1209
V O L U M E 2 2 , NO. 9, S E P T E M B E R 1 9 5 0 classified as tars ( 2 ) . This procedure was calibrated with technical sodium pentachlorophenate, which cancels out the effect of these substances.
producing the same red color and the same type of absorption curve. All the azure blue may be used up in this fashion, which is contrary to what is observed with methylene blue.
METHYLEYE BLUE METHOD
SUMMARY
Methylene blue hydrochloride combines with sodium pentachlorophenate quantitatively a t a pH of 10.9, producing a blue colored complex which is soluble in chloroform. The reaction ohcvs Beer’s law up to 1 mg. and the mayimum absorption band is at 600 to 640 mp as determined with a photoelectric colorimeter The blank, which is prepared with sodium hydroxide, chloroform, methylene blue, and water, is a magenta colored solution prior to being filtered through cotton. Upon being filtered, this solution turnr very pale blue, much lighter than that found with sodium pentavhlorophenate. A certain part of the color of the unknown is duo to this reaction, but is taken care of by setting the instrument to zero with the blank. Th(%reaction producing the magenta colored blank s e e m to be phyhicd and is probably a hydration phenomenon. Water is esseiitial for the production of the red color, and removal of the water results in a blue solution. It is believed that the alkali reacts with some impurity in methylene blue, such as methylene violet or azure blue ( 1 ) . Azure blue will react with an alkali,
Two methods have been developed for the determinatioii of sodium pentachlorophenate. The copper method is superior because of its wider range and greater simplicity. ’The methylene blue procedure is interesting from the standpoint of the reactions described with regard to this and similar compounds with alkalies, etc., which may be of some value in determining these substances. These methods were worked out for solutions containing sodium pentachlorophenate, but they can also be applied to textiles, wood, etc., if one takes advantage of the fact that this compound is soluble in water, alcohol, and benzene (water a t 25’ 26.1%, acetone 32 to 33%, alcohol 32 to 33%, and benzene 0.1%) (3). LITERATURE CITED
(1) Holmes, W. C., Stain Technol., 2, 71-3 (1927). (2) Monsanto Chemical Go., private communication. (3) Monsanto Chemical Go., Tech. Bull. 0-23 (Rept. 1. 1946). (4) Zbid., 0-24 (Maroh 15, 1945). RECEIYED February 24, 1949.
Determination of Tryptophan with p-Dimethylaminobenzaldehyde Using Photochemical Development of Color JOSEPH R. SPIES AND DORRIS C. CHAMBERS .4llergen Research Division, Bureau of Agricultural and Industrial Chemistry, U. S . Department of Agricultwre, Washington 25, D . C . zooi
.
,
Figure 1.
I
Standard Curves for Tryptophan
L . Color develop& with light 8. Color obtained by addition of sodium nitrite to previously illuminated test solutions C. Color developed with sodium nitrite
lW0 reactions are involved 111 pievious methods tor the determination of tryptophan (2). Reaction I is the condensation of tryptophan and pdimethylaminobenzaldehyde in 19 N sulfuric acid to form a colorless product, and rea@ion I1 is the development of a blue color by oxidation of this compound with sodium nitrite. The photochemistry of these reactions has been reported ( 4 ) . This nOte describes a method for the estimation of tryptophan, using light as the agent for inducing oxidation. This method may have special applications in some of the many c*omplexproblems related to tryptophan analysis, but it is not intended to replace previously described procedures using sodium nitrite as oxidant. The rate of reaction 11, caused photochemically, was rapid a t first but became slow as the reaction approached completion a s 70, 88, 91, 93% of the potentially available color (as compared with that obtainable with sodium nitrite) developed on illumination of test solutions for 5, 20, 60,and 120 minutes, respectively. Illumination caused slight destruction or alteration of the cahromogen, because after 5 minutes’ illumination 93 to 94% of the total color obtainable by sodium nitrite alone could be obtained by subsequent oxidation with sodium nitrite. This effect was not progressive, because whether illumination was for 5 or 120 minutes 93 to 94% of the total color was subsequently obtainable with sodium nitrite. The transmittancy-concentration relationship obtained u hen reaction I1 was carried out photochemically or with sodium nitrite is shown in Figure 1, where the log of the per cent transmittancy is plotted against weight of tryptophan. C was obtained using sodium nitrite, L was obtained by illumination for 20 minutes, and N was obtained by sodium nitrite oxidation of test solutions previous!y illuminated for 20 minutes. That C and.N do not coincide shows that light destroys or modifies some of the chromogen. The conformity of these results to Beer’s law, for transmittancies ranging from 12 to 82%, is shown because C, L , and N are straight lines
1210
ANALYTICAL CHEMISTRY
Table I.
Tryptophan C o n t e n t of Proteins Using Light a n d S o d i u m Nitrite to Develop Color Tryptophan Content' Sodium nitrite
Protein
Light
% Casein @-Lactoglobulin Ovalbumin, denatured Conalbumin, denatured CS-54R ( 1 )
APPARATUS, MATERIALS, AYD M h T H D D S
Difference
%
%
1.75 2.50
* 0.01(3)* * O.Ol(3)
1.70 2.52
2.9 0.8
1.55
*
1.46
6.2
0.02(3)
oxidation could be used in an analytical procedure with a reasonable degree of accuracy if use of sodium nitrite were not feasible.
3.01 12.3 3 . 3 8 * 0.04(3) 0.991 0.907 * 0.013(3) 8.5 0.561 CS-56R( I ) 0.524 * 0.005(2) 6.6 Same protein samples used for analyses using light and sodium nitrite. Results are on ash- and water-free ,basis and va!ues were corrected as described in ($). Values obtained using sodium nitrite were determined by Procedure 0 and were taken from Table XI11 ( 8 ) . b Numbers in parentheses show number of determinations made on each protein.
