V O L U M E 28, NO. 5, M A Y 1 9 5 6 solubility the recoinmendations of Elek (5') concerning the addition of phenol and propionic anhydride to the samples should be coilsidered. APPLIC.ATION O F IIETHOD
In 1W.i .\Ioi,g:in ( 7 ) rerognized that ethylene as well as ethyl iodide w a s a reaction product of hydrosyethylcellulose and hyd k d i c acid. This macle possible the first reliable determination of nintei,i:ils contailiiiig hydi,oxyetl-iyl groups as well as ethers arid este1.s of ethylene glycol. His work paved the way for many valuable aclvances in the analysis of glycol ethers. I n studying lose and Purves (10) antoq.1nted ~ i ~ d i ~ o x ~ e t l i y l c e l l u Tasker aI>.zcd thcir re:ic,tion produc+s for ethylene oxide content. They re1)iirtetl v:ilues as lo^ as 1.495;, but no degree of accuracy was inclicxtetl. Cohen and Haas ( 1 ) analyzed hydrosyethylcellulose w i t h rul)stitution values as low as 0.44 mole or about 10%. Glcior, \Ialilman, and Llrich ( 4 ) also used this method to measure the ainorint of substitution of h!~droxyethylcellulose. Haas, Coheii, ()glesby, and Karliri ( 6 ) prepared hydroxyethyl derivatives of nylon and found this method useful in determining the etIij.leiie oxide content of the poly(ethy1ene) glycol branches. RJ. analyzing a suitable low molecular weight analog of *\-hydroxyethyl nylon, they found that the carbon-nitrogen bond is not clcaved by this method; thus the combined ethylene oxide gi'oiip attsched directly to the amide nitrogen is not attacked. Colien and Haas (2) applied this method satisfactorily to hydroxj.ethy1 nylon and tosylated hydroxyethyl nylon. The modification.^ of apparatus and methods outlined in this p:rper may be used for Ion-substituted ethers of materials other thnn starch, such as cellulose. Ethers and esters of ethylene glycol or the types of material mentioned by lforgan ( 7 ) which have less than 552 substitution may be analyzed readily by this
895
.-
method. I n general alkyl groups of greater chain length than propyl, such as hydroxybutyl ether, do not give sufficiently volatile alkyl iodides to be distilled over from the constantboiling hydriodic acid. Satisfactory determinations were made on h> droxypropyl starch ether and the degree of substitution was calculated by using the appropriatp molecular weight value?. ACKNOWLEDGMEST
The author gratefully acknowledges the advice and counsel of C. C. Kesler and E. T. Hjermstad as well as help given by Joseph Dytrt in performing of experimental analyses. He also wishes to thank Harry Sunamaker of the State University of Iowa who gave valuable assistance in fahricating the glass apparatus. LITERATURE CITED (1) Cohen, S. G., Haas, (2) Cohen, S. G., Haas,
H. C., .J. -4m.Chem. Sw. 72, 3964 (1950). H. C., J . Polymer Sei. 11, 193 (1953). (3) Elek, A, IND. ENG.CHEM..ATL. ED. 11, 174 (1939). (4) Gloor, W. E., Alalilinan. B. H.. Ulrich, R. D . . I r d . E f i g . Chern. 42, 2150 (1950).
(5) Haas, H. C., Cotien. S. (2.. Oglesby, d. C.. Karlin. E. I:.. J . Polymer Sci. 15, 427 (1955). (6) Kesler, C. C., Hjeriwtad, E. T. (to Penivk and Ford. Ltd., IIIC.), U. 9. Patents 2,516,632, 2,516,633, 2,516,634 (July 2 5 . 1950).
(7) Morgan. P. K., ISD. ESG. CHEM..A s . k r . . ED. 18, 500 (1946). (8) Samsel. E. P., LIcHard. J. h.,Ibid., 14, 750 (1942). (9) Steyermark. -11, . X s . k ~ . .C H E M . 20, 3R8 (1948). (10) Tasker, C. W., Purven. C. B., J . A m . Chern. SOC.71, 1023 (1949). (11) Volhard, J., A n n . 190, 23 (1878); J . prakt. Chem. 117, 217
(1874). RECEIVED for review September
18. 1953. Accepted .iannarJ-1'8,143ti. Dirision of Carbohydrate Cheiiiistry. 128tIi Jleetinn, .iCS, 3Iinneapolis. Jlinn.. Segteniber 1955.
