absorption mixture after several analyses. S-carbobenzyloxy and O-carbobenzyloxy, but not &-carbobenzyloxy derivatives, could be :malyzed by the procedure described. Benzyl ester groups do not interfere with the determination of carbobenzyloxy groups (Table III), as no ca,rbon dioxide is formed during the H B r cleavage of the former. Carbobenzyloxy groups were determined in multichain polyamino acids ( 3 ) in which polyhenzyl glutamate side chains were connected to t-amino groups of a polylysine core and in which the terminal a-,tmino groups of the zide chains were blocked by carbobenzyloxy groups. From the number of carbobenzyloxy groups, the average length of the polybenzyl glutamate side chains could be derived and was in good agreement with the average length derived from amino acid analysis after total hydrolysis of the compounds.
Oxidized ribonuclease was carbobenzoxylated by the method of Anfinsen, Sela, and Tritch ( 1 ) and the number of the incorporated carbobenzyloxy groups was determined correctly. Finally, the samples used for analysis can be recovered in the decarbobenzoxylated form. The procedure can therefore be used for controlled decarbobenzoxylation of peptides and polypeptides in which amino or phenolic hydroxyl groups are carbobenzoxylated. LITERATURE CITED
(1) Anfinsen, C. B., Sela, M., Tritch, H., Arch. Riochem. Biophys. 6 5 , 156 (1956). ( 2 ) Ben-Ishai, D., Berger, A , , J . Org. Chem. 17, 1564 (1952). (3) Rerger, A , , Yaron, A , , “Polyaminn Acids, Polypeptides and Proteins,” M. A. Stahmann, ed., p. 13, The University of Wisconsin Press, Madison, 1962.
(4) Danlel, E., Katchalski, E., Ibid., D. 183. ( 5 j Fritz. J. 5.. Lisicki. Y . XI.. -ANAI. -CHEM.‘23, 58d (19il). ’ ( 6 ) Goodman, XI., Kenner, G. IT’,, Adoan. Protezn Chem. 12, 465 (1957). 17) , , Katchalski. E.. Grossfeld. I.. Frankiel. M., J . Am. i‘hem. Soc. 7 0 , ’ 2 h 4 (1048): (8) Katchalski, E., Sela, M., Advan. Protein Chem. 13, 213 (!958). (9) ;IledzihradszkS.-S~}i~~iger, H., Acta Chim. .Icad. ,9ci. H ~ t n g .37, 239 (1963). 0) Patrhornik, A , , Itogozinski, E. S., A N A L .CHEM.33.803 (1961 ). 1) Pat(-hornik, .i., Shalitin, Y.,Ibzd., 33, 1857 (1961). 2) Yogel, A . S L - “Practiral Organic Chemistry,” p 110, Longmans, Green, London. 1948. I
~~
,
I
A R I E H Y.4ROh
SARAH EHRI~ICH-~~OCOZI~’SKI ARIEHBERGER
Department of Biophysics The Weiemnnn Institute of Science Rehovoth, Israel WORK supported by Resemah Grant (FG-Is-101-60) from the U S, Department of Agriculture.
