measurable loss or change in partition coefficient. No properties of the pigments were changed by passage through the silica gel-methanol columns. When the fresh solutions of the isolated pigments were passed through the columns, the recovery exceeded goy0. When old solutions, kept 2 to 3 weeks at 4” C., were passed through the column, a 20 t o 40y0 loss 11-as noted. The nature of this loss is not understood. Apparently the pigment did not remain on the column. When these old solutions in petroleum mere shaken with methanol in determining partition coefficients, the sum of the pigments in the epiphase and hypophase was 20 to 40% less than the total pigment added. The method described is an example of true partition chromatography. When fresh petroleum extracts of grapefruit carotenoids xere passed through silica gel columns, treated only with petroleum. considerable pigments were lost and distinct separation into fractions according to partition characteristics was not achieved. The silica gel probably holds the methanol as a n immobile phase, and the pigments partition between the methanol and the mobile phase as they pass through the column. LITERATURE CITED
(1) Curl, A. L., Food Research 21, 689-93
(1956).
Table II.
Recovery of Carotenoids from Silica Gel-Methanol Column
Partition Coefficient, Petroleum- Petroleum- HexaneMethanol Methanol 95% Ratio ( 8 ) Name Methanol“ Ratiob Individual Pigments 1oo:o ,%Carotene Inf. 1oo:o Lycopene Inf. 1oo:o 1oo:o 82: 18 Cryptoxanthin 4.46 82: 18 Hydroxy-or... 73 :27 carotene 2.68 Cryptoflavine 1.74 64:36 12:88 10:90 0.11 Lutein 2:98 ... Violaxanthin 0.05 Recovery from a Mixture ... p-Carotene ... ... ... Lycopene .,. ... Cryptoxanthin ... ..* ... Lutein ... ... Average of several determinations. Kearest whole ratio. .
I
.
.4dded, Y
50.7
111.6 36.0 46 ,..
184 331
50.7 111.6 36.0 26.8
Recov- Recovered, ered, Y
%
49.8 104,s 33.6
98.2 94.4 93.4
43
...
94 0 ... 95.0 98.4
50.6 111.4 33.6 26.8
99.8 99.7 93.4 100 0
174 326
0
Curl, A. L., J . Aar. Food Chem. 1, 456-60 (1953). Curl, A. L., Bailey, G. F., Food Research 20, 371-6 (1955). Curl, A. L., Bailey, G. F., Zbid., 22,
__
63-8- f~ 1%7).,~
.
-
Curl, A. L., Bailey, G. F., J . Agr. Food Chem. 2 , 685-90 (1954). Goodwin, T. W., “Carotenoids” in Vol. I11 of “Modern Methods of Plant Analysis,” pp. 272-311, K. Peach and M. V. Tracey, eds., Springer Verlag, Berlin, 1956. ( 7 ) Karrer, P., Jucker, E., “Carotenoi ds,” Elsevier, Amsterdam, 1950.
(8) Petracek, F. J., Zechmeister, L.,
k i A L . CHEJI. 28, 1484-5 (1956). (9) Petracek, F. J., Zechmeister, L., J . Am. Chem. SOC.78, 1427-34 (1956). (10) Strain, H. H., “Chromatographic Adsorption Analysis,” Interscience, X e x York, 1942.
RECEIVEDfor review July 31. 1957. Accepted February 24, 1958. The U.S. Department of Agriculture does not recommend the products of one company over those of any othrrs engaged in the same business.
Chromatography of Esters on FIorisiI Detection as Ferric Hydroxamates F. B. O’NEAL and JACK CARLTON lyman Chemical laboratories, Georgia Institute of Technology, Atlanta, Ga.
b The chromatographic behavior of esters has been investigated employing Florisil, silicic acid, and alumina as adsorbents. Only the former allowed reproducible response of the various esters to the reagent adapted for their detection. This reagent, hydroxylamine-ferric ion, gave easily visible reaction products when applied to 0.01M solutions of the esters on Florisil.
