ANALYTICAL CHEMISTRY
1020
Table VII. Interference 5 mg.Fe 0 . 2 5 mg. V
0.5mg.V
Interfering Metals Added to Trivalent Chromium Standards Cr Added, 0.0 10.4
y
0.5 10.0
0.0
0.9 9.3
1 mg. Mo
10.4
9.9
5 mg. Mo
0.8
10.4
0.1 8.8
5 mg. Cu
0.0 10.4
0.1 9.2
5 mg. Cd
0.0 10.4
0.0 10.3
5 mg. H g
10.4 0.0 10.4 20 mg. of NaCl added before diphenylcarbaeide. a
y
ACKNOWLEDGMENT
9.9
0.0 10.4 10.4
a
Cr Found, 1.0
oxidation in alkaline media. The method is proposed for general use in the determination of microgram quantities of chromium by proper adaptation of existing procedures.
19.4 0.0 10.4
CONCLUSIONS
.4 new method for the oxidation of chromium, using permanganate and sodium azide, and the development of a color with diphenylcarbazide using a phosphate buffer to improve stability, gave higher recovery and more stable colon than the more energetic oxidizing agents previously used, such as penulfate or bismuthate and avoided losses due to precipitation which occur 17-ith
The author wishes to thank N.A. Talvitie for his many valuable suggestions during the course of the investigation and D. H. Byers and H. E. Stokinger for their review and helpful criticism of the work. LITERATURE CITED
Agnew, W. J., Analyst, 56, 24-8 (1931). Ege, J. F.,and Silverman, L., IND.ENG.CHEM.,ANAL. ED.,19, 693-4 (1947). Feigl, F.,“Qualitative Analysis by S p o t Tests,” 3rd ed.,pp. 2623,New York, Elsevier Publishing Co., 1946. Gentry, C. H. R., and Sherrington, L. G., Analyst, 75, 17-21 (1950). Hillebrand, W. F., and Lundell, G. E. F., “Applied Inorganic Analysis,” pp. 411-14,New York, John Wiley &Sons, 1929. Maunsell, P. W., New Zealand J. Sci. Techml., 26,9443 (1945). Perry, J. H., “Chemical Engineers’ Handbook,” 3rd ed., p. 174, New York, McGraw-Hill Book Co., 1950. Sandell, E. B., “Colorimetric Determination of Traces of Metals,’’pp. 189-99,New York, Interscience Publishers, 1944. Sandell, E. B., IND. ENG.CHEM.,ANAL.ED.,8,336 (1936). Urone, P. F., and Bnders, H. K., ANAL. CHEM.,22, 1317-21 (1950). Urone, P. F.,Druschel, M.L., and Anders, H. K., Ibid.,22,472-6 (1950). Welcher, F. J., “Organic Analytical Reagents.” Vol. 111. pp. 433-6,Ken, York, D. Van Nostrand Co., 1947. RECEIVEDfor review January 7, 19.52. Accepted l p r i l 14.1952.
Determination of Thiamine in Rice and Rice Products Rapid and Simple Method CARL M . LYMAN, ROBERT ORY, MARY TRANT, AND GENE RICH Texas Agricultural Experiment Station, Texas Agricultural and Mechanical College System, College Station, Tex.
