V O L U M E 2 6 , NO. 9, S E P T E M B E R 1 9 5 4 Table 11. Reproducibility with Different Variables Analyst A A B B B B B B
Electrode System Pt-calomel Pt-calomel W-calomel W-calomel Pt-calomel Pt-calomel Pt-calomel Pt-calomel
Readin Interval After I d d i t i o n , Sec
NasS01, Mg. 15 75.00 15 37.50 30 75.00 30 37.50 37.50 30 15 37.50 15 37.50 37.50 15 Arithmetical mean Average deviation Average deviation of mean
NarSOc Found,
% 100.07 100.29 100.12 100.43 100.12 100.28 100.12 100.12 100.19% &0.104% 0.085%
0 to 2% sulfate range i i 4~3.0%and for the 2 to 12% sulfate range &08% of the amount added. Several samples of impure toluene sulfonic acid containing 0.0 to 0.90% sulfuric acid have been analyzed. The results agree with gravimetric analysis data with the same degree of accuracy as was obtained with the synthetic samples. Samples of benzene sulfonic acid containing as much as 50% sulfuric acid have been assayed by this procedure. Phosphate, chromate, and chloride ions interfere with most benzidine sulfate precipitation procedures. The effect of these ions and a ferric salt upon the accuracy of the described method has been studied. -4nalyses of synthetic samples containing 50.0 mg. of sulfuric acid and varying concentrations of each ion have shorm that 5 mg. of phosphate (as NazHPOa), 10 mg. of chromate (as K2Cr04),500 mg. of chloride (as XaC1) and 10 mg of ferric chloride will not interfere with this method of analysis. If the concentration of these ions exceeds the stated limits they should be removed or rendered ineffective. I t is suggested t h a t this can be done by the procedures given by Telcher (9). It I S recommended that the titration be follon ed n i t h a tungsten-calomel electrode system. The authors have found that some platinum electrodes give as good results as tungsten, but others give much poorer results. The average potential break a t
1519 the end point with a tungsten-calomel electrode system per 0.1 ml. of 0.1M potassium nitrate solution is 114 mv This figure is obtained from titration data from the analyses of 252 benzene sulfonic acid samples. The largest potential break was 210 mv. and the smallest was 60 mv. The precision of the method was checked under various experimental conditions. Two analysts with separate reagents and equipment analyzed aliquots from a standard solution of sodium sulfate according to the conditions given in Table 11. Good reproducible results were obtained with an average deviation of 50.10%. The accuracy was determined by analyzing a standard solution of 0.2N sulfuric acid. Six samples gave an average of 99.87 i 0.15% sulfuric acid; theoretical value 100.00% sulfuric acid. The method has proved satisfactory for the determination of sulfate in three sulfonates. It can be used for the determination of sulfates in other mixtures with comparable results. The main advantage of this method over conventional gravimetric and volumetric procedures is the short assay time. ACKNOWLEDG3IENT
The authors gratefully acknowledge the help of C. H. Brackbill in obtaining experimental data. LITERATURE CITED
(1)
Callan, T. P., and Toennies, G., IND. ENG.CHEM., =lxua~.ED., 13, 450 (1941).
(2) Dmmmond,'J. C., Biochem. J . , 9, 493 (1915). (3) Fiske, C. H., J . B i d . Chem., 47, 59 (1921). (4) Hibbard, P. L., Soil Sci., 8, 61 (1919). ( 5 ) Ollgaard, E., Biochewz. Z., 274, 181 (1934). (6) Raisiss, G. W., and Dubin, H., J . Bid. Cheni., 18, 297 (1914). (7) Raschig, F., 2. angew. Chem., 16, 617, 818 (1903). (8) Ibzd., 19, 331 (1908). (9) Welcher, F. J., "Organic Analytical Reagents," Tola 11, p. 300, S e w York, D. Van Sostrand Co., 1947. RECEIVED for rpview September 21, 1953
Accepted J u n e 7 , 1954
Infrared Identification in Paper Chromatography T. Y. TORIBARA
and VICTOR DI STEFAN0
University o f Rochester, Rochester,
I
N. Y.,
and M a r q u e t t e University School o f Medicine, Milwaukee 3,
KFRARED spectrophotometry for the study of biological materials, especially those isolated by paper chromatography, has always appeared as an attractive possibility, but previously existing sample-handling techniques have been unsuitable. The quantities of material separated by a paper chromatogram are in the fractional milligram range. The conversion of such minute quantities to a suitable sample for infrared studies has been made possible through the development of the method for making dilutions of organic solids in solid potassium bromide by Stimson and O'Donnell ( 7 ) and Schiedt (4-6) Suitable records were obtained from these small samples by using the beam condensing system of Anderson and Woodall (2) which has provided a simple means for extending the analytical range of the spectrophotometer to samples as small as 10 y. The procedure of freeze drying a solution of the sample and potassium bromide was selected as the best method for quantitatively transferring and uniformly dispersing these small samples Fortuitously, freeze drying has been shown by Schiedt ( 4 ) to give the optimum particle size for the production of the most satisfactory spectral records. Because of the obvious advantages of this method, the procedure as adapted for small samples is described in some detail in the following sections.
