Spectrophotometric Assay of the Triphenylmethyl Group Robin M. Saundersl a n d H e n r y P. Schwarz Biochemistry Department, Philadelphia General Hospital, Philadelphia, Pa. John C. Stewart Twyford Laboratories, Twyford Abbey Road, London, England
A METHOD IS DESCRIBED for the determination of the triphenylmethyl (trityl) group in concentrations ranging 1 to 35 pg (0.004 to 0.14 Mmole). The analysis depends on acid treatment of trityl-containing compounds and subsequent colorimetric determination of the yellow triphenylmethyl cation produced. The method can be adapted for determination of the benzhydryl group. Trityl ethers are frequently used as intermediates in the synthesis of partially substituted sugars (Z), and N-tritylamino acids less frequently in the synthesis of peptides (2). A method for quantitative examination on a micro scale of reaction mixtures containing such compounds would clearly be of value. Previously trityl ethers have been assayed gravimetrically by precipitation as trityl chloride o r triphenylcarbinol ( I ) , and in one instance by a n ultraviolet absorption method (3). The former methods, however, suffer from lack of sensitivity and involve considerable loss of material in each assay, while the second is limited by the fact that several functional groups absorb in the ultraviolet region. Applegarth and Buchanan ( 4 ) introduced a technique for detecting trityl ethers of sugars on paper chromatograms in which spraying with dilute perchloric acid and subsequent heating produced yellow spots. We have noticed in thin layer chromatography of trityl ethers that a n immediate vivid yellow color is produced after spraying with 50% sulfuric acid. The yellow color is certainly due to the formation of the triphenylmethyl cation (5). We have utilized the fact that a yellow color is produced in acidic conditions to describe a very simple and highly sensitive assay of the trityl group. EXPERIMENTAL Samples containing the trityl ether or N-tritylamino group are soluble in organic solvents. Aliquots of the solution are placed in clean test tubes and the solvent is carefully removed with a stream of nitrogen. Ten milliliters of concentrated sulfuric acid are added taking care to wash down the sides of the tube, and mixed thoroughly. The color development is measured at maximum absorption 420 mp against a blank of concentrated sulfuric acid. The color can be measured at any time. Identical results were obtained with Coleman Junior and Beckman DU Spectrophotometers. Figure 1
1
100
-
80
.
60
-
40
.
(Y
E
H
-
I IE
3 G
P
20 -
01
1
I
300
400 500 Wavelength, m p
600
Figure 1. Absorption spectrum of triphenylmethyl cation in sulfuric acid shows the color absorption spectrum. Triphenylcarbinol is used to determine a calibration curve, linearity being observed to about 35 pg of trityl group.
Table I.
Quantitative Determination of Trityl Group in Various Compounds Trityl found Trityl compound ( of calculated amount) Trityl chloride 100 l-O-Trityl-2,3-di-O-benzyl-~102 glycerol (6) l-O-Trityl-3-O-benzoyl-~glycerol (p 102 Methyl 4-O-acetyl-2,3-anhydro-699 0-trityl-a-D-mannoside (8) Methyl 2-O-benzyl-3-O-tosyl-60-trityl-a-D-mannoside (9) 103 2,3,4-Tri-O-benzoyl-1,5-di-O98 tritybribitol (10) N-Tritylglycine hydrazide (1 1)s 100 N-Trityl ethyl glycine ( I 1 ) O 101 a These compounds were prepared in our laboratories (unpublished work), and were identical to the same compounds prepared by cited workers.
Present address, Western Regional Research Laboratory,
U. S. Department of Agriculture, Albany, Calif. ~~
(1) B. Helferich, Adoan. Carbohydrate Chem., 3, 79 (1948). (2) E. Gazis, B. Bezas, G. C. Skelakatos, and L. Zervas, “Peptides,” Proc. Vth European Symp., 1962, Pergamon Press, 1963, p. 17. (3) C. J. Malm, L. J. Tanghe, B. C. Laird, and G. D. Smith, ANAL. CHEM., 26, 188 (1954). (4) D. A. Applegarth and J. G. Buchanan, J. Chem. SOC.,1960, p. 4706. (5) L. P. Hammett, “Physical Organic Chemistry,” p. 54, McGrawHill, New York, 1940. 550
ANALYTICAL CHEMISTRY
(6) R. M. Saunders and H. P. Schwarz, J. Am. Chem. SOC.,88,3844 (1966). (7) E. Baer. I. B. Cushinn, _.and H. 0. L. Fischer, Can. J . Res... 21B., 119 (1943). (8) . , J. G. Buchanan and J. C . P. Schwarz. J . Chem. SOC.. 1962. p. 4770. (9) J. G. Buchanan and R. M. Saunders, Ibid., 1964, p. 1791. (10) D. A. Applegarth, J. Baddiley, and J. G. Buchanan, Ibid., 1965, p. 1213. (11) L. Zervas and D. M. Theodoropoulos, J. Am. Chem. SOC.,78, 1359 (1955). .
I
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-benzoyl-
1,5-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-inch stainless 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-
< 4000-
w
c Y
3000-
$20001000-
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