ICE constitutes a major part of the diet of a large percentage
’
of the world’s people, and beri beri is still a problem among many oriental peoples. Most of the thiamine in rice is lost during the milling process because it is concentrated in the brown outer coating, which is removed during the polishing, and unpolished rice is subject to rapid deterioration during storage. It thus becomes a practical necessity to polish rice in o r d s to prevent loss. It would appear possible to polish rice to the point where the product could be safely stored and still leave enough thiamine to supply the dietary needs for this vitamin, if thiamine could be determined without use of expensive scientific equipment. The present procedure was developed with the hope that it might meet this need. In contrast t o many other foodstuffs, most of the thiamine in rice is in the free form-approximately 85%, as indicated by experiments of the present investigators. This makes it possible to omit the enzymatic digestion of the sample, whereby thiamine is freed from cocarboxylase. The method reported here is based on the color developed with diazotized paminoacetophenone. The use of this reaction for the determination of thiamine was first described by Prebluda and McCollum ( 2 ) . The first part of the problem consisted in the establishment of conditions for the rapid preparation of extracts which may be used for the direct development of the color without
preliminary adsorption and elution procedures. The removal of impurities by the precipitation of barium sulfate in the extract proved important in this step. Before the establishment of the structure of thiamine, Williams and coworkers ( 3 ) found the use of barium hydroxide an effective means of eliminating certain impurities during the isolation of the crystalline vitamin from rice polish. Tests next established the conditions for the quantitative development of the color using reagents prepared and used a t room temperature. Because the amounts of the several reagents have been adjusted for use on rice extracts prepared under specific conditions, the procedure reported here may not be widely adaptable to other types of foodstuffs without further modification. The direct application of the color reagents t o rice extracts resulted in the development of a small amount of colored material extractable with xylene, which was not due to thiamine. This difficulty was eliminated by the use of mixtures of iso-octane and xylene in the place of xylene alone for the extraction of the colored compound. REAGENTS AND MATERIALS
p-Aminoacetophenone Solution. Dissolve 2.5 grams in 9 ml. of concentrated hydrochloric acid, and dilute to 100 ml. Sodium nitrite solution, 23 grams in 100 ml. of water.
V O L U M E 24, NO. 6, J U N E 1 9 5 2 Table I. Description of Sample Polished rice 1 Polished rice 2 Brown rice 1 Brown rice 2
1021
Recovery of Thiamine Added to 10-Gram Rice Samples Thiamine Found in Rice Y 12 11 8 8 34 25 31 25 31
Thiamine Added Y 13.3 13.3 20.0 20.0 20.0 20 0 20 0
20 0 20 0
Thiamine Found in Rice with Thiamine Added Y 25.3 25.2 28.2 27.6 53.2 48 6 51 6 47 5 50 7
Recovery of Added Thiamine
When this is done, more care is required in the separation of the octane-xylene extracts in order to have sufficient volume for color comparisons. PREPARATION OF STANDARDS
%
Add 0 . 5 , 1.0-, and 1.5-mI. portions of the stantlard thiamine solution containing 10, 20, and 30 micrograms of thiamine, respectively, to 160 nil. of water and develop the color as with the rice extract.
100 107 102 97 96
98
EVALUATION T E S T S
103 93 99
Av.
Sodium hydroxide-sodium bicarbonate solution, 60 grams of sodium hydroxide and 84 grams of sodium bicarbonate made up to 1 liter. Phenol-alcohol solution, 0.4 gram of phenol per liter of 95% alcohol. Sulfuric acid, 0.2 A'. Sodium hydroxide, 0.2 A'. Barium hydroxide, 0.3 LV. Bromocresol green indicator, 50 mg. dissolved in 10 ml. of alcohol and diluted t'o 100 ml. with water. Phenol red indicator, 50 mg. dissolved in 10 ml. of alcohol and diluted to 100 ml. with water. Xylene-octane mixture, 50% xylene and iso-octane (2,2,4-trimethylpentane, practical grade). Ashless filter paper tablets. Standard thiamine solution, 20 mg. of thiamine hydrochloride dissolved in water, and 1 ml. of 0.2 sulfuric acid added. Dilute to 1 liter, 1 ml == 20 micrograms of thiamine hydrochloride, PREPARATION OF EXTRACT
Boil 15 grams of finely ground rice for 15minutes with 150 ml. of 0.2 &Vsulfuric acid and one tablet of ashless filter paper broken into small pieces. On boiling, the mixture becomes viscous and then thin. Stir until it is t'hin enough to boil freely. Cool and add 90 ml. of 0.3 S barium hydroxide and 1 ml. of bromocresol green indicator. Add 0.2 N sodium hydroxide with a pipet until the color of the preparation first becomes definitely blur. This gives a p H between 5.0 and 5.4. On adding base, the indicator changes from yellorr to green and finally to blue. Dilute to 240 ml. in a graduated cylinder, mix, and filter through a fluted filter paper. The preparation filters rapidly, giving a transparent and almost water-xvhite solution. The indicator dye is absorbed on the precipitate and is thus removed. Collect 160 ml. of the filtrate, equivalent to 10 grams of rice. Start the thiamine determination within a short time after the filtration is complete; othern-ise some loss of thiamine may r ~ sult.
