were measured by the base line technique and combined with the known composition data to obtain the K factors for subsequent analyses. Absorption bands at 4.45 and 5.9 microns were used for acrylonitrile and methyl isopropenyl ketone, respectively. No correction was made for the ethyl vinyl ketone impurity in the methyl isopropenyl ketone, as its absorption coefficient is probably very close to that of methyl isopropenyl ketone. The methyl isopropenyl ketone percentage then, represents the total of methyl isopropenyl ketone plus ethyl vinyl ketone. For butadiene, the sum of the two bands at 10.35 microns (trans) and 10.90 microns (vinyl) gave more consistent results than the 10.35 micron band alone, probably indicating some variation in the ratio of 1,4-addition to 1,2-addition of butadiene in the polymerization process. Using the sum of the two bands gives a firstader correction for such variations. (It k here assumed that the ratio of trans- to Cis- 1,4 polymerized butadiene i: essentially const.ant.) The K factors thus determined appear in Table VI. The values for K M vary considerably more than those for Kv. However, the composition measurements for acrylonitrile, are probably more accurate than those for methyl isopropenyl ketone, so a greater fluctuation is expected. Another source of error may be in variations of the spectrometer zero which would affect the deep C 4 absorption of methyl isopropenyl ketone much more than the weaker C=N. Unknown latex samples were prepared and their infrared spectra scanned in the same manner as the standards. Although the film thickness does not enter into the calculation directly, more accurate results are obtained if the thickness is held within reasonable limits. For this reason the film thickness was adjusted by stretching, or the 6lms were recast, until the absorbance of the C=O absorption at 5.9 microns
-
Table VI.
K Factor Determinations
Ye
% ButsMIK dlenea (C14 (DifDu- Trac- ferer) ence) K v Ky Run mas 1 23.8 14.6 60.2 0.356 2.96 0.364* 3.01* 2 19.6 18.6 59.8 0.341 3.24 0.365 3.54 3 15.2 22.2 60.6 0.353 3.52 0.366 3.47 4 15.9 14.0 68.6 0.350 3.29 0.345 3.26 5 9.8 10.0 79.1 0.358 3.31 0.362 3.35 6 24.5 23.8 49.4 0.354 3.01 0.338 2.95 Av. (1.354 3.24 a Butadiene figure corrected for a small percentage of inorganic material found in $dyer. b Fdm of each standard were cast in dudicate. K factors obtained from both SlmS are given. Table VII.
Infrared Analysis of Pilot Plant Latex
Butadiene,
Sample % A 59 B 6 0 c 59 D 60.5 E 59.0 F 61
G 6 0 H 61 I 58
J K L
59 6 0 61
VCN, %
21 20 21.5 20.5 21 19 20 20
22 21 19 19.5
MIK, % 20 20
19.5 19 20 20 20 19 20 20
21 19.5
was between 0.7 and 1.0. In som2 cases where films could not be removed easily from glass, they were cast on silver chloride p l a h and s c ~ n n e ddirectly. Table VI1 shows a typical set of analyses on a series of pilot plant runs in which
Table VIII. Reproducibility of Analysis of a Typical Latex
Film No.
