without apparent loss. Analysis. T h e sample is transferred t o t h e analytical column by connecting t h e sampling tube t o a magnesium perchlorate tube and then t o t h e gas pipet and the chromatograph. T h e gas pipet and the perchlorate drying tube should first be flushed with helium. Desorption and transfer of the sample are acconiplished by inserting the sampling tube in a n oil bath maintained a t 200” C. Simultaneously, the magnesium perchlorate tube is inserted in a bath maintained at 100” C. and helium is passed through the system, sweeping the sample through the drying tube and gas pipet and thence to the analytical column. The flow of helium is allowed to continue so as to develop the chromatogram. As the chromatogram is developed, the sample may be retrapped for further investigation by passing the gases through a cooling coil and then on to
a sampling tube maintained a t 0” C. The cooling coil may be kept at room temperature or a t lower temperatures, depending on the nature of the sample and the operating temperatures used for the analytical column (Tables VI and VII). The pollutants are identified by comparing their retention times on one or more columns with those of known compounds. The amount of each component can be estimated by reference to appropriate calibration curves. Figure 6 is a chromatogram of a sample of automobile exhaust gas collected on a sample tube (0.2 gram of charcoal). Water was retained by magnesium perchlorate tube. The sampling is satisfactory and the existence of 19 compounds can be readily recognized. However, because of lack of a comprehensive catalog of retention times of the compounds, the individual components were not identified. ACKNOWLEDGMENT
The authors wish to acknowledge the cooperation of Kem-Tech Laboratories in providing certain equipment and technical assistance. They also thank B. R. Sant for assistance during the latter part of the work. LITERATURE CITED
(1) Bacarella, A,, David, F. D., Grun-
wald, E., ANAL. CHEM.27, 1833 (1955). (2) Dimbat; RI., Porter, P. E., Stross, F. H., Ibid., 28, 290 (1956). (3) Ingram, W. J., Dieringer, L. F., Am. Ind. Hva. Assoc. Quart. 14, 121 (1953).-“ (4) Jacobs, AI. B., “Analytical Chemistry of Industrial Poisons, Hazards and Solvents,” 2nd ed., Interscience, Yew York, 1949. (5) Keulemans, 8.I. A I . , Kwantes, A., Zaal, P.. Anal. Chim. Acta 13, 357 (1955). Quiram, E. R., SIetro, S. J., Lewis, J. B., ANAL.CHEM.26, 352 (1954). Shepherd, AI., Rock, S. AI., Howard, R., Stormes, J., Zbid., 23, 1431 (1951). Silverman, L. A., “Encyclopedia of Instrumentation for Industrial Hygiene,” p. 7, Publication Distribution Service, University of Michi an, lZnn Arbor, 1956. (9) Smith, F., Rees, 0. IT., Hardy, V. R.. J . ,4m. Chem. Soc. 54. 3520 (1932). Turk, A,, Am. SOC.Testing Materials, Tech. Bull. 164 (1954). Turk, il., Ann. iV. Y . Acad., Sci. 58, 193 (1954). Turk, A., Sleik, H., PIIesser, J. P.,
6.
Am. Ind. Hyg. Assoc. Quart. 13, 23 (1952). West, P. TTT., Sen, B., i i s . 4 ~ .CHEY. 27, 1460 (1955). West, P. W.,Sen, B., Z. anal. Chem. 153, 177 (1956). RECEIVEDfor review October 11, 1957. Accepted March 18, 1958. Investigation supported by Research Grant S-43, Study Section of Sanitary Engineering and Occupational Health, Division of Research
Grants, Public Health Service.
High Frequency Technique for Continuous Recording in Chromatographic Analysis of Bile Acids GlLLlS JOHANSSON and K. J. KARRMAN Department of Analytical Chemistry, University o f Lund, Lund, Sweden ARNE NORMAN Department of Physiological Chemistry, University of Iund, Iund, Sweden
b The high frequency technique may be utilized in chromatographic work to record substances in the effluent. The effluent passes a cell which forms a part of a high-frequency resonant circuit, and changes in the conductivity are recorded as a function of time. The method has been used in the separation of conjugated and free bile acids by reversed-phase partition chromatography. The general applications of the method are discussed.