Quantitative Gas Liquid Chromatography of Phenols by Complete Trimethylsilylation of Hindered Phenols in Presence of Acidic Oxides SIR: A method is provided for complete conversion of phenols, including hindered compounds such as 2,6xylenol, to trimethylsilyl ethers in the presence of acidic oxidm such as silica, alumina, and titania. .\ neutral drying agent such as sodium sulfate aids in the reaction of 2,6-xylmol to its ether with hexamethyldisila:sane, but does not result in complete conversion. Acidic catalysts appear to be essential. I3asic oxides such as those of nickel and copper are ineffective ,ind even retard formation of trimethylsilyl ethers. In the original work on the gas liquid chromatographic separation of phenols as trimethylsilyl ethers, Langer, Pantages, and Wender (8) used trimethylsilyl chloride or hexamethyldisilazane as the trimethylsilylating agent. They were able to separate m-cresol from p-cresol. Pyridine, which promotes the reaction as a proton acceptor ( 6 ) and as a polar solvent (II), is now employed widely in the trimethylsilylation of hydroxyl groups. Ailthough trimethylsilyl chloride in pyridine has been used (4, 5 ) , hexainethyldisilazane admixed with trimethylsilyl chloride and pyridine is more efficient, particularly in the case of hindered compounds. This mixture has been used for alcohols (7, 1 d ) , carbohydrates (5, IO), bile acids ( 9 ) , and phenols (2-4). For routine quantit,ative analysis of cresylic acids it is desirable to avoid the use of solvents because these act as diluents and cause peak broadening,
lowering of sensitivity, and interference with chromatographic separation. Small quantities of reactants, simple apparatus, and short reaction times are also highly desirable. I n view of the results obtained by Grant and Vaughan ( 3 ) for the direct reaction of phenols with hexamethyldisilazane, a series of experiments with known mixtures was carried out. The effect of acidic catalysts was observed upon the reaction of hindered phenols (1) quch as 2,6-xylenol. The degree of conversion of phenol4 to trimethylsilyl ethers was measured by qtrip chart peak integration. Percentages were based upon areas obtained from mixtures of known compoiition. EXPERIMENTAL
Reagent. was purchased ChemResearch,
Hexamethyldisilazane from Penninsular Inc., Gainesville,
Fla. Phenol, Mallinckrodt reagent grade, was used. The alkylphenols were obtained from Consolidation Coal Co. These were 99 to 1 0 0 ~ opure as determined by infrared spectrophotometry, gas liquid chromatography, and freezing point. Titanium dioxide (Raker) ; aluminum oxide (Woelm, hlupharm Chemicals, N e w Orleans, La.) ; calcium sulfate, anhydrous (“Drierite”) ; and a 1,inde Molecular Sieve, 5.1, 8 mesh, \yere used. Sodium sulfate, anhydrous; silira (sea sand) ; molybdic anhydride (almit 100%) ; manganese dioxide ; anti
nickelous oxide are Fisher certified chemicals. Apparatus. A flame ionization chromatograph of conventionnl design was used with a Research Specialties Corp. electrometer, Model 605-3. .in 0.010-inch i.d. caliper cal)illary, 150 ft. long, coated with dioctyl sebacnte was used with a helium flow rate of 2 ml. per minute (mmsured a t 125” C.). Samlde injections of 0.1 to 0.2 PI. were made with a split ratio of 100:1 a t a n inlet helium pressure of 8.n-ith a bypass flow of 200 ml. per minute. .\ Sargent AIR recorder set a t 5-mv. input was used, coul)lcd with a n Instron Integrator for digital rcwlout of chromatographic. peaks. The still used for I)rcliaration of the trimethylsilyl ether> consisted of a female 19,’38 standard taper joint scaled about 3 inches from the cmd as the pot, and a matching rnalc joint sealed t’o a borosilicate tube of equal diameter (overall length, 1-1 inch(,*) as the air condenser. Eoth meriil)(w are kelit in a drying oven at 110” C. until immetliatcly bcforc us(’, Procedure. Onr gram (or 1 ml.) of sample, 1.5 nil. of hesamethyldisil:iz:inc, 0.5 gr:tni of oxide or other catalyst, :ind 0.5 gr:tni of drying agent (if u w l ) :ire Iil:iineil i n the ,till ant1 reflused for 45 niinutcs on :L200” C. sand h t h . The, Iiot is voolcd u n t i w the ttip and :I samiilc irijecatc’tl inimcc1i:itrly i n t o t h c c ~ 1 i r o i i i ~ ~ t ~ i ~ r : ~ ~ i l i . I’r~tkarcas arc’ intryr:itcd. I’crc~ntages (1 f in(li vid 1131 ( Y 1 iii 1 invn t 3 :I rc 1) a s d ripcin pc:ik a r w frwtions of the, ciitii of thc pcvrks (ct,hvrs i i i i r c u ~ t ( ~I>li(xiioIs), (1 Rcsl )onw f nc.t o r r()rrwa t ic )ni ivo rv 110t
+
VOL. 36, NO. 7, JUNE 1964
1389
used since only small corrections would be necessary for the ethers. Although t h e factor for the 2,6-xylenol ether was found to be 0.90 relative to the parent compound (as determined by preparative scale work), corrections are secondary for the relatively small amounts of unreacted phenols.
