interferences. If necessary, the isobutyl alcohol content could be determined quantitatively a t a l o r e r temperature. The chromatographic and chemical results agree Fell. Only 12 minutes is required per chromatographic analysis, whereas 40 minutes is required by the chemical method.
Table VII. Retention Time of Isomers of Butyl Alcohol and Chlorobutane
Retention Time," Minutes Compound At 85" C. -It65"C. n-Butyl alcohol 10.5 25 6.5 10.5 sec-Butyl alcohol 8.0 17.0 Isobutyl alcohol I-Chlorobutane (n-butyl chloride) 6.25 10.0 2-Chlorobutane 4.5 ... l-Chloro-2methylpropane 4.5 7 2-Chloro-2methylpropane 3.0 4 a All measurements were recorded in conventional manner employing a PerkinElmer A column with helium as carrier gas metered at 100 cc. per minute.
niatographic method, obtained from six determinations of the same sample, is 0.037,. The extreme variation from the mean is 0.077,. If a n isomer of butyl alcohol or chlorobutane has the same retention time as the normal isomer, anomalous results Ivill be obtained. The retention times of some butyl alcohols and chlorobutanes a t two different temperatures are listed in Table VII. The retention times of 1-chlorobutane and isobutyl alcohol (2methyl-1-propanol) differ by 15 seconds; thus, they cannot be differentiated easily by this method. The isobutyl alcohol content of the reactant, n-butyl
ACKNOWLEDGMENT
The authors are indebted to W. J. Lambdin for performing the mass spectrometric analyses of the 1,2dichloroethane samples. LITERATURE CITED
:;, in "Vapour Phase Chromatography, D. H. Desty, ed., p. 256, Academic Press, SeTT York, 1957. (2) Green, S. K., Zbid., p. 388. (3) James, A. T., Martin, A. J. P., Annliist 77. 915 (1952'1. (4j-jaGes, A'. T., h a r t i n , A. J. P., iochenz. J . 5 0 , 679 (1952). A (5) James, A. T., Martin, A. J. P., G. H., Zbid., 5 2 , 238 (1952). OL 1 ( 6 Smith, ) James. D. H.. Phillius, C. S. G., J. 4 12 10 8 Chem. doc. 1953, 1600.* ' Retention time, minutes ( 7 ) Percival, IT. C., ASAL. CHEM. 2 9 , m 9.57'1 -" (1 ,-"-. ,. Figure 4. Chromatogram of refined 1 (8) Pollard, F. H., Hardy, C. J;: in chlorobutane 'Vapour Phase Chromatography, D. H. Desty, ed., p. 115, Academic Press, A. n.Butyl alcohol (1) 1) Evans, D. E. hl., Tatlom, J.
\
,
-
8.
1 -Chlorobuiane ( 1 6)
alcohol, normally is insignificant and is removed in the refining process. Therefore the method is essentially free of
Ivln,
LO",.
ebe, A. K., J. Phys. Chem. 60, 685 RECEIVEDfor review dugust 22, 1958. Accepted January 12, 1959.
Gas Chromatographic Analyses of Products from Aldol Condensations G. W. WARREN, W. J. LAMBDIN, J. F. HASKIN, and V. A. YARBOROUGH Development Department, Union Carbide Chemicals Co., Division of Union Carbide Corp., South Charleston, W. Va.
b In the aldol condensation of various aldehydes, a mixture of products often i s formed. To determine the optimum operating conditions for satisfactory laboratory processes, reliable methods were needed for the analysis of the different mixtures. A mass spectrometric procedure was developed, but was discarded in favor of a more rapid and precise gas chromatographic method. Gas chromatographic methods were developed for the analysis of three different aldol condensations: acetaldehyde with butyraldehyde, isobutyraldehyde with butyraldehyde, and isobutyraldehyde with propionaldehyde. The major com1016
ANALYTICAL CHEMISTRY
ponents in these mixtures can be analyzed with a precision of about f 4 Q / o o f the contained amounts with limits of detectability which vary from about 100 p.p.m. for butyraldehyde to 1000 p.p.m. for 2-ethyl-2-hexenal.