Modified Ultraviolet Spectrophotometric Micromethod for Determination of Amino Acids and Peptides as Copper Complexes ARTHUR CHERKIN', HOWARD WOLKOWITZ2, and MAX S. DUNN Chemical Laboratory, University of California, Los Angeles, Calif.
The uiefulness of the Spies spectrophotometric niicromethod for determining amino acids and peptides has heen confirmed. The method has been simplified, the blank value has been reduced approximately two thirds, and the accuracy with small samples (below 1 micromole) has been increased.
HE need for a micromethod to determine tyrosine and other amino acids in mixtures of amino acids and peptides led to a study of the Spies (1) ultraviolet spectrophotometric procedure. This method appeared to offer distinct advantages in that it is simple and rapid, requires small samples (0.5 t o 5 micromoles), distinguishes betw-een free amino acids and peptides, and yields reasonably accurate values for copper-reacting nitrogen. As in prelimina1.y experiments the results Kith samples below 1 micromole were less dependable t,han had been anticipated, the sources of error were ascertained and an improved method was devised. EXPERIMENTAL
Tlir following method of analysis was employed by Spies (11 1 2
Present address. Don Baxter, Inc., Glendale, Calif. Present address, dmerican Meat Institute, Chicago, Ill.
To 5.0 ml. of buffer solution in a 28-m1., glass-stoppered Erlenmeyer flask were added 5.0 nil. of a water solution of an appropriate concentration of the test substance; 0.1 ml. of copper chloride solution &*asthen added, and the flask was shaken and let stand for 10 minutes a t a convenient room temperature, the same temperature ( & 1") being used for all determinations. The suspension was centrifuged in a capped 12-ml. tube for 5 minutes, and the clear solution was carefully decanted into a clean flask. Transmittancy of the solution was determined a t 230 m+. Control solutions were made by adding 5.0 ml. of the test solutiori to 5.0 ml. of buffer solution. A control analysis of freshly prepared alanine solution was made with each series of tests to check for possible slight variations in the absorbancy due t o the distilled water or other causes. Spies' procedure was modified by addirig 0.1 ml. of distilled water to the control solution to compensate for the 0.1 ml. of cupric chloride solution added to the test solution, employing aliquots of only one solution (all reagents evcept cupric chloride) in preparing both the control and test solutions, centrifuging (International centrifuge Model 1C) a t 3000 r.p.m., approsimately 1800 X gravity, and increasing the centrifuging time from 5 to 20 minutes. Absorbances were determined using 1-em. cells. The effect of centrifuging times from 8 to 60 minutes on absoiba w e s \Vas investigated.
ANALYTICAL CHEMISTRY
896 Table I.
Ultraviolet Absorbances of Copper Complexes of Certain ..imino Acids and Peptides
Substance'" Alanine llrginined Aspartic acid Cvstine Glutamic acid Glycine Histidined Hydroxyproline Isoleucine Leucine Lysin ed llethionine Phenylalanine Proline Serin e
Threonine Tryptophan Tyrosine Valine Alanvlglycine Glycilleucinee
.*lanine Eiiui\ alence b a t 235 Z I p ,
Absorbance 11axi mu m ,
WaveLength Rangec.
5-c
Alp
ZIp
100 112 93 84 103
235 235 238 239 236 232 255 245 238 237 235 237 240 240 23 6 238
234-238 534-238 233-2338 234-24'3 233-238 229-233 225-255 235-250 134-240 "34-239 233-2338 233-239 "35-233 23.5-253 233-188 233-39 2320 233-236 234-238 207-2380 207-2360
93 92
51 100
107 108 108 88 87 97 103 ca. 1101 ca. 871 102 72
73
i
235 235 208
210
other compounds 3OOpM. It was assumed t h a t the latter substances react with copper to form principally complexes of the general formula Cu.12. and cystine and peptides Cu.4. C Absorbances of all compounds except tryptophan and peptides were within IYc of t h a t a t 235 mp. .d . Hydrochloride was used, b u t absorbance was calculated for free aiiiino acid. e Kot tested by Spies. I Accurate determination was difficult, owing t o high absorbancc and impossibility of adjusting the blank t o zero absorbance of the control. 8 Absorbances could not be measured accurately, especially a t loiver \rave lengths.
