Combined Determination of Riboflavin and Thiamin in Food Products R. T. CONNER AND G. J. STRAUB Central Laboratories, General Foods Corporation, Hoboken, N. J.
N
UMEROUS chemical and microbiological methods for the separate determination of thiamin and riboflavin have appeared in the literature. The procedure
The aliquot to be taken from the supernatant liquid will be dependent upon the amounts of thiamin and riboflavin present in the extract. A single aliauot may be used if it cantins about
described below makes i t possible to determine both vitamins on the same sample, and has enabled the authors t o make complete analyses on as many as 8 to 12 samples in the course of a 7-hour day. The number is limited only by the estraction units in operation and available personnel. The method is an extension of the one proposed for thiamin (t),which includes a review of the literature on the determination of this vitamin. Following the discovery by Knhn and Wagner-Jauregg (9) that riboflavin concentrates give off a yellow-green fluorescence unon aebivation .~~ bv lieht of certain wave leneths. numerous &&,hods were proposed for determining the vita&, 'based upon measurement of the fluorescence of riboflavin or of its dearadation ~~~~~
~~~~~~
~
I
~~~~
.. . . . ~ ~ ~ ~ ~ ~
~
~~~
earth pre asation. ThiGtLmin was then eluted with a mixture of meth ?alcohol, pyridine, and water, and the fluorescence of the resding mlution determined visually or by means of aphotocell as described by Cohen (1). In 1935 Koschara (7) proposed a method based on measurement of t,he light absorption of the vitamin extract rather than on its fluorescence. Prior to measuring its light ahsorptiou, Kosehsm treated the estract with potassium permanganate to oxidize interfering pigments, and destroyed excess potassium permanganate with hydrogen peroxide. Recently Hodson and Norris (6) proposed a method in which 0.25 N sulfuric acid was used to extract the riboflavin. Following the extraction, the solution was filtered, and the filtrate wns I
I
I
1
while stmmous chloride reducegthe interfering pigments. Th$ riboflrwin wa6 then reoxidised by bubbling air through the solution and its fluorescence measured by the use of a fluorophotometer. Ferrebee (3) has recently introduced two fuller's earth preparations, Floridin and Supersorb, for the adsorption of riboflavin. The vitamin was eluted from these adsorbents with a solution of 20 per cent pyridine in 2 per cent acetic acid, and the eluate treated with potassium permanganate and hydrogen peroxide to measuring the fluoreseenoe. Other methods (4, 1S-15) ased on the above prooedures have been proposed.
1'
Description of Method The sensitivity of riboflavin to light makes it necessary to carry out the entire analysis under controlled illumination. The authors used Eastman safety lights equipped with series OA Wrattan light filters. For the combined determination of thiamin and riboflavin, a 3- to 5-gram sample of the finely pulveriaed food material is prepared as described in the thiamin method (9). The weighed sample is placed in the specially designed extraction tube (8) and is extracted with 50 00. of 0.04 N sulfuric acid on a boiling water bath for approximately 1 hour, with continuous stirring. A t the conclusion of the extraction, each stirrer is carefully washed down with 5 cc. of distilled water delivered from a graduated pipet and the tube is cooled to room temperatm with running water. Ten cubic centimeters of a five per cent solution of clarase (9)are added to each tube and the contents are thoroughly mixed with a glass stirring rod. The tube, containing the stirring rod, is then incubated at 45' C. for 2 hours. Following the incubation period, each stirring rod is carefully washed down with exactly 1 ce. of distilled water and the extraction tube is cooled to room temperature. Each tube is then centrifuged at high speed until & clear supernatant liquid is ohtained.
FIGURE I
column containing the Decalso is then detached and transferred to a 125-cc. elass suction flask where it is readqfor elution of the thiamin with 25 per c e n t potassium chloride as previously outlined (S). The lower column containing the Supersarb is detached from the vacuum receiver, which is washed with hot water and dried by pulling air through it. The column is-then attacxed to the receiver again and the riboflavin eluted with 25 cc. of a solution of 20 ner cent pyridine in 2 per cent acitic wid added from an automatic pipet, the flow through the column being adjusted to 1 cc. per minute. (The pyridine solution should have a low fluorescence and be stored in a dark bottle.) Additional small portions of the eluant are then passed through the column until the volume of the filtrate is approximately 50 cc. as indicated by the graduated readings on the vacuum receiver. In analyzing most food materials, the filtrate is then made up to exactly 50 CC. with the pyridine-acetic acid mixture. However, for concentrates of riboflavin it may be necessary to make up t.he filtrate to a larger volume, so that a 1&cc. aliquot will contain from 0.5 to 1.0 miorcgram of the vitamin. A l h c . aliquot of the riboflavin filtrate is pipetted into a 50-00. brown bottle or tlaask, 1 ec. of a 4 per cent solution of
INDUSTRIAL AND ENGINEERING CHEMISTRY
386
Vol. 13, No. 6
standard solution must be treated in the s m e manner as the sample, since the fluorescence of riboflavin solutions is dependent upon the solvent employed as well as upon the hydrogen-ion concentration. A blank correction for the standard solution is obtained by determining the fluoreti cenee of 15 cc. of a solution taken from a mixture of 1 ce. of 4 per cent potassium permanganate, 3 cc. of 3 per cent hydrogen peroxide, and 15 ce. of the pyridine-acetic acid reagent. A "complete blank" (e), consisting of all the reagents used throughout the method, is also run through the entire procedure and serves as a blank for the thiamin determination &s well as for the riboflavin. In the case of riboflavin the value for the blank is subtracted from fluorescence readings of the sample.