T
dimerization of aldehydes, or cross-condensation if two different aldehydes are involved, is a very general type of reaction known as the aldol condensation. Usually, the initial product loses water giving a n a,p-unsaturated carbonyl compound. I n crosscondensations, in particular, a mixture of products often is obtained. Gas HE
chromatographic procedures, as developed by James and Martin (3-5) and Phillips (8),have been used quite successfully for the separation of many mixtures ( 1 , 2 ,6) and for the analysis of other nonhomologous mixtures of oxygenated compounds (9) ; therefore, use of this new analytical technique to resolve the products of aldol condensations of 3- and 4-carbon aldehydes appears attractive. Mass spectrometric methods have been used (Y), but they are not as rapid or precise and are not as easily and economically converted to on-stream analyses of products. Analytical differentiation of molecular isomers is accomplished mass spectro-
metrically by use of the distinctive fragments which are produced by electron bombardment. Simultaneous equations often must be used; whereas, in gas chromatograms, separate bands free from interference usually can be obtained if the proper operating conditions are used. Gas chromatographic procedures, therefore, seem preferable.
P
I
PROCEDURE
I
30
I
f
28
26
I
24
I
I
22
20
I
' ' 14
I
I
I
I
I
I
I
12
10
8
6
4
2
0
Retention t i m e , minutes
Figure 1 . Chromatogram of products from aldol condensation of acetaldehyde and butyraldehyde Column, di(2-ethylhexyl) phthalate (30%) on Celite 545, 2 meters X 4.7 mm. Temperature, 140' C. Flow, 100 cc. of He per minute. Recorder ottenuation in parentheses A. 2-Ethyl-2-hexenal ( 4 ) E. Unidentified ( 4 ) F. Butyraldehyde ( 4 ) B. 2-Vinylcrotonaldehyde (4) C. 2-Hexenal ( 4 ) G. Unidentified ( 4 ) D. 2-Ethylcrotonaldehyde ( 4 )
Table 1.
Gas Chromatographic Analysis of Aldol Condensation Products of Aldehyde Mixtures
AV.,
yo by K t .
Component
Approx. Minimum Detectable Concn., P.P.M.
Av. Dev., 70 of Contained d m t .
4 cetaldehyde and Butyraldehyde 44 9 3 2 27 4 2 3 24 3 4 8 2 7 12 3 0 3 20 0 0 4 25 0
2-Et hylcrotonaldehyde 2-E thyl-2-hexenal 2-Heuenal 2-Vinylcrotonaldehyde Butyraldehyde Unidentified"
150 1000 200 500 100
...
Ieobutyraldehyde and Butyraldehyde 30 0.5 20 0.5 36 1.4
Water Ethyl alcohol Isobutyraldehyde Butyraldehyde Isobutyl alcohol 2-E thyl-4-methyl-2pentenal 2-Ethyl-2-hexenal Unidentified
800
1000
1000
1000 1000
28 20
0.9 0.5
12.0 52.5 1.7
2.9 2.5
500 500 500
17.6
The Perkin-Elmer Model 154 (modified) Vapor Fractometer was used for all determinations. For the analysis of products from the aldol condensation of acetaldehyde and butyraldehyde, the column, 2 meters by 4.7 mm. in inside diameter, consisted of di(2-ethylhexyl) phthalate (30%) on Celite 545 (80 to 100 mesh) and was operated a t 140' C. with a flow rate of 100 cc. per minute of helium. For the aldol condensation of isobutyraldehyde with butyraldehyde, the products were analyzed with the 2meter column described. The same operating conditions n-ere used. The products from the aldol condensation of isobutyraldehyde with propionaldehyde were analyzed n i t h a 2meter column, 4.7 mni. in inside diameter, paraffin (30%) adsorbed on Celite 545 (80 to 100 mesh), with a helium flow rate of 100 cc. per minute a t 120' C. Samples (0.01 ml.) were introduced with a hypodermic syringe and a 16-mm. S o . 27 hypodermic needle through a silicone-rubber diaphragm into the stream of helium. The detector response was recorded on a Brorrn 0- to IO-mv. strip chart recorder. The signal was attenuated b y a k n o m factor t o maximize each deflection on the chart paper. The base line rvas not changed when the signal was attenuated. The areas under the curves were measured with a n Ott compensating polar planimeter. Components were identified by comparison of retention times and by mass spectrometric analysis of fractions trapped at the proper time from the effluent of the chromatograph.
Isobutyraldehyde and Propionaldehyde Propionaldehyde Isobutyraldehyde 2-Methyl-2-pentenal 2,4-Dimethyl-2-pentenal Two different peaks.