Buta-
diene
VCN, k
hlIK, %
5% 1
2 3 4 5 6
60.4 60.3 60.5 61.1 60.0 602
19.6 19.3 19.2 i9.5 19.8 19.2
20.0 20.4 20.3 19 4 203 20 6
the monomer charge was 60% butadiene acrylonitrile, and 20% methyi isopropenyl ketone. Because infrared absorption was the only analytical method used to analyze the pilot plant s%mples,thcre wcre no direct data indicating the accuracy of tne method. However, considering the probable errors in the K factors and errors in measurement on the spectra it wems probable t h s t the butadiene prrcentages are within =kayoof tlic nc.tud value and thc acrylonitrile and nwtliyi isopropenyl ketone figures within f1%. Six analyses of a single mmple shonn in Tab!e VI11 indicate that the reproducibility of the method is within tht: estimated error. LITERATURE CITED
(1)Brown, S. C., Miller, W. W., Rev. Sn’. Znstr. 18,496 (1947). (2)Eidinoff, M. L., ANAL. CHEM.22, 529 (1950). (3) Govans, W.J., Clark, F. E., Ztrid., 24, 529 (1952). (4) Hekche;, 1,. W.,Ruhl, H. D., Wright, N., J. Opt. Soc. Am. 48,36 (1958). (5) Kemp, A. R., Peters, H., ANAL. CHEM.15,453 (1913). (6) Kobeko, P. P., Moskvina, E. J., J. A w l . Chem. 19, 1143 (1946). (7) Pep, J. J., Kniel, I., Czuba, M., Jr., ANAL.CHEM.27,755 (1955). (8)Van Slyke, D. D., Plazin, J., Weisiger, J. R., J . BioZ. Chem. 191,239 (1951). RECEIVEDfor review March 23, 1959’ Acce ted July 1, 1959. Division of Rubber &emistry, ACS, Los Angeles, Calif., May 1959.
Qua ntita tive Determination of Reduc ing Suga rs after Separation by Paper Chromatography ATHINEOS J. PHlLlPPU laboratory of Physiology, University o f Athens, Athens, Greece .A method is described for the quantitative determination of reducing sugars after chromatographic separation. The experimental error of the method amounts to 0.8 to 5.470, depending upon the quantity of the sugar.
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methods have been described recently for the quantitative
ANY
determination of reducing sugars after separation by paper chromatography. The method now proposed has the advantages of simplicity, accuracy (the coefficient of variation being 0.8 to 5.4% depending upon the quantity of the sugar), and sensitivity; as little as 20 y of glucose, galactose, and fructose and 40 y of lactose can be determined.
EXPERIMENTAL
From 0.001 to 0.001 ml. of the aliquot containing the reducing sugars to be determined is dropped on two separate filter papers. After a simultaneous development, the regions of the spots are identified by spraying one of the filter papers with the detection solvent of Bryson and Mitchell (1). The correVOL. 31, NO. 10, OCTOBER 1959
1615
~
RESULTS A N D DISCUSSION
Table 1.
-
Detn. 1 2 3 4 5 6
Mean Std. dev. Coeff. of variation, %
Reproducibility Experiments
(Glucose concentrations in micrograms) Absorbance X 100 50 100 150 200 250 300
350
400
450
29 42 87 111 139 161 191 220 243 133 164 197 219 248 31 45 81 107 134 192 224 247 161 81 107 28 41 139 196 223 248 161 29 44 85 113 112 134 164 195 220 248 87 31 41 137 193 220 248 167 83 110 29 45 39 43 83 110 136 163 194 221 247 f1.55 f 1 . W f2.76 f2.54 f2.68 f2.45 f2.37 f2.0 f 2 . 0 5.4
4.4
3.3
sponding regions of the nonsprayed paper are cut and dipped in tubes containing 10 ml. of methanol. The tubes are placed in a water bath at 65" C. for 20 minutes for a complete elution of the sugars from the filter paper. The methanol of each tube is transported in another tube 20 cm. in length and 2.5 cm. in diameter and 3 ml. of the following solution are added: 1-propanol: aniline: orthophosphoric acid = 75:0.5:
2.3
2.0
1.5
1.2
0.9
0.8
0.5 v./v. The tubes are placed in a boiling water bath for 20 minutes. To each tube are added 5 ml. of methanol and the yellow-brown color appearing during boiling is measured in a spectrophotometer at 400 mp. A blank is run on a piece of sugar-free paper dipped into methanol. Standard curves are obtained by plotting concentrations of known a m ple~ against density.
