V O L U M E 25, N O . 10, O C T O B E R 1 9 5 3 minations require k/12 to the time required for assays by the modified Fischer-retort method. Slow deterioration of the f u r ~ due e to periodic heating and cooling can be minimized by maintaining the furnace at a moderate temperature, a8 100" to 150" C., between tests. Breakapt' of borosilicate glasb test tubes is negligible. In 1000 tests no Lest tubes were broken or cracked duriug - the heating- operation; . however, 3 tubes were broken during the cleaning operation. The test tubes may be cleaned by soaking in benzene, folloned by washing with soap and water and a tube brush or st.eel wool, if necessary.
1553 ACKNOWLEDGMENT
The ashistance of W. S. McAuley, J. C. Curran, and J. J. Cummins, lvho c,onducted most of the experimental lvork, is gratefully acknowledged. LITERATURE CITED
(1) Stanfield. K. E., and Frost, I. C., U. S. Bur. Mines, Rept. Inzest. 4477 (1949).
(2) Winchester, Dean C., U. S. Bur. Mines, B d . 729, 13 (1923). RECEIVED for review .\lay 29, 1953. Accepted July 7 , 1953.
Quantitative Determination of 1,2=Glycolsin Mixtures EDWARD A. ADELBERG Department of Bacteriology, University of California, Berkeley, Calif. URISG
the course of investigations on the biosynthesis of
D isoleucine and valine, it became necessary t o determine quantitatively the a,p-dihydroxy acid precursors of these compounds Lvhen present simultaneously in filtrates of Sez~rospora cultures ( 4 ) . The method employed consists of chromatographirig t,he filtrates on paper, eluting the identified dihydrosy compound spots, and determining the eluted compounds colorimetrica1l.y after their conversion to carbonyl-containing fragments by periodate oxidation. I n the present work three compounds were used: o,8-dihSrdroxy--p-eth~lbut?-ric acid, a,p-dihydroxy-pmethylbutyric acid, and tartaric acid. When carried out with the precautions described helou, the method affords 1 0 0 ~ oreof these compounds \vith a maximum error of &lo%. The mcthod should be applicable t o any compound ivhich can be cleaved by periodate t o carbonyl-containing fragments, including otlicr glycols and such conipounds as serine and threonine.
on thc slicctt, so that each spot could be compared with a blank immetiiatcly adjacent t o it. Elutioii Mas accomplished by cutting out each rectangle, wetting one edge ~11thwater, and attaching t o a paper wick down which r a t e r was flowing by capillarity from a chromatography trough. The rectangles were modified t o provide a drip tip a t the bottom; Klett tubes were clipped in place below t o catch the drops from each rectangle. It was extremely important t o wash the wick immediately before use by allowing water t o descend through it, since the watei front coming off the wick repeatedly brought down a concentrated band of carbonyl material, yielding high and variable blanks. The wick must not dry out betaeen washing and attaching the paper t o be eluted. Elution n as allowed t o proceed until all bromophenol blue color has been washed off the papers; this required 4 t o 5 drops.
600 I
I
"
r\
I
I
EXPERIMEfiTAL
Solutions. Sodium metaperiodate, 40 micromoles per ml. (8.6 nig. per ml.). I'otassiuni iodide, 200 micromoles per nil. in 6 S hydrochloric acicl ( 3 3 nig. per ml.). 1Iake up immediately before use. Sodium thiosulfate, 20 micromoles/ml. ( 5 m y . of sodium thiosulfate pentahvdrate per nil.). 2,~-I)initrophenylhydra~iric, saturated Poiution in 2 -Yhydrochloric acid. 3 S sodium hydroxide. Chromatography. T-ntreated Whatman S o . 1 sheets were used in all cases. For the isoleucine and valine urecursors. the solvent system was a 70% ether-30% benzene mixture, made 3 M with foiniic acid and saturated with water ( 1 ) . In this system the isoleucine precursor has an R, of 0.7 and the valine precursor an ,?i of 0.4. Tartaric acid was found t o move satisfactorily in water-saturated n-butanol made 3 with formic acid ( R j = 0.27). It was found essential to treat the solvents with ferrous sulfate solution; otherwise, losses of up to 30% of the glycol oceuried because of peroxide artion during chromatography. Since the compounds used in these experiments were all organic acids, they were detected by spraying lightly with bromophenol blue in ethanol ( S ) , the dye having been found not t o interfere with subsequent steps in the procedure. For nonacid g1j cols a method of detection has been described by Buchanan et ai. ( 2 ) . If the procedure described below were t o apply in such cases, the spots t o be eluted would have t o be located in unsprayed areas by comparison with sprayed guide strips. Elution. A modified rectangle was penciled around each spot with the aid of a stencil; use of the stencil ensured the elution of strictly comparable areas of paper in all cases, including areas used as blanks. The size of the rectangle was so chosen that ample margins were left around all spots. Blanks were outlined with the same stencil, at the same R, as the compound being determined. One blank was provided between each two spots
500 W
$400 w (L
I-
300
c w
< 200 02
04
06
08
u , M GLYCOL
IO
12
"8"
Figure 1. Glycol Determinations 0 Sample solutions treated with periodate and de-
termined direatly 0 Samples chromatographed, eluted, and detsrmined as described in text A . d-Tartaric acid B. a,p-Dihydroxy+-methylbutyric acid Values plotted are net after subtracting blanks, in caee of chromatographed material
Oxidation. T o each Klett tube, containing the drops of eluted material, 1ml. of water was added. Each tube then received 0.1 ml. of metaperiodate solution a t 30-second intervals, followed at exactly 5 minutes in each case by 0.1 ml. of potassium iodide solution. Preliminary experiments showed that maximum color resulted with an oxidation period of 5 minutes a t room temperature. The addition of the acidified potassium iodide solution stopped
ANALYTICAL CHEMISTRY
1554
mittance with an appropriate reagent blank and was used with the green (No. 54) filter. The results obtained with two different compounds are shown in Figure 1. Paper blanks have been subtracted in each case; blanks gave Klett values from 15 to 45, averaging about 30. This compares with a Klett value of about 125 for 0.1 micromole of tartaric acid, and about 80 for 0.1 micromole of a,@-dihydroxyp-methylbutyric acid.
