V O L U M E 2 8 , NO. 9, S E P T E M B E R 1 9 5 6
1481
DISCUS SlON
T h e determination of fluorescence intensity has the advantage of being simple and fairly rapid by comparison with other methods for the determination of the concentration of amino acids directly on the chromatograms. I n the total color density methods ( 2 , 5 , 1 4 ) , the average color densities of a number of ninhydrin-sprayed strips of chromatographically separated amino acids are plotted on graph paper and the areas representative of each amino acid are determined. T h e absorption method requires the use of a base line (dependent on the colorless area) and certain assumptions with respect to the position of the dividing line between overlapping bands. These difficulties are avoided by the measurement of the fluorescence, which is proportional to the amount of material present. The maximum color density method of Block ( 1 ) also avoids this problem and, like the fluorescence method, is applicable t o two-dimensional paper chromatography. The maximum color density and the total color density methods require more expensive reagents and a piece of apparatus, the densitometer, which is not as commonly found in the laboratory as the Beckman spectrophotometer. Good accuracy ( 5 to 10% error) may often be obtained, although errors considerably larger than the mean are occasionally encountered (Table 11). For qualitative location of spots, the method is similar in sensitivity t o the ninhydrin method. When the amino acids in the mixture to be analyzed can be separated in one dimension, the quantitative analysis can be carried out easily and rapidly. However, when two dimensions are required, the determination may become tedious because of the number of chromatograms necessary for mixtures containing a large number of amino acids in widely varying amounts. While rather time consuming for an absolute, complete protein analysis, the fluorescence method seems to be a rapid means of comparing relative amounts of amino acids in a series of samples. LITERATURE CITED
(1) Block, R . J., ANAL.CHEM.22, 1327 (1950). (2) Block, R. J., Science 108, 608 (1948). (3) Block, R. J., Durrum, E. L., Zweig, G., "Manual of Paper Chromatography and Paper Electrophoresis," Academic Press, New York, 1955. (4) Brand. E., Ann. S. Y . Acad. Sci. 47, 187 (1946). (6) Bull, H. B., Hahn, J. W., Baptist, V. H., J . -4m. Chem. SOC. 71,550 (1949).
(6) Enders, C., Sigurdsson, S., Biochem. 2. 316, 303 (1944). (7) Friedman, L., Kline, 0. L., J . B i d . Chem. 184, 599 (1950). (8) Johnson, G., Guadagni, D. G., U. S.Patent 2,475,833 (1949).
Table I. Fluorescence Intensities of Xylose Derivatives of Various Amino Acids as Function of Quantity of Amino Acid 0 025
Amino Acid Alanine Glutamic acid Leucine Valine Glycine Lysine
8 10 9 17
Amino Acid, pmole 0 10 0 125
0 075
0 05
Fluorescence Intenaity 24 34 40 26 41 43 17 20 23 10 14 16 24 :3;3 38 42 53 58
17 16
12 8 18 30
0 15
0 20
48 52 30 22 46 6.5
68 72 42
Table 11. Amino Acid Composition of Insulin and pLactoglobulin by 3Ieasurement of Fluorescence Intensities Amino Acid
Glutamic acid Aspartic acid Glycine Serine Leucine isoleucine Phenylalanine Valine
+
Alanine Lysine Aspartic acid Leucine isoleucine Glutamic acid Valine
+
(9) (10) (11) (12) (13) (14)
Moles per 105 Grams of Protein Found Reported values Diff., Insulin Brand (4) 1;;
90 60 120 60
75
I50 50
2
1%
50 67
0-Lactoglobulin Stein, Moore ( I : ) 85 80 86 77 84 86 170 158 140 1:30 60 49
C
9
IO 50 9 4 20 12
R 1" 9
4 8
?'
Levy, -4.L., Chung, D., h . 4 ~ CHEX . 25, 396 (1953). McFarren, E. F., [hid., 23, 168 (1951). Olcott, H. S., Dutton, H. J., I n d . Eng. Chem. 37, 1119 (1945). Pardee, A. B., Shore, V. G., unpublished data. Redfield, R. R., Biochim. et Biophys. Acta 10, 344 (1953). Redfield, R. R . , Barron. E. P . G., Arch. Biochem. and Biophys.