A
series of ethyl esters through ethyl decanoate was used in the study of the chromatographic behavior of some fatty esters. The iron hydrosaniate test proved suitable for the detection of esters on Florisil, but i t failed to give reproducibly the characHOMOLOGOUS
teristic reaction product when applied t o other columns. When the ethyl esters were chromatographed on Florisil, the R values generally increased as the number of carbon atoms in the acid chain increased. This was particularly regular for the lower members of the series. When acetates (ethyl through amyl acetate) were chromatographed, there was also a steady increase in R value. EXPERIMENTAL
Materials and Reagents. Florisil (Floridin Co., Tallahassee, Fla.), 100 mesh, was ground for 3 hours in a ball mill and used without further treatment. Adsorbent characteristics, in t h e manner of LeRosen a n d his
associates (4) are: -4 = 7000, DH = 150. Silicic acid, hlerck reagent. Aluminum oxide, hlerck reagent, for chromatographic purposes, ground to pass 170-mesh sieve. Esters employed were Eastman white label or a n equivalent grade prepared and purified in this laboratory. Hydroxylamine hydrochloride, 12.5 grams dissolved in 100 ml. of methanol. Sodium hydroxide, 12.5 grams dissolved in 100 ml. of methanol by refluxing. After 5 ml. of this solution were mixed with 5 ml. of the hydroxylamine solution, sodium chloride was filtered off and the filtrate x-as used as preliminary reagent in the ester test (reagent A). It was stable for approximately 4 hours. Ferric perchlorate was prepared by VOL. 30, NO. 6, JUNE 1958
1051
NUMBER OFCARBON ATOMS
Figure 1. Variation of R value with hydrocarbon chain length for the homologous series of ethyl esters
NUMBER OF CARBON ATOMS
Figure 2. Variation of R value with hydrocarbon chain length for a short series of acetates
Adsorbent, Florisil; developer, benzene Adsorbent, Florisil; developer, benzene
Table 1.
R Values for the Homologous Series of Ethyl Esters Ester R Ethyl formate 0.23 Ethyl acetate 0.16 Ethyl propionate 0.18 Ethyl n-butyrate 0.20 Ethyl n-valerate 0.21 Ethyl n-hexanoate 0.19
Ester Ethyl n-heptanoate Ethyl n-octanoate Ethyl n-nonanoate Ethyl n-decanoate Ethyl laurate Ethyl palmitate
R 0.23 0.30 0.30 0.30 0.31 0.35
Table II. R Values for Acetates, Esters of Polybasic Acids, and Unsaturated Esters
Ester Ethyl acetate n-Propyl acetate n-Butyl acetate n-Amyl acetate Benzyl acetate Ethyl oleate Ester Ethyl cinnamate Diethyl oxalate Diethyl malonate Triethyl citrate Diethyl maleate Di-n-butyl phthalate
R 0.16 0.22 0.26
0.31 0.25 0.28
R 0.17 0.08
0.08 0.08
0.12 0.11
Table 111. R Values of Some HaloEsters and Branched-Chain Esters
Ester Isobutyl propionate Methyl chloroacetate a-Chloroethyl chloroacetate 0-Chloroethyl chloroacetate n-Propyl chloroacetate yChloro-n-propyl chloroacetate Ester Is0 ropy1 chloroacetate
R 0 27 0 34 NR5 0 34 0 46 0 35
R 0 35 0 42 0 46 XR NR 0 85
P-Ckloroisopropyl ohloroacetate Ethyl a-bromopropionate Ethyl a-bromoisobutyrate Ethyl isovalerate Ethyl trichloroacetate XR,no reaction observed on column. 5
1052
ANALYTICAL CHEMISTRY
dissolving 0.8 gram of C.P. iron wire in 10 ml. of warm 70% perchloric acid. After the solution was cooled and made up to 100 ml. in a volumetric flask with ethanol, 5 ml. were added to 1 ml. of 70% perchloric acid. This mas the second streaking solution (reagent B). Procedure. Borosilicate glass chromatographic tubes, KO. I , precision tapered (Scientific Glass Apparatus Co., Bloomfield, 1c-. J.), were packed with adsorbent t o a height of 75 =!= 5 mm. under a n applied vacuum of approximately 5 mm. Solutions of t h e various esters were prepared t o provide 0.01M concentrations in benzene, and 0.5-ml. aliquots were taken as the proper sample size. The sample was introduced onto the top of the column and worked into the column with three or four small increments of the developing solvent, benzene. The space above the adsorbent was filled with benzene and development proceeded under the vacuum until the solvent reached the bottom of the column. The tube Tvas disconnected, and the adsorbent was extruded and streaked with the reagent. Bfter reagent A was applied, the column was baked for 2 minutes under an infrared lamp. The column was then overstreaked with reagent B. Purple zones appeared where the ester was adsorbed on the column. RESULTS A N D DISCUSSION