Wis.
In-the course of work on the calcification mechanism it wa8 suspected that an organic phosphate compound could be a possible intermediate in the deposition of bone salts. Indeed, such a compound was found in protein hydrolyzate8 of both bone and calcifiable cartilage (3). The two-dimensional chromatogram of L) cartilage hydrolyzate is represented schematically in Figure 1. The unknown spot is the suspected organic phosphate. T o gain some insight into the nature of this unknown material, a sample was isolated for study by infrared analysis. I n order to obtain larger quantities of the unknown substance than would be separated by the usual two dimensional chromatography, the isolation wa3 carried out as two separate single-dimensional steps. ISOL4TION OF SAXfPLE BY PAPER CHROIIATOGRAPHY
Six milligrams of calcifiable rachitic cartilage from the tibia of albino rats were placed in an ampoule containing 5 ml. of 6.V hydrochloric acid. The ampoule was sealed and placed in boiling nat'er for 21 hours. The hydrolyzate was filt'ered, evaporated to dryness, and reconstituted with distilled mater. This process was repeated once again to remove the hydrochloric acid. Finally, the residue was dissolved in a convenient quantity of distilled water (usually 1 ml.) and transferred by means of a
1520
ANALYTICAL CHEMISTRY
capillary pipet along a pencil line 4 inches from the bottom and running across an 18 X 24 inch sheet of Whatman No. 1 filter paper. A one-dimensional chromatogram was developed in collidinelutidine and a narrow strip was cut from the left side of the sheet and sprayed with ninhydrin (1 mg. of triketohydrindine per ml. of butyl alcohol) to determine the positions of the various bands. From a consideration of Figure 1 i t is obvious that the unknown should be found in the topmost band along with the leucines and tyrosine. Separation of these materials was effected by elution of the top band in distilled water and the subsequent development of a similar chromatogram in phenol. Because the unknown compound moves slowly in phenol relative to tyrosine and the leucines, it was found in the bottom band (Figure 2).
on the outside of the tube ceased. Complete drying was effected by continuing the high vacuum pumping ( 7 to 9 hours total time). No attempt was made in this work to shorten the drying time by application of heat. I n the Eastman Kodak spectroscopy laboratory ( 1 ) samples are dried completely in I to 2 hours. PRESSING PROCEDURE
The solid left after freeze drying was light and fluffy. This material was scraped from the tube and a suitable amount was weighed for pressing into a pellet according to the method described by Anderson and Woodall ( 2 ) . The die was not evacuated, and transparent disks were not obtained.
! A
I
0
Figure 2. Schematic Depiction of Separation of Unknow-n, Tyrosine, and Leucines o n One-Dimensional Chromatogram Developed i n Phenol
W
D
Narrow strip has been cut from left side of sheet and sprayed with ninhldrin t o 10calize bands +
Figure 3. Freeze Drying Apparatus
PHENOL-
-
Figure 1. Schematic Two Dimensional Chromatogram of Bone or Rachitic Cartilage Hydrolgzates
WAVE NUMBERS IN GM-'
Dotted lines indicate topmost band which occurs after development of one-dimensional chromatogram i n collidine-lutedine
The lowermost band was cut away and its contents were eluted with distilled water. Extraction of the eluate was carried out with ether over a 24-hr. period in a continuous extraction apparatus. To remove the ether, the solution was evaporated to dryness. The residue was then reconstituted with 2 ml. of distilled water containing 20 mg. of potassium bromide and transferred to the flask of the freeze-drying operation.
I
I
l
l
I 0
--3
2
4
5
6
7
8
Figure 4.
DILUTION OF SAMPLE WITH POTASSIUM BROMIDE
The freeze-drying apparatus is shown in Figure 3. The sketch shows only one sample holder, but any number may be built into the manifold. The sample holder was made from a male 29/26 medium length standard-taper joint with dimensions to provide easy removal of the sample. All connecting tubes should be of large diameter (tubing 13 mm. in outside diameter works well). I n principle it is a sublimation apparatus in which frozen water is sublimed under a high vacuum into the trap immersed in dry ice-acetone. The solution was cooled rapidly by immersion of the tube in a dry ice-acetone mixture. T o spread the solution in a thin layer, the sample tube was tilted and rotated during the freezing process. If too thick a layer of frozen material is obtained, spattering may occur during the sublimation. The sample tube was then placed on the manifold and evacuated. The vacuum may be supplied by an ordinary motor-driven pump such as supplied by Welch or Central Scientific Co. I n this work the standard unit for sublimation purposes consisting of a twc-stage mercury diffusion pump with a Cenco Hyvac forepump was used. The sublimation was rapid enough to prevent liquefaction of the sample when the sample tube was exposed to the laboratory atmosphere. Moisture in the air condensed on the outside of the sample tube and froze. When most of the sample water had been removed (usually 2 hours), condensation
u o
9 1 0 l l WbVE LENGTH IN MICRONS
1
2
,
1
l
3
l
~
~
1
4
I
5
Spectrum of B-Alanine WAVE NUMBERS IN CM-'
WAVE LENGTH IN MICRONS
Figure 5.