35,443 (1952). (15) Rice. R. G., Kertesz, Z . J., Stotz. E. H., J . A m . Chem. SOC.69, 1798 (1947). (16) Shore. B., Shore, V. G.. Plasma 2, 621 (1954). (17) Stein, W. H.. IIoore, S., J . B i d . Chem. 178, 79 (1949). (18) Wadman, W.H., Thomas, G . J., Pardee, A. B., A x . 4 ~ .CHEM. 26,1192 (1954).
RECEIVED for rex~ieuFebruary 1.3, 1956. Accepted >lay 5 , 1956. Aided b y grants f r o m t h e Vniversity of California Cancer Research Funds, Lederle Laboratories, and T h e Rockefeller Foundation.
Determination of Acetylenic Compounds via Hydration SIDNEY SlGGlA Central Research Laboratory, General Aniline &
Film Corp., Easton, f a .
The hydration reaction of the triple bond, when a mercuric ion catalyst is used in an acidic medium, converts the acetylenic compound to a ketone, which is then determined using hydroxylamine hydrochloride. This approach makes possible the determination of acetylenic compounds, for which earlier methods are unsatisfactory. The precision and accuracy are, in general, within 3 ~ 2 % ;depending on the type of compound determined.
M
ANY methods have been developed for determining monosubstituted acetylenes of t h e type HC=CR (1-3, 6,
6, 10, 12-14), All these methods involve replacing t h e acetylenic hydrogen atom by a metallic ion and titrating either t h e acid
formed or the excess of metallic cation uoed. These methods cannot be used for determining disubstituted acetylenic comAlso, in the case of monosubstituted acetylpounds (RC-CR). enes, these methods sometimes cannot be applied because of interfering substances, which react n-ith the metallic ions used in the analysis. Wagner, Goldstein, and Peters ( 1 6 ) describe a method for determining mono- and dialkyl acetylenes of four or five carbon atoms by reaction with methanol, using mercuric oxide and boron trifluoride as catalyst. The acetylenic compound is thus converted to the ketal. The ketal is distilled into hydroxylamine hydrochloride reagent, which hydrolyzes the ketals to the ketones and then forms the oxime of the ketone. T h e reactions used in this method are as follows:
1482
ANALYTICAL CHEMISTRY
RC=CR’
+ 2MeOH HgO + RC(0Lle)zCHzR’ BF,
0 RC(OMe)zCHzR’
+ HzO
!I
-L
RCCH2R’
+ 2MeOH
0
II
RCCHzR’
+ KHzOH. HC1+
SOH
I1
RCCHzR’
+ H20 + HCl
T h e hydrochloric acid fornied in the last reaction is titrated, and the amount of acetylenic compound present in the sample is calculated from this value. The results obtained by this method are about 92% of the theoietical values. The above procedure could not be used for the acetylenic compounds under investigation because of the high boiling points of the acetals formed, which made distillation impossible, the instability of the acetals, vhich resulted in decomposition a t the
distillation temperatures, or the low accuracy inherent in the method. Koulkes (8) used the reaction between the acetylenic triple bond and mercuric acetate t o determine several disubstituted acetylenic compounds. The niercuric acetate presumably adds onto the triple bond, and the excess of the acetate is determined by addition of sodium chloride and titration of the acetic acid liberated This approach is fast, but impurities which react with mercuric acetate interfere: ethylenic compounds, soine of which also add niercuric acetate, and inorganic and s o n e organic halides, which comples n i t h the mercuric ion. Some organic compounds such as carbosylates and sulfonates, form precipitates, and others are ieadily oxidized by the nierctiric ion. I n the procedure described, the hydration reaction is used, with a mercuric catalyst in a strongly acidic medium, and the ketone