30
0.5 4 0
500 800 500 800
10
70.0
2 0
6 5
25 5
Figure 2. Chromatogram of products from aldol condensation of isobutyraldehyd e and butyra Idehyd e E 0
++
'd VI
uTI aJc rla
'H aJh
5
k
k k c, O .d W
u n
C
aJk
V
d
r2
I
32 30
28
26
24
22
20
18
16
14 12 10
R e t e n t i o n time, m i n u t e s
I
l
8
6
l
4
l
)
2
0
Column, di(2-ethylhexyl) phthalate (30%) on Celite 545, 2 meters X 4.7 mm. Temperature, 140' C. Flow, 100 cc. of He per minute. Recorder ottenuation in parentheses A. 2-Ethyl-2-hexenal (2) 6. 2-Ethyl-4-methyl-2-pentenal (2) C. Unidentified ( 1 ) D. Isobutyl alcohol E. Butyraldehyde F. Isobutyraldehyde (1 1 G. Ethyl alcohol H. Water 1. Air
4 VOL. 31, NO. 6, JUNE 1959
1017
About 30 minutes of instrument time is required per analysis. DISCUSSION AND RESULTS
Butyraldehyde
]I
.4cetaldehyde
3-H ydroxyhexanal
alkali
//
C3H,-CH=CH-C
+
\
HzO
H
2-Hesenal 3-Hydroxy-2-ethylbutyraldehyde
It
H 2-Ethylcrotonaldehyde CpH5
0
CH,-C\
//
\
0
+
alkali
CH3--C
H
H'
Acetaldehyde
Aldol (3-hydroxy-
Acetaldehyde
butyraldehyde) II'
0
Ha0
I' CH,-CH=CH-C
/
+ CHz=CH-CH,-C 3-Butenal
Crotonaldehyde (2-butenal)
0
GHi-C
\H Butyraldehyde
0
+
+ H,O
\H
\€I
/
0
//
C8H7-C'
OH
alkali
H' Butyraldehyde
3-Hydroxy-2-ethylhexanal
It 2-Ethyl-2- hexenal 0
0
Acetaldehyde
3-Butenal
3-Hydroxy-2-vinylbutyraldehyde
CH3-CH=C-C
/
// \
0
CH=CH, H 2-Vinylcrotonaldehyde 1018
*
ANALYTICAL CHEMISTRY
+ HzO
The analysis of a composite of fractions from a distillation of products from the aldol condensation of acetaldehyde and butyraldehyde is given in Table I. The following major reactions are possible: The minimum detectable concentrations of these possible products (under the conditions used) generally vary with the retention time, increasing with increasing time (9). As shown in Table I, the minimum detectable concentration yaries from 100 p.p.m. for butyraldehyde to 1000 p.p.m. for 2-ethy1-2hexenal. A typical chromatogram is given in Figure 1. The results of a gas chromatographic analysis of the reaction product from the cross-condensation of isobutyraldehyde and butyraldehyde are also given (Table I). Reactions of a type similar to those given in Equations 1 to 4 are possible. The resolution of the two 8-carbon aldehydes, 2-ethyl-4-methyl2-pentenal and 2-ethy1-2-hexena1, is excellent; each of these isomers can be detected in a concentration of 500 p.p.m. Resolution of other components is fair; but, because of their low concentration, each is determined within the normal limits of precision. Increased resolution of the lower-boiling components could be obtained by using a lower column temperature; however, the conditions used for this method are optimum with respect to resolution of all components. 2-Ethyl-4-methyl2-pentenal and 2-ethyl-2-hexenal vere analyzed with a precision of +2.9 and 3 ~ 2 . 5 % respectively, ~ of the contained amount. The limits of precision for all components are listed in Table I; a typical chromatogram is shown in Figure 2. The results of a gas chromatographic analysis of a sample of reaction product from the aldol condensation of isobutyraldehyde and propionaldehyde (Table I) show that 2-methyl-2-pentenal and 2,4-dimethyl-2-pentenal can be analyzed with a precision of 1 2 . 0 and +G.57,, respectively, of the contained amount. Figure 3 is a representative chromatogram obtained for analysis of these condensation products. Calibration factors were used to convert area to weight per cent. These factors ranged from 0.7 for water t o 1.OG for 2-ethyl-2-hexenal, For best results these factors would have to be redetermined on any other instrument used for this analysis; therefore, they are not included in the data given here. Use of the proper operating conditions-e.g., higher temperatures, multiple columns, different flow rates-
should make possible the analysis of higher-boiling and more complex aldol condensation products with equal speed and precision. LITERATURE CITED
( 1 ) Desty, D. H., “Vapour Phase Chromatography,” Academic Press, S e w York, 1957. (2) James, A. T., Biochenz. J . 52, 242 I 1 8.52’1 \----,
I
I
26
24
I
22
20
I
18
I
16
I
I
14
I
12
10
I
I
I
I
I
8
6
4
2
0
R e t e n t i o n time, m i n u t e s
Figure 3. Chromatogram of products from aldol condensation of propionaldehyde and isobutyraldehyde Column, paraffin on Celite 545, 2 meters X 4.7 mm. per minute. Recorder attenuation in parentheses A. 2,4-Dirnethyl-2-pentenal (8) B. 2-Methyl-2-pentenal (8) C. lsobutyraldehyde ( I )
Temperature, 120’ C.