As a means of evaluating possible sources of error such as destruction of thiamine during the procedure, incomplete development of color, or incomplete extraction of the colored compound from the extract, recovery tests were carried out by adding standard thiamine solutions to a numbsr of samples of rice and then analyzing the materials with the added thiamine. The data in Table I show good agreement between the values obtained on duplicate determinations and satisfactory recoveries (93 to 107%) of thiamine added to rice samples. I n obtaining the data reported in Table I the color measurements were made bv the use of a Reckman spectrophotometer in order that the reliability of the color development might be more accurately measured. The wave-length selection setting of the instrument was a t 520 mp. For processing control a simple block comparator is entirely adequate. Under the conditions reported here, rice with as little as 1 microgram of thiamine per gram yields a distinct color which can be readily estimated visually. I n order to evaluate the new method further, samples of brown rice were analyzed with the simplified colorimetric procedure, with the thiochrome method ( 1 ) after preliminary enzymatic digestion. and with the thiochrome metliod omitting the enzymatic digestion, Comparison of the values obtained with the thiochrome method carried out in the two ways gives a measure of the proportion of the thiamine in rice Lvhich is present in the free form.
99.4
Table 11. Thiamine Content of Brown Rice Samnle A
B
C
Thiochrome Method With enzymatic Without enzymatic digestion digestion 3.8 3.6 4.1
3.1 3.2 3.4
Simplified Colorimetric Method 3.2 3.0 3.4
DETERMINATION OF THIAMINE
To 160 ml. of the estract in a 1-liter bottle or Erlenmeyer flas!i, add 100 ml. of the phenol-alcohol solution. Add 4 drops of t,he sodium T hydroxide until tht, phenol red indicator and then 0.2 -I solution changes from yellox t o red. Mix the following reagents in a small flask and add the mixture immediately to the extract: 1.6 nil. of sodium nitrite solution, I .O nil. of p-aminoacetophenonr solution, 20 ml. of Tvater, and 50 ml. of sodium hydroxide-sodium bicarbonate mixture. .4dd 8 ml. of t,he xyleneoctanc misture and shake vigorously at 1.5-minuteintervals for 1 hour. Soparate enough of the toll layer (sylene-octane) for a roloi, comparison by pouring into a 30O-ml. Erlenmeyer flask and pipetting off the top layer, vdiich collects in the neck of the flask. Occasionally there is a slight cloudiness due to small droplets of \rater. This can be quickly rcmoved t)y adding a little anhydrous sodium sulfate and then pouring o f f t,he liquid into a clean tube. X separators funnel may be usetl, but this is less convenient. The amount of thiamine prcwnt in the unlinovn is determined by faomparing the color with standards prepared with 10, 20, and :30 micrograms of t,hiamine. For processing control, a simple block comparator is adequate. For more accurate work a photoelectric colorimeter equipped with a color filter xhich should have a maximum transmitt,ance at ,520 nip or a spectrophotometer may be used. If desired, the entire procedure can be carried out on half quant.ities of materials, including the rice samples and all reagents.
The representative data given in Table I1 show that more than 80% of the thiamine of ground rice is present in the free form, and that the values obtained for the free thiamine by the thiochrome method and by the simplified colorimetric procedure are in good agreement. DISCUSSION
It is recognized that the smsll amount of thiamine present in rice in the phosphorylated form is not estimated by this method. For the purpose for which the method was developeti-processing control-this is of no consequence. LITERATURE CITED
IIennessy, P. J., and Cerecedo, L. R., J . Am. Chem. Soc., 61, 179 (1939). (2) Prebluda, H. J., and McCoilum, E. V.,Science, 84, 488 (1936). (3) ITilliams, R. R., Waterman, R. E., and Keresxtesy, J. C., J. Am. Chem. Soc., 56, 1187 (1934).
(1)
RECEIVED for review September 8, 1951. Accepted Kovember 29, 1951. Investigation supported in part b y grant-in-aid from the Williams-Waterman Fund for the Ccmbat of Dietary Diseases, Research Gorp., New York, N. Y.
