RESULTS
Listed in Table I are results for determination of the trityl group in various compounds which differ considerably in other types of substituents. The accuracy of the method as measured by comparison with per cent calculated is high and the other groups present do not interfere. A group which doe!; interfere is the benzhydryl group [(C6H&CH+],although in practice it is very unlikely that these two groups would occur in the same reaction mixture. The benzhydryl group can actually be assayed in an analogous manner except that the color development is measured at 440 mp, and in concentrations ranging 1 to 30 pg (0.006 to 0.18pmole). Using this procedure we have easily been able to construct elution curves for column chromatography of reaction mix-
tures containing trityl compounds, so avoiding the timeconsuming steps of evaporating fractions to dryness, weighing, and running chromatograms. ACKNOWLEDGMENT
We thank J. G. Buchanan of the University of Newcastle, England, for specimens of methyl 4-0-acetyl-2,3anhydro-6-O-trityl-cr-~-mannoside and 2,3,4-tri-O-benzoyl1,5-di-O-trityl-~-ribitol. RECEIVED for review December 6, 1966. Accepted January 20, 1967. This work was in part carried out under Contract NYO 1864-21 with the United States Atomic Energy Commission.
Anomalous Calibration Curves in Gas Chromatography of High-BoilingCompounds M. M. E. Metwally, C, H. Amundson, and T. Richardson Department of Food Scieiice and Industries, University of Wisconsin, Madison, Wis. WHILEATTEMPTING to es Lablish calibration curves to quantify 2-nonadecanone ethylertethioketal and hexadecylaldehyde ethylenethioacetal extracted from reaction mixtures, we observed abnormal behavior of synthetic standards when analyzed by gas chromat13graphy. When varying volumes of a standard solution wert: injected, the anomaly was characterized by a failure of tht: calibration curve (area response us. concentration) to extrapolate through the origin and an apparent displacement of the calibration curve along the ordinate dependent upon the concentration of the standard solution. When the same volumes of different concentrations were injected, the calibraxion curves extrapolated through the origin; however, the slopes changed as the volume injected was varied. This reporf describes our attempts to resolve this anomaly and describes the effect of the solvent on the calibration curves. EXIPERIMENTAL
Hexadecylaldehyde ethylenethioacetal (m.p. 39" C) and 2-nonadecanone ethylene thioketal (m.p. 41 " C) were synthesized by the technique of Fieser ( I ) . Elemental analyses agreed very well with the theoretical elemental composition, and gas chromatography indicated only one peak for each standard. The compounds were chromatographed on an Aerograph 1520 equipped with flame ionization detectors. A 5-fOOt X 1/8-inchstainless steel column packed with 10% FFAP on 8OjlOO mesh IIMCS-treated chromosorb W was employed. The nitrogen carrier gas and hydrogen flow rates were 30 ml/min. Initially, the injector temperature was 275" C, detector temperature was 240" C, and column temperature was 210" C. Subsequently, the injector temperature was lowered to about 200" C in attempting to resolve the problem. Standard solutions were prepared so that a 10-pl syringe could be used to give concentrations of approximately 40 pg of standard. To obviate needle error the
(1) L. F. Fieser, J . Am. Chern. SOC.,76, 1945 (1954).
6o
5000-
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w
c Y
3000-
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Figure 1. Calibration curves of 2-nonadecanone ethylenethioketal showing concentration dependence Solvent, iz-hexane; column temp., 210°C; injector temp., 215OC; detector temp., 240°C
solution volume was read prior to injection by pulling all the solution into the syringe barrel, and the volume injected into the gas chromatograph was read by difference. RESULTS AND DISCUSSION
Figure 1 shows the type of calibration curve consistently obtained when either the thioketal or thioacetal was dissolved in chloroform or n-hexane and injected at increasing levels into the gas Chromatograph. Straight lines were obtained for the standards; however, they did not extrapolate through the origin. In addition, when the concentration of the standards was lowered, there was a displacement of the straight line with approximately the same slope toward the origin. Calibration curves in the 0- to 3-pg range also failed to extrapolate through the origin. Suspecting adsorption on the column, VOL. 39, NO. 4, APRIL 1967
551
:;:y I
Yl
-t
6000
1: n J 4000 4 50001 1:4000
Ir'
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0
5
10
15 20 25 pq/PEAK
3000 30001
30
40
Figure 2. Calibration curves of 2-nonadecanone ethylenethioketal showing effect of injection level on slope Solvent, n-hexane; column temp., 215 "C; injector temp., 205 "C;detector temp., 235 "C
decomposition, or incomplete volatilization in the injection chamber, we tried different column packings (carbowax 20m), glass columns with FFAP, on-column injection, glass injection liners, lower injection temperatures, etc., with the same apparent concentration dependent calibration curve. Figure 2 illustrates three calibration curves obtained when the injection volume used for each curve was constant and the standard concentration was varied. Although the curves do go through the origin, it can be seen that the slope of each calibration curve decreased as the injection level decreased. The problem was solved when we went to higher-boiling solvents to prepare our standard solutions. For example, Figure 3 shows normal calibration curves at 2 different concentration levels when xylene was used as the solvent. Similar results were obtained when other high-boiling solvents such as isoamyl alcohol and dimethylsulfoxide were used. Apparently the low-boiling hexane and chloroform flashed
552
ANALYTICAL CHEMISTRY
2000
1000
Ioook 0 IO 20 30 40 0 pq/PEAK
5
Figure 3. Calibration curves of 2-nonadecanone ethylenethioketal dissolved in xylene Column temp., 215"; injector temp., 200°C; detector temp., 240°C
out of the needle leaving a small but significant residue in the needle of the syringe while the higher-boiling solvent obviated this. RECEIVED for review November 3, 1966. Accepted February 16, 1967. Work supported in part by the American Dairy Association, the Co-operative State Research Service, United States Department of Agriculture, and the Research Committee of the Graduate School, with funds supplied by the Wisconsin Alumni Research Foundation. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station.