n RCECR’ t HzO
HgS04 RCCHzR’
fornied is a measure of the acetylenic material. This approach is subject to less interference than the mercuric acetate addition method, as the hydration vi11 still proceed if a portion of the cat&st is consumed by other componentc. The accuracy of this system is, in general, 100 i 27,, and a variety of acetyTable I. Determination of Acetylenic Compounds lenic compounds are deterReflux Time, N i n . Mole Vole % minable (Table I). Found Compound Hydration Oxiniation Used Recovery % Purity Ncthod hcidic or alkaline impurities Butynediola 15 1.5 0 01398 0 01339 95.8 98.9 + 1 .i 30 30 in the sample do not interfere, 0 01398 0 01391 99.5 .. 30 30 0.01398 0 01381 98.8 .. as the system is neutralized 30 30 0 00702 0 00682 97.2 .. 30 45 0.01404 0 01415 100.8 .. prior to oximation. E t h j leiiic 30 45 0.01404 0 01422 101.3 .. 30 unsaturated componnds do not 43 0,00702 0 00700 99.7 .. .. 45 (io 0.01271 0 01277 100.5 .. .. interfere, as they do not foiin 60 I; 0 0.01398 0 01395 99.8 .. 120 60 0.01398 0 01388 99.3 .. carbonyl compounds under the conditions of the reaction. 18 hr. a t 1 2-Propyne-I-olb 30 0 01473 95.0 99.0 & 1 A ‘I room temp.] 0.01551 The only interferences n-hich 30 0.01551 0 01387 8 9 . 4 . . 3P can be envisioned are froin car30 0 01526 0.01651 98.4 .. bonyl compounds or carbonyl18 hr. a t room temp., forming compounds such a i GO 30 0 01517 0.01551 97.8 .. .. 30 60 0.01551 0 01512 97.5 .. acetals or vinyl ethers. However, these can be determined 1-Butyne-3-olb 60 f)O 0 01321 0 01125 85 2 8 4 . 1 j= 1 B 60 GO 0 00881 0 00732 83.1 .. by running the oximation 3-Butyne-1-olb 30 0 01317 B analysis alone on a sample GO 0 01376 without first running the hyfi0 0 01414 60 0 01355 dration; this should yield just 60 0 01364 .. 60 0 01402 .. the carbonyl compound. The hydration analysis should then E thynylcyclohexanolc 30 30 0 01381 0 01280 92.7 . . d .. 60 ‘io 0 00810 0 00781 96.4 .. .. yield the total of carbonyl 60 UO 0 00810 0 00793 97.9 .. .. compound and acetylenic comPhenylacetylene 6 GO f 0 0001985 0 0001915 9 6 . 5 97 2 & 1 B pound; the acetylenic compo60 f 0 0005958 0 0005744 96.4 .. .. nent can then be determined 1-HexyneC 75 60 0 01223 0 01088 89.0 8 9 . 1 =k 1 C ;1 0 0 01083 88.6 .. 90 by subtraction. Some aldehydes are not stable (oxidize) 3-Hexynec 750 00 0 01381 0 01235 89.2 91.5 & 1 C 0 01263 75 60 .. 91.3 .. n-ith mercuric ion and samples D 3-Octynec 900 DO 0 00936 0 00875 9 3 . 5 9 3 . 0 z!= 1 containing large amounts of 150 60 0 00868 92.7 .. .. aldehydes or acetals should be 3-Octyne-1-olc 900 60 0 00806 0 00678 8 4 , l 8 7 . 5 zt 1 C examined thoroughly to make 0 00676 .. 150 83.7 GO .. sure that the corrections applied are valid. A. Acetylation method (for alcohols) (11). B. Acetylenic hydrogen method (1.5). .-k potentiometric titration is C. Bromination, method (9). D. Hydrogenation method ( 4 ) . used to measure the hydrochloric acid liberated in the a Recrystallized from ethyl acetate. oximation step. The end b Laboratory samples distilled once through helix-packed column. C Analyzed as purchased from Farchan Research Laboratory. points are not sharp, but, in d Could not be determined by method A , B , C, or D. e Analyzed as purchased from Eastman Kodak. general, the precision is within f Dinitrophenylhydraaine method used because acetophenone could not be measured b y oximation method. f 27‘ and sometimes within Q 20 ml. extra methanol used in hydration s t e p because of insolubility of compounds. f 1%.