P
methods have been described for recording the amounts of substances in the effluent from chromatographic columns. These are generally less time-consuming than chemical analyses and leave all the subHYSICAL
stances intact. The present investigation further develops a method based on the high frequency technique. As compared to low frequency conductance methods, the high frequency technique offers several advantages. Because the electrodes do not come in contact with the substances being analyzed, decomposition and electrode absorption are avoided. More detailed discussions are given by earlier workers (1, 4, 6, 13). Baumann and Blaedel ( I ) developed a method to record the conductivity of the stationary phase by clamping electrodes on the column. I n the present method, the conductivity of the eluent is measured as it passes through a n external cell.
APPARATUS
High Frequency Device. T h e high frequency apparatus, which has been described previously (9), was rebuilt to gain higher mechanical and electrical stability. The voltage drop across the grid bias resistor of a crystal oscillator was compensated and measured with a vacuum tube voltmeter. The oscillator was operated a t 5 Me. The electronically regulated power supplies and the vacuum tube voltmeter were housed in the same case with the oscillator circuit. T o obtain the highest possible long-time stability, the line voltage was stabilized by a magnetic transformer. The apparatus required a warm-up period of 2 to 3 hours. The amount of electrolyte in the measuring cell was continuously registered by a n automatic recorder, either VOL. 30, NO. 8, AUGUST 1958
1397
30 ppf. could be added in parallel to the tuning capacitor to obtain a reasonably high Q value for the circuit, which was necessary for high sensitivity. Tubular antenna cables with lower capacitance were reiected because thev lacked stability. Calibration. The cell was calibrated with Dotassium chloride solutions. Water (1.1 X mho) was used as a reference liquid-Le., when the cell was filled with water and the circuit was tuned to resonance, the meter mas set a t zero. Then a potassium chloride solution was introduced to replace the water and two readings were taken. The first reading was taken without tuning the circuit to resonance. This reading represents the conditions encountered in chromatographic analysis. The second reading was taken with the circuit in resonance. By repeating this procedure with potassium chloride solutions of different strengths, the curves in Figure 4 were obtained. The abscissa represents the low frequency conductance of the potassium chloride solutions as measured with a Philips resistance bridge, the left ordinate the response of the high frequency conductance, and the right ordinate the change in capacitance. The linearity is improved when no resonance tuning is made. Over the first part of the curve, the linearity is very good. The linear range extends t o 65 micromhos (0.0005N potassium chloride).
'/
MICROMHOS Figure 2. Calibration curves for the cell
I. A/
A
Figure 1. A, B. C, E. D. a.
Chromatographic cell
Semicircular electrodes Perspex holders Supporting metal tube Width of interchangeable electrode
a Speedomax (Leeds & Northrup Co., Philadelphia, Pa.) or a Varian instrument (Varian Associates, Palo Alto, Calif.), using a span of 10 mv. The voltage drop across the microammeter in the vacuum tube voltmeter was divided by an attenuator. The attenuation ratio was adjusted to give 10 mv. to the recorder for full-scale deflection of the panel instrument. Measuring Cells. Several types of cells were investigated t o find one with good sensitivity b u t still of small volume. Some cells had rather large tailings probably resulting from the formation of still zones, especially a t slow flow rates. The best results were obtained with a cell consisting of a straight glass tube connected to the chromatography column with a British Standard). spherical joint (S Figure 1 shows the cell employed in this work. The electrodes, A and B, attached to the glass tube are semicircular with a diameter of 6 mm. and a length of 70 mm. Because wall thickness also influenced the sensitivity of the apparatus ( 1 4 , a very thin cell wall (0.3 mm.) was used. The electrodes are fixed by two Perspex pieces, C and E (Imperial Chemical Industries, Ltd., England). A metal tube, D,serves as support and shield. The grounded electrode, A , is interchangeable; by varying the size of this electrode, the operational range may be altered (6). A set of calibration curves for different values of grounded electrode width (a) and wall thickness are shown in Figure 2. Dilute potassium chloride solutions were used for calibration of the cell. The volume required to wash out the cell completely and refill it should be 2 ml. (the cell volume), if the two liquids pass through the cell n4th sharp boundaries. Actually the two phases are mixed lvhen passing through the cell. This intermixing depends on the flow rate. A setup was made so that, by turning a stopcock, either distilled water or 0.001N hydrochloric acid 1398
ANALYTICAL CHEMISTRY
W a l l thickness 0.3 mm., a = 6 mm. 11. W a l l thickness 0.5 mm., a = 5 mm. Ill. W a l l thickness 0.5 mm., a = 2 mm. IV. W a l l thickness 0.3 mm., a = 0.5 mm. 10 r
SAMPLES AND REAGENTS
. 01
i '
1
1
1
,
53
MILLILITERS Figure 3. Intermixing of boundaries in cell
The conjugated bile acids n-ere synthesized according to Bergstrom and Xorman ( 2 ) . The preparation and analyses of the samples have been given (12). The free bile acids were purified by crystallization and had the same analytical data as previously reported (12). Chloroform, technical grade, mas carefully washed 11-ith sulfuric acid, 5% sodium carbonate, and water,
The base line is drawn when the cell i s fllled with water and the line a t upper level when it is fllled with 0.001 N hydrochloric acid
could flow through the cell. The flow rate could be adjusted by another stopcock. When the water was replaced by 0.001N hydrochloric acid with the flow rate a t 0.72 ml. per minute, 6.5 ml. was required before a constant value was obtained (Figure 3). When the hydrochloric acid solution was replaced by water with a flow rate a t 0.31 ml. per minute, only 3.7 ml. was required before the base line was reached. The tailing was less pronounced a t low flow rates. The cell was connected to the high frequency apparatus by a coaxial cable. When cable RG8/U was used, it mas possible to use a cable length of only 25 em. With a special low capacity cable, RG63/U, the length could be 50 em. The cable length is limited by the capacitance it introduces in parallel with the measuring cell. Not more than
LO r
7
LO
30
v, I-
J
0
>
-20
20
"p
10
cl'o
'r?
2c3
350
MICROMHOS
Figure 4. Calibration of potassium chloride solutions
cell with
--Response without tuning to resonance - - - -Response when tuning to resonance - .- .Capacitance change
dried over potassium carbonate, and distilled. Methanol was dried and distilled over potassium carbonate. Butanol (Merck) for chromatography. Iso-octyl alcohol (6-methyl-1-heptanol) and heptane, technical grade. Hyflo Supercel (Johns-Nanville Co., New York, N. Y.) was washed with dilute hydrochloric acid and water, dried a t 110" C., and stored in a desiccator with dimethyldichlorosilane. It was finally washed with methanol and dried a t 100" C. CHROMATOGRAPHIC PROCEDURES
The reversed-phase partition chromatography of Howard and XIartin (7') was used for separation of bile
'1
acids (3, 11, 16). The phase systems used are listed in Table I. The phase systems were equilibrated a t 23" C. The chromatographic tube had an inner diameter of 13 mm. and a height of 500 mm. A stopcock and a spherical joint (S 1/12) were attached to the lower end of the column, which was charged with 4.5 grams of hydrophobic Hflo Supercel. The cell was filled with the mobile phase by sucking through a male joint attached to the female joint a t the top of the cell (Figure 1). When the auxiliary male joint is removed, the cup is left filled. Thus, the column was easily attached without allowing air bubbles to enter the cell. The mobile phase was alloxed to run through the column until a constant reading mas obtained (usually about
TC
10
25
is
50
125
EFFLUENT, ml. Figure 5. Chromatography of 0.3 mg. of taurocholic acid (TC), 3.0 mg. of glycocholic acid (GC), and 4.5 mg. of glycodeoxycholic acid (GD) Calibration curve I
Phase system C
Figure 6.