Table I shows the reproducibility of the determinations when 4OO-mp a m ples of known glucose concentration are measured after chromatographic development. The method can be employed for sugar concentrations from 20 up to 500 y with good accuracy by using a Hilger spectrophotometer. The standard deviation and coefficient of variation of other reducing sugars, such as galactose, lactose, and fructose, are approximately the same as those of glucose. The method is generally reproducible and the whole procedure is much facilitated by the simple way of eluting sugar from the filter paper. The accuracy of the method of sugar elution is shown from the coefficient of variation, ranging from 0.8 to 5.4%. LITERATURE CITED
(1) Bryson, J. L., Mitchell, T. J., Nalure 167,864 (1951).
RECEIVED for review December 5, 1958. Accepted June 11, 1959.
Paper Chromatography of the Saturated Fatty Acids M. A. BUCHANAN The institute of Paper Chemistry, Appleton, Wis.
b l n a new procedure for the paper chromatography of the saturated fatty acids, the developer reacts with the unsaturated acids to form products which readily separate from the saturated acids. Hydrogen peroxide and formic acid are added to the usual acetic acidwater developers used for reverse-phase chromatography of the fatty acids. The new procedure permits the separation of small amounts of saturated acids from large amounts of the unsaturated acids, and is suitable for tentative identification of the even-numbered straight-chain acids from lauric acid to lignoceric acid.
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species of wood contain a p preciable amounts of fatty acids, resin acids, and sometimes other acids. r%verse-phase paper chromatography has proved to be a useful tool for the tentative identification and separation of the fatty acids. Stearic, oleic, linoleic, and linolenic acids are readily separated on paper treated with mineral oil ( I ) developed at. room temperature with acetic acid-water (6,7,8),but these conditions are not suitable for the detection of some of the saturated acids which are present in wood extracts. Kaufmanu (2) has reported the following ANY
critical pairs of acids which are not separated by the usual conditions: pafitic-oleic, myristic-linoleic, and h u ric-linolenic. In addition, the saturated acids are often minor components, and the detection of small quantities is difficult. The saturated acids can be separated from the unsaturated acids before chromatography, but this is an added step, and often chromatography is of value for testing the efficiency of the desired separation. Chromatography at -30" C. has been suggested (4, 8) for the separation of the critical pairs, and treatment with bromine has been used (7) to form addition products with the U n s a t u r a t e d acids on the chromatogram. They can then be separated from the saturated acids. Recently, Mangold, Gellerman, and Schlenk (6) reported a new procedure in which peracetic acid is added t o the acetic acid developer. The peracid reacts with the double bonds of the unsaturated acids to form oxygenated products with high R, values. They found the oxidation of the unsaturated acids to be quantitative. In these laboratories, 1 to 1 mixtures of fordaic acid and 30% hydrogen peroxide have been added to the acetic acid developer. Formic acid was used because it reaets
readily with hydrogen peroxide to form the peracid (9, IO). The exact composition of the peracid developers has not been determined, but i t seems likely that they consist of acetic acid, performic acid, formic acid, and water, but some peracetic acid and/or unreacted hydrogen peroxide may be present. Fatty acid mixtures also have been pretreated with peracids, and the resulting mixture has been chromatographed with the usual acetic acid developers. EXPERIMENTAL PROCEDURES
Chromatography with Peracid Developers. Strips of Whatman No. l
filter paper (7.25 X 24 inch) were drawn through a solution consisting of 7 grams of water-washed USP heavy mineral oil in 100 ml. of USP ether ( 1 ) . After hanging in the hood lor a few minutes, the treated papers were ready for spotting. Approximately 0.005 ml. of a solution of the fatty acids in chlorofmn (or any other suitable solvent) was spotted 3 inches from the end of the oil-treated paper. The concentration of the solutions used for spotting was varied according to the proportion of saturated acids in the sample. For the chromatograms reported in Tables I and 11, the solutions of known acids contained 10 grams per liter of the individual satu-