1483
V O L U M E 2 8 , NO. 9, S E P T E M B E R 1 9 5 6 Acetylenic compounds with substitutents on t h e carbons adjacent to the acetylenic linkage (RICHCICCHR~) do not hydrate
I
R1 R: r:iI)idly, owing to the hindrance caused by substituents. an:ilj-sis of
OH OH I CzHjC-C=C-CC2Hs I
An
v a s attempted by this
I
CH, CHa method, but conversions of only about 50% JT-ere obt,ained under the conditions described; 70% conversions were obtained Then the hydration time v a s doubled. t-nfortunatell-, not enough compounds could be obtained n-ith substituents adjacent to the xcetylenic linkage to compare the rates of hydration with the type of substituent. Attempts were made to analyze acetylenic bromine compounds of tlie type RC-CCHtBr. However, the bromine atonis on these compounds are so labile that they are removed by the niercuric c:lt:ilyst to form the mercuric bromine complex; and the catalytic xction of the mercuric ion is much decreased; this type of compound cannot be determined by this method. The chloride compounds of t h e same structure will probably behave in the same iiuiiner, but this has not been tested. In the case of phenj-lacetylene, the ketone formed on hydration is acetophenone. This ketone is one of the very fen- that cannot be determined by oximation in an aqueous or partially aqueous mediiini, because of the eqiiilibrium v-hich is present in t h e oximation system. Water is z product of the osimation reaction, and an aqueous system keeps the reaction from going to rompletion. I n the case of phenl-lacetylene, after hydration 311)-excess mercuric ion is removed by bubbling hydrogen sulfide through the solution and filtering off the mercuric sulfide. Then the ncetophenone in the solution is determined b y the 2,4-dinitrophenylhydrazine method of Iddles and Jackson (7‘). This xmie technique should be applicable t o other acetylenic compounds which yield ketones that are not readily determined by osiiiiation. This technique is applicable to samples containing small amounts of acetylenic compounds! as the 2,4-dinitrophenylhydrazine method requires only 4 X IO-’ mole of ketone for nptiniuni operating conditions. This precipitation approach is less applicable to the hydroxyacetylenic compounds because of tlie solubilizing effect of the hydroxyl groups. Before t h e 2,4-dinitrophenylhydrazineapproach was tried on the acetylenic compounds, blanks n-ere run, in which all the steps were included. This v a s to make sure that no extraneous precipitate formed on addition of the hydrazine, which n-odd affect thc results. Known samples of acetophenone were run through t h e entire procedure to establish the conditions for complete recovery of the ketone. Then the acetylenic compounds n-ere used. REAGENTS AND PROCEDURE
Reagents. Hydroxylamine hydrochloride (0.LY) in 1 to 1 methanol-water. T h e catalyst is made from 0.5 gram of mercuric sulfate, 2 ml. of sulfuric acid, and 63.4 nil. of mater Alcoholic sodium hydroxide (1.OAV). Sodium hydroxide is dissolved in as little water as possible. T h e sodium carbonate is filtered off, and the solution is diluted viith methanol t o the desired volume. This solution need not be standardized. Aqueous sodium hydroxide, 0.55 (standard), is used. Apparatus. Glass and calomel electrodes, with a Model H-2 Beckman p H meter. Procedure. d sample containing 0.05 to 0.20 mole of acetylenic compound is dissolved in methanol and diluted to 100 ml. in a volumetric flask; 10-ml. aliquots are used for t h e determinations. Ten milliliters of sample solution are added t o 20 nil. of catalyst in a 200-ml. three-necked flask connected t o a reflux condenser. Glass stoppers are inserted in the two unused necks of the flask. The mixture is refluxed for 1 hour and then cooled in ice with the condenser still attached. After cooling, t h e condenser is washed with 10 ml. of 1 t o 1 methanol-mater and allowed t o drain. The flail; is disconnected from the condenser and glass-calomel