The relative color intensities obtained on 20 minutes' illumination of tryptophan, casein, ovalbumin, and a cottonseed allergenic polysaccharidic protein CS-54R ( 1 ) were 97, 99, 100, and 99%, respectively, of the color intensity obtainable by a 60minute period of illumination of each substance. These data indicate that the photochemical development of color, reaction 11, is slightly more rapid for proteins than for free tryptophan and for greatest accuracy in an analytical method the exact time of maximum color development for tryptophan and each protein should be determined as was done previously for r e action I1 with sodium nitrite ( 2 , S). In the present work, however, a 20-minute pertod of illumination was adopted as sufficiently accurate to illustrate the method. Results of tryptophan analysis of several representative proteins using light to develop the color are compared in Table I with values obtained using sodium nitrite. The difference between values varied from a minimum of 0.8% with p-lacte globulin to a maximum of 12.3% with conalbumin, and the average difference for six proteins was 6.2%. The precision using photochemical oxidation was * 1%, while that using sodium nitrite was *0.88%. These results indicate that photochemical
Apparatus, materials, and general procedures have been described in detail (2-4). Reaction I for free tryptophan and for proteins can bc carried out by any appropriate procedure. The apparatus and procedure for illumination of. test solutions have been described ( 4 ) C, Figure 1, is the same as described in Figure 4 of ( 2 ) . 1, and .Y were obtained as described below using Procedure E ( 2 )for reaction I.
Procedure S. To 1.003 mg. of tryptophan and 250.8 mg. of p-dimethylnminobenzaldehyde (30 mg. per 10 ml.) in a 125ml. glass-stoppered Erlenmeyer flask were added 83.6 ml. of 19 N sulfuric acid at 25", solution A. In another flask 100 ml. of 19 N acid were added to 300 mg. of p-dimethylaminobenzaldehyde, solution B. Required volumes of solutions A and B were mixed immediately in 25-ml. glass-stoppered flasks to give test solutions containing 10 to 120 micrograms of tryptophan and 30 mg. of p-dimethylaminobenzaldehyde in a volume of 10 ml. The solutions were reserved in the dark at 25" for 19 hours. Each solution was then illuminated for 20 minutes a t 25" and transmittancies were read using a blank solution similar to the test solution, except that tryptophan waa omitted. The lowest transmittancy obtained over the wave length ranging from 580 to 620 mp was used for calculation of results. Results are plotted as L. To each solution was then added 0 1 ml. of 0.04% sodium nitrite solution and after standing for 30 minutes t r a n s mittancies were read. Results are lotted as N . For the proteins, Procedure 0 ($was used with approximately individual optimum times for reaction I (5). Solutions were illuminated for 20 minutes. Transmittancies were converted to weight of tryptophan from L,Figure 1. LITERATURE CITED
Spies, J. R., Bernton, H. S., and Stevens, H., J. Am. Chem. Soc., 63,2163 (1941). (2) Spies, J. R., and Chambers, D. C., ANAL.CHEM., 20,30(1948). (3) Ibid., 21,1249-66 (1949). (4) Spies, J. R.,and Chambers, D. C., J . Am. Chem. SOC.,70, 1682 (1948).
(1)
RECEIVED November 2, 1949. Paper V in series entitled "Chemical Determination of Tryptophan." For previous paper see ($).
Determination of Cholesterol Adaptation of Schoenheimer-Sperry Method to Photoelectric Instruments FRANCIS F. FOLDES A N D B. CRAIG WILSON D e p a r t m e n t of Anesthesia, Mercy Hospital, Pittsburgh, Po. HIS paper describes a modification of the SchoenheimerT Sperry method (I), simple enough for clinical w e and accurate enough for research purposes. A similar adaptation,
0.500, and 0.750 mg. were prepared with each series of determinstions. APPARATUS
published by Sperry (6),has limited distribution and is a t present out of print. The authors have tried to eliminate some of the difficulties of the method which in the past have been made ita introduction into the routine of any laboratory a tedious and timeconsuming procedure.
The a paratus was that described by S erry ( 4 ) , except that 15-ml. gfrtss-stoppered, heavy-duty borosi6aate glass centrifuge tubes (obtained from MacAllaster-Bicknell, Cambridge, Mass.) were substituted for the stirring rods and preserving jars.
REAGENTS
EXPERIMENTAL
With three exceptions, the reagents used are the m e aa described by Sperry (4). Instead of redistilled alcohol and acetone, C.P. acetone and absolute alcohol are entirely satisfactory. The 0.4yoaqueous digitonin solution has been replaced by an 0 . 5 7 digitonin solution in 50% alcohol, as recommended by Sobel and Mayer ( 2 ) . The use of alcoholic di i t o i h solution results in an evenly dispersed, fine precipitate. $he precipitate is not sticky, and the washed precipitate easily dissolves ill glacial acetic acid without the development of the sediment referred to by Sperry ( 4 ) . The stork standard used contained 2 mg. of cholesterol per ml., and three different working standards containing 0.250,
Ih attempting to adapt the method to the spectmphotometer and photoelectric colorimeter it soon became evident that, the development of the color and the determination of its intensity are the most sensitive points of the reaction. The time relationship of the color development and the relationship between the color ,intensity and the Beer-Lambert law seemed to be the moat important in this respect. In his earlier publications Sperry recommended that if the color is developed a t 25' C. it should be read a t 30 minutes ( 9 . 4 ) . In his