(GD), 3.0
EFFLUENT, ml. Chromatography of 2.5 mg. of glycodeoxycholic acid mg. of glycolithocholic acid (GL), and 4.0 mg. of litho-
cholic acid (L) Phase system
F
Calibration curve II
51
1:
5
lo 0
15
Im
u5
EFFLUENT,mi. Figure 7. Chromatography of 4.0 mg. of taurodeoxycholic acid (TD) and 3.0 mg. of taurolithocholic acid (TL) hase system D
Calibration curve IV
Table I.
Chromatographic Systems
hZobile Phase RI1. A. Methanol
Water C. Methanol Water
180 120 150 150
D. Water F. hlethanol
300 165 135
W'atrr
Stationary Phase hI1. Chloroform 45 Heptane 5 Iso-octyl 15 alcohol Chloroform 15 1-Butanol 100 Chloroform 45 Heptane 5
50 ml. was required). Then the mixture to be analyzed mas added, and the effluent was collected in test tubes, by using an automatic fraction collector. The flow rate mas adjusted to 0.3 ml. per minute. All chroniatographic work was done a t 23" C. in a constant temperature room. There is a slight warming of the percolate between the electrodes. I n a chromatographic column with a constant flow rate, this does not c a u v interference. The slight temperature rise, hon ever, causes precipitation of butanol drops along the cell wall when water saturated with butanol is used as the mobile phase. This solvent separation makes the recording erratic. To avoid the precipitation of butanol, the effluent from the column was first passed through a water-cooled condenser (100 mm. high and 2 mm. in inner diameter). The condenser mas fitted to the column and cell with spherical joints. RESULTS
The high frequency technique has been used to indicate the elution of substances in the chromatographic separation of taurine- and glycine-conjugated bile acids and the corresponding free acids. K i t h phase system C, the taurineconjugated bile acids are eluted with the front. The glycocholic and glycodehydrocholic acid are eluted a t 30-4050 ml., cholic acid a t 8Cb-100-120 ml., glycodeoxycholic acid a t 100-120-130 nil., and dehydrocholic acid a t 110130-150 ml. The figures give the beginning, peak, and end volume of each bile acid band a t the total volume (nil.) of effluent a t these points. Figure 5 s h o m a chromatogram of taurocholic, glycocholic, and glycodeoxycholic acid, The open circles connected by a broken line show the titrations values of each fraction. The solid line shons the high frequency recording. The small amount of eluted taurine conjugated bile acid gives a very high peak. If 10 mg. of cholic acid mas chromatographed hyith the same phase system and cell as in Figure 5, no significant recording could be obtained. Glycodehydrocholic and glycocholic acids give recordings of about the same order of magnitude. They can be separated using phase system F, where glycocholic VOL. 30, NO. 8, AUGUST 1958
1399
Table II.
High Frequency Response of Some Bile Acids Dissolved in
60%
Ethanol
to improve the stability of the apparatus.
Calibration Curve I Acid
0 0001'tl
0 00111f
0 002M
0 01M
0 02M
Volt Taurocholic Glycocholic Cholic Deoxycholic Lit hoc holic Dehydrocholic
0 79 0 40
7 6 2 77
14 9 4 23
0 92 0 97 1 57 1 26
1 51 1 63 2 27 2 00
ACKNOWLEDGMENT
The authors wish to thank Lennart Stigmark, Institute of Physics, Lund, for valuable help with the construction of the apparatus. LITERATURE CITED
acid is eluted with the front and glycodehydrocholic acid a t 20-30-40 ml. Figure 6 gives a separation of glycodeoxycholic acid a t 10-15-20 ml. and glycolithocholic acid a t 25-35-40 nil. The unconjugated lithocholic acid is eluted after the conjugates a t 70-80-90 ml., but no recording was obtained. For recording taurine conjugates with phase system D, the cell with the smallest sensitivity was used. A chromatogram of taurodeoxycholic and taurolithocholic acids is given in Figure 7. DISCUSSION
The chromatograms show that the response depends on the nature of the substance, mainly as a function of the conductivity. The free bile acids have a dissociation constant of the order of 7 X lo-' in 50% ethanol-water solution (IO). The conductance of these acids is so low that they could not be detected in the effluent concentrations encountered (Table 11). The conjugates are stronger acids. Their dissociation constants in water range from lo-* to 10-5 ( 8 ) . The bile acids with sulfonic substituents are the strongest acids. Thus it is possible to record
taurine conjugates in much smaller concentrations than can be determined by titration. It is difficult to predict the conductivity of a compound in organic solvent systems because of the complex nature of these systems. In general, the acids and bases must have a dissociation constant of at least 10-6 in water to be detected by the method outlined, The high frequency technique will find many applications in chromatographic work, especially in partition chromatography where organic solvents with low conductivity are used. The method is suitable for the analysis of substances with dissociation constants greater than Sonaqueous solvents may be used if the solvent mixture is such that ion pairs can be formed. The amount of salt or acid in the solvent must be kept low; othernise the sensitivity will be decreased. I n this application, the demands on the stability of the high frequency apparatus are extremely high. Many precautions must be taken to avoid appreciable drift: Temperature change of the crystal will cause amplitude variations, and the tubes must be in excellent condition. Kork is being carried out
Baumann, F., Blaedel, W. J., AKAL. CHEM.28, 2 (1956).