Spectrum of Unknown Material
An example of the type of record that can be obtained is in Figure 4 where 50 y of @-alaninewas dispersed in 10 mg. of potassium bromide and carried through the procedure. Figure 5 is a spectrogram of the unknown from the chromatogram dispersed in potassium bromide. From an analysis of this spectrogram together with supporting chemical and chromatographic evidence, a t least a tentative assignment of galactoseamine phosphate has been made for the unknown component. Direct spectrographic
6
1521
V O L U M E 26, NO. 9, S E P T E M B E R 1 9 5 4
(2) Anderson, D. H., and Woodall, N. B., ANAL.CHEM.,25, 1906
comparison of the unknown with either galactoseamine phosphate or galactoseamine has not yet been possible, as neither of these substances was available in pure form.
(1953). (3) Di Stefano, V., Neuman, W. F., and Rouser, G., A r c h . Biochem. and B i o p h y s . , 47, No. 1, 218 (1953). (4) Schiedt, U. (reported by H. Hausdorff), A p p l . Spectroscopy, 7, NO.2, 75-84 (1953). (5) Schiedt, U., 2. Naturforsch., 8b, 66-70 (1953). (6) Schiedt, U., and Reinwein, H., Ibid., 7b, 270-7 (1952). (7) Stimson, AI. M., and O'Donnell, M. J., J . Am. Chem. Soc., 74, 1805 (1952).
ACKNOWLEDGMENT
The authors Lvish to acknowledge valuable technical assistance from R. G. Smith and D. H. Anderson of the Spectroscopy Laboratory, Eastman Kodak Co.
RECEIVED for review April 2, 1954. .4ccepted June 3, 1954. Based on work performed under contract with the United States Atomic Energy Commission a t the University of Rochester Atomic Energy Project, Rochester, N.Y,
LITERATURE CITED
(1
.-lnderson, D. H., personal communication.
Colorimetric Determination of Ascorbic Acid New Developments Concerning the Reaction with Diazotized 4-Methoxy-2-Nitroaniline MORTON SCHMALL, C. W. PIFER,
E. G. WOLLISH, ROBERT DUSCHINSKY, and HAROLD GAINER
Nutey, N. 1.
Hoffmann-La Roche Inc,,
A
(IV). With n-araboascorbic acid (isoascorbic acid) the corresponding L-erythronic acid lactone (111) is formed in an analogous manner, which upon alkaline hydrolysis yields the identical sodium salt (IV). When the absorbancies of the pure isolated dye and of the color produced by an equivalent of ascorbic acid subjected to the method described ( 4 ) were compared, it was found that the reaction proceeded with a yield of 90%. Considering the rather complicated mechanism, this yield is surprisingly high. The appearance of a negative charge a t the a-nitrogen atom is in accord with the known a-sodium salt of phenylhydrazine (3). IDEYTIFICATION O F BLUE COMPOUYD AND P R E P 4 R i T I O I Resonance forms such as ( V ) undoubtedly contribute to the OF 4YALOGS formation of an intense color. This form (V) also includes Two of the authors (Duechinsky and Gainer) identified the enolization in the group -NHCO- of (IV) which increases the final reaction product as the deep blue disodium salt of oxalic number of double bonds in conjugation. Compounds of analacid 4-methoxy-2-nitrophenylhydrazide (IV). The mechanism ogous type, such as l-(p-nitrophenyl)-2-acetylhydrazine are of formation of the oxalyl compound is still not completely knoa-n to produce a deep red color with alkali ( 2 ) . -4number of clarified. Whatever the mechanism may be, it appears that related acylated 0-, m-, and p-nitrophenylhydrazines which were ssvorbic acid (I) and the 4-methoxy-2-nitrobenzenediazonium prepared are given in Table I. For the production of an intensely cation (11) undergo an oxidation-reduction reaction, probably colored sodium salt, @-acylationas well as ortho- or para- nitraafter forming an adduct, while the substituted benzene diazonium tion, is essential. moiety forms a hydrazide. The ascorbic acid part suffers, in Compounds 1, 2, 4, 6, 10, and 11 were obtained from ascorbic addition to oxidation, an opening of the furan ring. The resultant acid and the requisite diazonium salt as indicated above. The product is the 4-methosy-2-nitrophenylhydrazideof the aformyl compounds 3, 9, and 12 were prepared either by formylaoxalate of D-threonic acid lactone (111). Further action of tion of the hydrazine or by refluxing of the corresponding oxalic alkali on (111)yields finally the blue disodium oxalate derivative acid monohydrazide in acetic or propionic acid. A mixture of compounds 5 and 7 was obtained when 2nitro-4-methoxy-phen~-lhydrazine was heated 0 0 !I I \ with ethyl oxalate. The diacetyl compound ('~--COH SO?