No satisfactory streak reagent has been reported for adsorption chromatographic techniques for esters. I n the initial search for a reaction which might be developed into a suitable streak reagent, two reactions listed by Feigl were considered for adaptation. The first involved the colored molybdate-xanthate complex (1); while designated for primary and secondary alcohols, it could detect esters because they are partially saponified under the conditions of the test. However, this test required streaking the column four times, which itself was undesirable. Csing silicic acid as adsorbent, various orders of streaking with the detection system produced no positive results. The hydroxamate-ferric ion complex
had been successfully used by Feigl for the identification of esters ( 2 ) and had been investigated by Goddu, LeBlanc, and Kright (3) as a reagent for the quantitative estimation of esters spectrophotometrically. Employing reagents very similar to those suggested by the latter group, various adsorbents were used to determine the chromatographic behavior of esters. Silicic acid and alumina both proved unsatisfactory when the reagent was unable to bring out the position of the ester on the column. Possibly sodium hydroxide, present in the reagent to saponify the ester, was lost by reaction with the adsorbent. Florisil gave reproducible results, and R values for the various esters were determined easily. The lower members of the ethyl ester series did not require as much heat, but 2 minutes under the infrared lamp was the best average to bring out a color reaction among all the esters. The heat lamp was set 33 nim. above the columns and a temperature of 135' C. was measured a t its surface. Table I lists the R values of the homologous series of ethyl esters used. Table I1 lists the R values of a short series of acetates, as well as R values for several esters of di- and tribasic acids. The esters derived from di- and tribasic acids were all strongly adsorbed a t the top of the column and had R t values (Rt refers to the trailing edge of the zone) of zero. Unsaturation in the aliphatic chain decreased the R value, as in the case of ethyl cinnamate and ethyl oleate (Table 11). Figures 1 and 2 summarize graphically the data in Table I and the short series of acetates in Table 11, respectively. In the ethyl ester series, ethyl formate (the first member) is displaced slightly and ethyl hexanoate is below the expected R value. There is a general leveling off after the octanoate is reached. Ethyl laurate, ethyl palmitate, and ethyl oleate were the only esters used with hydrocarbon chain lengths greater than ten carbon atoms. Figure 2 indicates a very regular increase in R value m-ith increase in the hydrocarbon chain of the alcoholic half of the molecule.
Table I11 lists cstw-. art1 their R values, which were uscd to tleterniine whether or not branching or substitution in the hydrocarbon chain affected adsorption on the column. Because the carbonyl oxygen is considered the principal adsorbing atom, through hydrogen bonds to electron acceptor sites on the adsorbent, the presence of bulky atoms or groups of atoms on iicighboring carbon atonis could partiall:- block adsorption of the adsorptive on the column. The chlorine atom could be involved in steric hindrance of the carbonyl oxygen, it could itself be adsorbed, or by virtue of its electronegativity it may exert inductive influences on the adsorbing atom which n-oultl lessen
affinity of the molecule for the adsorbent. The size of the chlorine atom does not suggest any particular potential for it as a blocking atom, nor is there evidence that the chlorine atom has more than very slight affinity for adsorption columns. Electronegativity of the chlorine atom would reduce the electron density about the carbonyl oxygen, thus lowering adsorption of the adsorptive. This is reflected in the R values measured for the halogenated esters, and i n particular ethyl trichloroacetate. ACKNOWLEDGMENT
The authors wish to express their
appreciation to the Research Corp. for financial assistance niaking this work possible. LITERATURE CITED
(1) Feigl, F.,, “Spot Tests in Organic Analysis,” p. 173, Elsevier, Ken-
York, 1957. ( 2 ) Zbid., p. 237. (3) Goddu, R. F., LeBlanc, F., Wright, C. hl., ANAL. CHEW 27, 1251
(1955).