Bergstrom, S., Korman, A., Acta Chem. Scand. 7, 1126 (1953).
Bergstrom, S., Sjovall, J., Zbid., 5, 1267 (1951).
Blaedel, W,J., RLalmstadt, H. V., k A L . CHEM.22, 734 (1950). Clayton, J. C., Hazel, J. F., XcSabb, W. lI.> Schnable, G. L., Anal. Chim. Acta 14,269 (1956).
Hall, J. L., Gibson, J. A, Jr., ANAL.CHEY.23, 966 (1951). Hon-ard, G. il., Martin, A. J. P., Biochem. J . 46, 532 (1950). Josephson, B. A., Biochem. Z. 263, 428 (1933).
Karrman. K. J.. Johansson. G.. Mikrochim. Acta 1956. 1573. (10) Kumler, IT. D., Halveritadt, I. F , J . Bid. Chem. 137,765 (1941). (11) Sorman, A , , Acta Chem. Scand. 7 , 1413 (1953). (12) Sorman, A , , Arkiv Kemi 8 , 331 (1955). (13) Oehme, F., Chem.-Ztg. 80, 162 f19.56).
(14) R&i&; 'C. N., McCurdy, H. W., Jr., A s . 4 ~ .CHEX.25, 86 (1953). (15) Sjovall, J., Acta Phusiol. Scand. 29, 232 (1953).
RECEIVED for review October 28, 1957. Accepted February 21, 1958. Supported bv grants from Statens Naturvetenskapliga Forskningsrgd (Swedish Natural Science Research Council) and Statens bledicinska Forskningrgd (Sn-edish Medical Research Council).
Analysis of the Nonvolatile Acids in Cigarette Smoke by Gas Chromatography of Their Methyl Esters LOUIS D. QUlN and MARCUS E. HOBBS Duke University, Durham,
N. C.
b The nonvolatile acid fraction of cigarette smoke, after conversion to a mixture of methyl esters with diazomethane, was analyzed by gas chromatographic techniques. Of the 16 esters detected, 1 1 have been identified. Lactic, glycolic, succinic, 'and malonic acids constitute about 7570 of those identified, among which only succinic acid has been reported previously as a smoke constituent. Although some acids containing certain other functional groups cannot be 1400
ANALYTICAL CHEMISTRY
analyzed by the techniques described, the method may b e of value in the partial analysis of complex acid mixtures from sources other than cigarette smoke.
T( 4 ) ,
steam-volatile acids of cigarette smoke have been examined in debut little is known about the tail nonvolatile acids. In a survey of the literature up to 1954, Kosak (12) listed succinic, fumaric, citric, and phenolic acids as possibly present in HE
cigarette smoke. Nicotinic and glutamic acids ( 3 ) and three a-keto acids (glyoxylic, pyruvic, and a-ketoglutaric) (6) have been detected since this report. The present paper is concerned with the detection and determination of a number of nonvolatile acids in cigarette smoke. The presence of succinic acid has been confirmed, whereas the other acids detected have not been reported previously. Gas chromatography is the basis for the analytical method used. It is not