the reaction by converting excess metaperiodate (as well as formed iodate) to iodine. The latter was then titrated to colorless with thiosulfate solution; 1.5 and 2.0 ml. were required. The use of potassium iodide and thiosulfate as described was found necessary because the colorimetry depends on the formation of 2,4dinit~ophenylhydrazones of the products of periodate oxidation. However, if an excess of either periodate or thiosulfate was present, the addition of 2,4dinitrophenylhydrazine solution resulted in the formation of a heavy precipitate. The procedure was thus designed to ensure that no reagent is present in excess a t time of addition of 2,Pdinitrophenylhydrazine solution. Colorimetry. Each tube next received 0.1 ml. of 2,Cdinitrophenylhydrazine solution, following which all tubes were diluted to 5.0-ml. volume with water. Fifteen t o 20 minutes a t room temperature were allowed for hydrazone formation. One milliliter of sodium hydroxide solution was then added to each tube a t 30-second intervals. Fifteen minutes were allowed t o elapse, during which time the rate of color-fading decreased to its minimum: the tubes were then read in a Klett colorimeter a t 30second intervals. The colorimeter was adjusted to 100% trans-
ACKNOWLEDGMENT
This work was supported by a contract between the Office of Kava1 Research and the regents of the University of California. LITERATURE CITED
(1) Adelberg, E. A., Bonner, D. M., and Tatum, E. L., J . BWZ. Chem.. 190. 837 (1951). (2) Buchanan, J: G., Dekker, C. A,, and Long, A. G., J . Chem. SOC.,1950, 3162. (3) Lugg, J. W. H., and Overell, B. T., Nature, 160, 87 (1947). (4) Tatum, E. L., and Adelberg, E. A., J . Bid. Chem., 190, 843 (1961).
RECEIVED for review .4pril 10, 1953. Accepted June 22, 1953.
Titration of N-Carboxy-alpha-amino Acid Anhydrides in Nonaqueous Solvents ARIEH BERGER, MICHAEL SELA,
AND
EPHRAIM KATCHALSKI
The Weizmann Znstitute of Science, Rehovoth, Israel Figure 1 no special steps were needed, as carbon dioxide is practically insoluble under the experimental conditions used. The products of the titration of S-carboxyamino acid anhydrides with sodium methylate in the presence of methanol were investigated in order to elucidate the course of the reaction. For example, LV-carbox?rphenylalanine anhydride( I ) ( 5 ) b a y react with sodium methylate and methanol to give either the sodium salt of aV-carboxyphenylalaninemethyl ester(II), or the sodium salt of S-carbomethoxyphenylalanine(II1).
to the interest in ,V-carboxy-a-amino acid anhydrides as 0 starting materials in the synthesis of poly-a-amino acids (8) WING
and low molecular weight peptides ( 1 , 7 ) , a convenient method for their quantitative determination was required. It has been reported that acid anhydrides could be titrated in nonaqueous solvents with sodium methylate, using thymol blue as indicator (6, 11). This method was adapted for the quantitative determination of AT-carhoxy-a-amino acid anhydrides in various organic solvents. The color of the indicator changes sharply
CaHsCHZ-CH
COOCHB SH-CO
f
-
HC1 CeHoCHt-CH
\
\
(11)
SHCOOCHa CsHbCH2-CH
/ \
HCl CeH6CHt-CH
/
--L
‘COOH
(VI from yellow to deep blue upon the addition of an equimolar quantity of the titrant. From 0.1 to 0.5 millimole of the amino acid anhydrides could be estimated with an accuracy of +t2Y0 by the procedure described below. When using a micrometer syringe, from 0.003 t o 0.03 millimole could be estimated with the same accuracy. This titration seems to be particularly useful in polymerization studies of N-carboxy-a-amino acid anhydrides, since the amount of unreacted anhydride present a t any instant may be determined conveniently. The course of a typical polymerization reaction followed by this method is given in Figure 1. As the carbon dioxide evolved during polymerization interferes with the titration, it should be removed. In the particular case shown in
By determining the amounts of phenylalanine methyl ester hydrochloride(1V) ( 4 ) and N-carbomethoxyphenylalanine(V) (1.3) formed on acidification of the reaction mixture, it was found that 1 mole of ( I ) yields 0.96 mole of (11) and 0.03 mole of (111). Under similar conditions 1 mole of e,N-carbobenzoxy-a,-V-carboxylysine anhydride (3) gives 0.86 mole of the sodium salt of e,N-carbobenzoxy-a,N-carboxylysine methyl ester and 0.11 mole of the sodium salt of c,N-carbobenzoxy-a,N-carbomethoxylysine. REAGENTS AND MATERIALS
Solvents. Benzene, purified grade, dried over sodium; methanol, absolute; dioxane and butylamine, refluxed with po-