(4) LeRosen, A. L., Monaghan, P. H., Rivet, C. -4,, Smith, E. D., Zbtd., 23, 730 (1951).
RECEIVEDfor review August 14, 1957. Accepted January 27, 1958.
Use of Substitute Standards in Infrared Differential Spectrophotometry W. H. WASHBURN and M. J. MAHONEY
Abbott laboratories, Norfh Chicago, 111. ,The accuracy in infrared differential analysis for a specific impurity is often limited b y the presence of small amounts of the impurity in the standard material. This limitation can b e overcome to a great extent by substituting for the reference standard a known pure compound of different chemical structure, but having nearly identical absorption characteristics in the area of analytical interest. The important factors in selecting a proper substitute are discussed.
can be largely eliminated if another compound, obtainable in a pure state and possessing the same absorption characteristics as the major component in the region of analytical interest, is substituted for it in the reference beam. The choice of a suitable substitute reference is dependent upon a number of factors. The relative importance of these factors was investigated and is reported in this study. RECOMMENDED PROCEDURE
D
.IXALTSIJ has frequently been applied to the determination of small amounts of known impurities in relatively pure materials. I n the differential method. employed when there is overlapping of absorption bands of major and minor components, the absorption due to the major component is essentially subtr:tcted from the total absorption. The remaining absorption is due to the minor component (1-9). If, however, sonie of the impurity to be measured-for example, lyc-is prebent in the niaterial to be used as standard, all determinations based on this standard will be 1% lower than the true value. The accuracy is thus dependent on the availability of a standard sample free of the impurity to be measured. Frequently, due to difficulties in purification, such a standard is not available. This problem is often encountered when the major Component and the impurity are isomeric. This limitation of differential analysis
IFFERESTIAL
-4 11 qualitative and quantitative measurements were obtained using a PerkinElmer Model 21 double beam infrared spectrophotometer equipped with sodium chloride optics. \Then a reference library of spectra is scanned to select a possible substitute standard, i t is necessary to consider the following points. The peak absorption of the substitute should be within 1 0 02 micron of the peak absorption of the major component. The slope of the absorption band of the substitute standard should be closely parallel to that of the absorption band of the major component through an interval a few hundredths of a micron on either side of the analytical peak of the minor component. These rather close specifications require that one obtain the pure reference compounds and check the absorption requirements on one’s own spectrometer. Solvent, cell thickness. and concentration, if not already decided upon, should be established a t this point as though the determination n-ere to be a conventional differential analysis (1-9).
A further examination of the possible substitutes is now undertaken by espanding the abscissa or wave length scale from the usual 2 inches per microii to 16 inches per micron. With nothing in the reference beam, the sample (standard in question) is now run through the interval of interest, a t the concentration and cell thickness determined above. I n the case where the impurity peak lies on the short wave length side of the major band, this interval is roughly from a point 0.10 micron shorter wave length than the peak of the minor component, to the peak of the major component. When the impurity peak lies on the long wave length side of the major band, the interval of interest is reversed. Next, the several substitutes being considered are superimposed on this run, a t concentrations such that all substitutes absorb somewhat less intensely than the sample (about 0.10 t o 0 . 2 5 absorbance unit less than the sample in the range of 0.02 to 0.03 micron on either side of the peak absorption point of the minor component). The best substitute will be the material absorbing most nearly parallel to the sample run over this 0.04- to 0.05-micron portion of the interval, and also absorbing more strongly than the sample a t a point about midway between the minor component peak and the major component peak of the sample. The slight undercompensation of the major component from 0 . 0 2 t o 0.03 micron on either side of the peak absorption of the minor component ensures against loss of the minor component absorption band in the differential curve. The slight overcompensation of the major component at a point a few hundredths of a micron VOL. 30, NO. 6, JUNE I958
1053