Figure 1. Silylation of phenols in the presence of sodium sulfate
RESULTS A N D DISCUSSION
Drying agents alone were added to a known mixture of phenols and reagent yielding results shown in Table I. Sodium sulfate, a neutral drying agent, gave complete conversion with all phenols except 2,6-xylenolJ as shown in Figure 1. Free phenols can be distinguished from silyl ethers by their tailing peaks-Le., the 31.7-minute 2,6xylenol peak in Figure 1. The retention times of all six phenols, as measured by known standards, differ substantially from any of the ethers and from each other, eliminating the possibility of interference. Calcium sulfate, a slightly acidic compound, on the other hand, resulted in quantitative formation of all the ethers. To prove that drying ability was not the deciding factor, 5-4 Molecular Sieve, a superior desiccant, was tried. It gave a result similar to that obtained with sodium sulfate (Table I).
Table 1.
Acidic oxides were then tried without the use of drying agents. The results are shown in Table 11. Complete conversion of phenols resulted in all cases. When a small drop of concentrated HCl and 0.5 gram of sodium sulfate were added to the silylation mixture (S), all the phenols present were converted to ethers. A set of relatively basic oxides admixed with drying agent gave the results shown in Table 111. Cupric oxide and manganese dioxide had no apparent effect but gave incomplete conversion of 2,6xylenol as did sodium sulfate
Effect of Drying Agents on Trimethylsilylation of Phenols
Added, Component
9%
NapSOc
CaSO,
5 8 , Mol. Sieve
Phenol o-Cresol &Cresol 2,5-Xylenol 2,4-Xylenol 2,6-Xylenol
10.0 16.8 11.8 9.6
10.4 17.2 12.2 9.9
11. 0
11.1 35.2
10.4 17.3 ii.9 9.5 10.7
10.5 17.1 12.0 10.0 11 .o 36.3 (3.7% unreacted)
Table II.
40.7
40.2
( 4 .2y0 unreacted)
Effect of Acidic Oxides on Trimethylsilylation of Phenols
Added, Component
772
Ti02
Si02
MOO*
ALOI
Phenol o-Cresol m p-Cresol 2,5-Xylenol 2,4-Xylenol 2,6-Xylenol
10.1
10.7 6.6 12 7 6 3 10 5 53 5
10.6 6.4 12 8 6 3 10 4 53 3
10.7 6.5 12 7 6 3 10 5 53 3
10.2 6.5 12 8 6 2 10 I 53 3
+
Table 111.
Component Phenol o-Cresol m p-Cresol 2,5-Xylenol 2,4-Xylenol 2,G-Xylenol
+
1390
6.5 12 9 6 5 10 9 53 2
Effect of Basic Oxides on Trimethylsilylation of Phenols
Added, 70 10 6 12 6
1
5 9 5
10 3
53 2
ANALYTICAL CHEMISTRY
CaO-XazSOc 10 6 12 6 10 49
7 6 8 4 3
2
MnO2-Na.BOd
NiO-CaSOa
10 8 6 6
10 6 13 6 7 21
12 8
6 3 10 9 48 1
9 4
3
0 9 7
alone. However, nickel oxide drastically inhibited conversion of 2,6xylenol, even in the presence of calcium sulfate. These observations tend to reinforce the generally held concept of acid catalysis in the silylation of hydroxy compounds by hexamethyldisilazane. The concept of formation of protonated nitrogen to give a n ammonium intermediate of hexamethyldisilazane coupled with nucleophilic displacement by the OR- group of the alcohol or phenol may represent the correct mechanism for the reaction (6). Currently the effect of these catalysts upon more hindered phenols such as 2, Bdi-tert-butyl-phenol, 2,4,6-trimethylphenol, and 2,4,6-tri-tert-butyl-phenol is being studied, in a n effort to extend the applicability of this approach. LITERATURE CITED
(1) F e d m a n , S., Kaufman, M. L., mender, I., J . Org. Chem. 27, 664 (1962). (2) Friedman, S., Steiner, W. A., Wender, I., Fuel 40, 33 (1961). (3)