D.
E.
Flow, 1 0 0 cc. of He
Propionaldehyde (1 ) Air
Aiaalyst (3) James, -4.T., Martin, A. J. P., A. T., Martin, A. J . P., Bio77, 915Is,(1952). (4)Jame , chem. J. 50, Si? (1953). Smith, ( 5 ) James, A . T., 1\lartin. -2. J. P.. G. H., Ibid., 52, 238 (1952). ( 6 ) James, D. H., Phillips, C. S. G., J . Chem. Soc. 1953, 1600. ( 7 ) Lambdin, IT.J., Kourey, R. E., Yarborough, V. A., unpublished data. (8) Phillips, C. S. G., Discussions Faraday Soc. 1949. s o . 7 . 241. (9) ITarren,‘ G,-JT.,’Haslrin, J. F., Kourey, R. E.,. Tarborough, T’. h., A h - a ~ . CHEM.,submittc:d for publication.
RECEIVED for review -4ugust 22, 1958. Accepted January 12, 1959.
Analysis of Mixtures of Amino Acids by Gas Phase Chromatography C. G. YOUNGS Prairie Regional laboratory, National Research Council o f Canada, Saskatoon, Sask., Canada
b A number of amino acids were quclntitatively separated by gas phase chromatography, The amino acids were converted to their N-acetyl butyl esters prior to chromatography and the esters fractionated on a column of firebrick coated with a hydrogenated vegstable oil. Quantitative determinations of glycine, alanine, valine, leucine, isoleucine, and proline were obtained both for synthetic mixtures of the pure acids and for protein hydrolyzates. Several additional peaks were obtained during the analyses of the protein hydrolyzates. These materials, which were probably additional constituent amino acids, came off the column after the above six amino acids.
G
PHASE chromatography has made possible the quantitative separation of the components of numerous complex mixtures. The rapidity, ease of operation, and the small sample size required by this new technique have opened many new avenues of research in fields such as fats and oils. I t s extension to the
AS
separation of mixtures of amino acids would greatly facilitate much of the work being done on proteins from a nutritional, biochemical, or chemical aspect. The separation of amino acids by gas phase chromatography is, in effect, trvo problems. The acids must be converted quantitatively to a volatile derivative and a suitable column and operating conditions must be found n hich give a sharp separation of these derivatives. K o r k is being done on the conversion of amino acids to aldehydes with ninhydrin and separation of the aldehydes by gas phase chromatography (2, 7 , 8 ) . Bayer. Reuther, and Born ( 1 ) have prepared the mcthjl esters of several amino acids by the Fischer method (6) and qeparated the esters by gas phase chromatography. None of these reports give quantitative data. Both the preparation of aldehydes and esters pose difficult problems in quantitative recovery of the derivatives. Cherbuliez et a / . (3, 4 ) have reported that LYacetylation of amino acid esters overcomes a number of the difficulties inherent in the Fischer ester distillation
method and this paper deals with the gas phase chromatography of these latter derivatives. EXPERIMENTAL
The X-acetylated ethyl esters of a mixture of glycine, alanine, valine, and leucine were prepared and injected into a Beckman GC2 chromatographic unit, Reasonable retention volumes were obtained a t 220’ C. with a good separation and symmetrical peaks. The .Y-acetylated ethyl ester of glycine tended to crystallize from the mivture of acetylated esters and a homogenous sample could not be taken for injection. This problem was overcome by conversion to the butyl esters of the amino acids rather than the ethyl esters. K h e n this was done the derivatives of all the amino acids remained as a viscous oil and by using the .&--acetylated butyl esters, a satisfactory chromatographic procedure was developed. Of the columns tested. the one consisting of hydrogenated vegetable oil on firebrick gave the best resolution with these derivatives. Preparation
of
N-Acetyl
VOL. 31, NO. 6, JUNE 1959
Butyl
1019