pounds except tyrosine and tryptophan obeyed Beer's la\$-from zero to the highest concentration tested. The authors' data are shown in Table I. The sharp rise in the blank absorbance value observed by Spies ( 1 ) is eliminated by application of the authors' modified piocedure and the ware length of maximum absorbance of the copperalanine complex is shifted from 230 to 235 mp, in agreement with the corrected curve (Figure 2) of Spies. Shifts of the absorbance maxima ranging from 1 to 30 mp were observed for the copper complexes of 17 other amino acids and two peptides. On the other hand, the copper alanine equivalences found for these compounds at 235 mp diverged by less than 10% from those at 2'30 mp reported by Spies. The reduction in the blank absorbance values is explained, apparently, by the more complete removal of suspended cupric hydroxide. It was found, by inspecting the clarified solutions in a Tyndall beam, that filtration on a funnel fitted with paper or medium porosity fritted glass was as effective as centrifugation in removing suspended cupric hydroxide. The filtrates, hon-ever, gave erratic absorbance values, apparently due to absoi hing mateiials extracted from the filter paper or the fritted glass. The present authors' results confirmed those of Spies in ~iiis~iccessful attempts to determine combined copper of amino acids and peptides as the copper salt of alanine by an ultraviolet spectrophotometric adaptation of the visible photometric procedure of Spies and Chambers ( 2 ) . ACKNOWLEDGMENT
The authors are indebted t o .Joseph R. Spies for valuable sug-gestions ( 3 ) . LITERATURE CITED (1) SpFs, J.
RESULTS A Y D DISCUSSION
Photometric readings (Beckman DU spectrophotometer) deviated 0.1 to 570,with 10% deviation in three cases. The blank absorbance values at 230 mp were reduced from an average of 0.086 obtained with Spies' procedure to as low as 0.024 and an average of 0.034 with the authors' modified method. ill1 com-
R.,J . B i d . Chem. 195, 65 (1952).
( 2 ) Spies, J. R., Chambers, D. C., J . Bid. Chem. 191, 787 (1951). (3) Spies, J. R., private communication, Sept. 28, 1953.
RECEIVED for review January 6, 1955. Accepted January 21, 1956. PapeXo. 102. Work aided by grants from S d t and Co., G . S. Public Health Service, and the University of California. Taken in part from a thesis by Arthur Cherkin submitted in partial fulfillment of the requirements for t h e degree of doctor of philosophy. February 1953.
Investigation of the Franke Method of Determining Free Calcium Hydroxide and Free Calcium Oxide E. E. PRESSLER, STEPHEN B R U N A U E R , and D.
L.
KANTRO
Portland Cement Association, Research and Development Laboratories, Chicago,
The Franlie method (or acetoacetic ester method) for the determination of uncombined calcium hydroxide and calcium oxide in the presence of hydrated and anhydrous substances w-as investigated. Repeated extractions gave the correct values for calcium hydroxide in the presence of a calcium silicate hydrate in which the calcium was firmly bound; however, some calcium was removed from another calcium silicate hydrate in which the binding was weaker. A modification of the method gave accurate results for free lime in anhydrous substances, such as portland cements. The precision of the Franke method was about the same as that of the Lerch and Bogue method (or glycerol-alcohol method), but the former presented certain adbantages.
I
111.
N T H E investigations of the kinetics and thermodynamics of
the hydration of the calcium silicates, conducted a t this laboratory, one of the problems \vas to obtain a reliable method for the determination of calcium hL-droxide in the presence of hydrated calcium silicates. This problem has received considerable attention in the past two decades because of its importance in the investigations of portland cements, pozzolanic cements, and other industrial materials. The main difficulty is that the analytical methods proposed for the determination of uncombined calcium hydroxide involve its solution in some organic solvent, and this is usually accompanied by the solution of some of the combined calcium. The difficulties may be illustrated with two examples taken from the literature. I n 1938 Bessey ( 4 ) reported uncombined calcium hydroxide values for four hydrated portland cements