electrodes are inserted into the flask through the two side necks. T h e acid is just neutralized (pH 7 ) with 1.ON alcoholic sodium hydroxide. Fifty milliliters of hydroxylamine hydrochloride are added, the mixture is again refluxed for 1 hour and cooled in ice, and the condenser is washed Kith 1 to 1 methanol-water. T h e mixture is transferred t o a 400-ml. beaker, using 50 ml. of 1 to 1methanoln-ater to wash the flask. As much of the solid residue as possible is left in the flask during transfer. T h e liberated hydrochloric acid is titrated potentiometrically with standard 0.5N sodium hydroxide, using the glass and calomel electrodes. T h e end point is determined from a plot of milliliters of reagent us. pH. If carbonyl compounds are present in the sample, they should be determined using the hydroxylamine hydrochloride analysis (see discussion above). 2,4-DINITROPHENY LHYDRAZONE METHOD
Reagents. Catalyst as described above. A saturated solution of 2,i-dinitrophenylhvdrazinea t 0’ C. in 2 5 hydrochloric acid. Procedure. A sample is dissolved in methanol and diluted to 100 ml., so that a IO-ml. aliquot contains approximately 4 X lo-‘ mole. Ten milliliters of sample are added to 20 ml. of mercuric. sulfate-sulfuric acid catalyst and refluxed for 1 hour in a thrcenecked flask n i t h glass stoppers in the txvo unused necks. After the hydration reaction period, the flask is cooled in ice with the condenser attached, and the condenser is washed with 10 ml. of 1 to 1 methanol-water. At this point there is a white precipitate in the flask, 11-hich does not appear to affect the results. With the condenser still in position, hydrogen sulfide is passed into the solution t o precipitate mercury as the sulfide. When this reaction is complete (5 to 10 minutes), the sulfide is filtered o f f through a S o . 30 Whatman filter paper and the flask and paper are n-ashed with a 1 to 1 solution of methanol-i+-ater. T o the filtrate are added 50 nil. of 2,4-dinitrophenylhydrazine solution, and the mixture is allowed to stand 0.5 to 1 hour. T h e resulting solution is warmed on a hot plate n i t h constant stirring to coagulate the precipitate. K h e n the supernatant liquid is clear, the precipitate is filtered off through a Gooch crucible ~ i t h an asbestos mat, washed with water, dried a t 100’ C., and weighed. If the resultant hydrazone exhibits a significant solubility mith the alcohol present (this must be predetermined), the solution is boiled for a few minutes to remove as much alcohol as possible before filtration. Acetylenic compounds containing hydroxyl groups cannot usually be determined by this method, because of the solubilizing effects of these groups. ACKSOWLEDGMEBT
T h e author wishes to acknowledge the contributions made by William 5’. Curran and Richard Rheinhart to the successful completion of this paper. LITERATCRE CITED
(1) -Utieri, V.J., “Gas Analysis and Testing of Gaseous Materials,” pp. 330-2, American Gas Association, New York, 1945. ( 2 ) Barnes, L., Molinini, L. J . , -kN.4L. CHEIr. 27, 1025 (1955). (3) Chavastelon, AI., Compt. Tend. 125, 245 (1897). (4) Gould, C. W ,Drake, H. J., .&TAL. CHEM.23, 1157 (1951). (5) Hill, A . J., Tyson, F., J . Ani. Chem. Soe. 50, 172 (1928). (6) Hyzer, R. E., A N A L . CHEM.24, 1092 (1952). (i) Iddles, H. A , Jackson, C. E., ISD. ESG.CHEM.,AXAL.ED.6, 454 (1934). (8) Koulkes, AI., Bull. SOC. chim. France 1953, 402-4. (9) Lucas, H. 3 . . Pressman, D., IND.ENG.CHEM.. ANAL.ED. 10. 140-2 (1938). (10) AIarssak, I., Koulkes, 31.,Mem. services chim. &at (Paris)36, S O . 4, 421-6 (1951). (11) Ogg, c. L., Porter, TV. L., TVillits, C. 0 , ANAL.CHEM.17, 394 (1945). (12) Robey, R. F., Hudson, B. E., Wiese, H. K., Zbid.. 24, 1080 (1952). (13) Ross, W. H., Trumbull, H. L., J . Am. Chem. SOC.41, 1180 ( 1919). (14) Siggia, S., “Quantitative Organic Analysis via Functional Groups,” pp. 49-58, Wiley, Sew York, 1949. (15) Siggia, S., Hanna, J . G., ANAL.Cmiv. 21, 1469 (1949). (16) Wagner, C. D., Goldstein. T., Peters, E. D., Zbid., 19, 103-5 (1947). RECEIVED for re7 iem December 15, Ig.55.
.iccepted .May 16, 1956.