Critical Study of Steps in Procedure OF LIQRT ON RIBOFLAVIN 3 AT VARIOUS PH VALUES.
investigators had rea t r i b o f l a ~ nis sensitive t o authors studied the extent d whether i t is influenced
4s
FIGURE 2. MULTIPLE A
OF RIBOFLAVIIY B Y ~rlvwrx r TABLE I. DESTRUCTION PH VALUES
Nature oi Illumination
Diffused daylight
Artifioihl light
Loss of Riboflavin Following Di5crent pN of Riboflavin InterYBls' Solution 1 day 2 days 5days 7 days % % % % 2.2 40 96 98 .. 4.0 04 96 96 .. 6.0 82 94 98 ..
8.0
72
2.2
12
4.0 6.0 8.0
15 23 l6
96
37 l6 38 37
.. .. ..
.. 0
VANLYU~
96 16 60 58
55
.. ..
..
..
..
28
~y m e p n
01 me SVIUCIVII.
Solutions oi pure riboflavin were prepared in pbosphattcitric soid buffers to give pH values ranging from 2 to 8. The solutions were placed in white glass bottles having tight screw caps, and a layer of toluene was added to prevent mold growth. One set of solutions was exposed to the diffusedlight of the laboratory, another set to artificial light, and a control set was kept in a dark room. The amount of riboflavin present in each solution was determined fluorometricallv at the beginninr of the exneriment
78 79 6s
0 0 0 0
conducted at the same nH value. This was accomnlished by
Initial content of riboflavin in each solutia~nW B Q approximately 50
micrograms.
-
l.ft,tl?R." --- int,ervai ------ .-I of a potassium permanganate is added and. L-. minute, 3 cc. of B 3 per cent solution of hydrogen peroxide. The pre by diluting a 30 per latter oan be solution of hydrogen peroxide &uperoxal) a few minutes before the determination is to be run. The potassium permanganate solution should be prepared fresh daily. The reaction mixture is shaken vigorously and then allowed to stand until all efferveEicence has ceased, as the presence of bubblm Will introduce an error in the fluorescence readinss. Fifteen cubic centimeters of the solution are pipetted into cuvette of the fluoraphotometer, and fluorescence readings are made after adjustin the instrument (S). A Corning glass filter No. 511 is usef for transmitting the incident light and No. 351 for the fluorescent light. Both filters are of 2-mm. thickness. Although in the range 0.2 to 2.0 micrograms a linear relationship exists between the amount of riboflavin and galvanometer readings, it has been found advisable to make a daily determination on a standard solution of riboflavin in pyridine-acetic acid instead of depending entirely upon a calibration e w e . The standard solution is made up to contain 0.0856 microgram of riboflavin per ec. and should be kept in a dark-colored bottle away from light. No destruction of the vitamin occurs under these conditions. For determining the fluorescence of the standard solution, a 15-cc. aliquot Is treated with 1 ec. of the 4 per cent solution of potassium permanganate and 3 ee. of 3 per cent hydrogen peroxide. Fifteen cubic centimeters of this solution, representing 1 microgram of the vitamin, are then pipetted into the cuvette of the fluomphotometer and its fluorescenceis determined. The
a
From Table I, it will be seen t h a t 1 day's exposure t o the diffused light of the laboratory caused a n extensive destniction of ricoflavin, particularly in the samples at pH values above 2.2. After 5 days' exposure, practically none of the original riboflavin remained in any of the solutions. In the solutions exposed t o artificial illumination, the destruc-
TABLE11. ADSORPTION THIAMIN AND RIBOFLAVIN BY UECALSO vitamin Content of Solution l i t e r Passing through Deodao Beforo Adsorption Column Thiamin Riho5avin Thiamin Riboflsuin .wcrogl.oms
5.00
5.00 5.00
?ABLE LLL.
Amount of Riboflavin
bh~".--A
MiGlOgrO?M
3.00
5.00 10.00
.w;mog,am
.Micromans
0 0 0
4.95
3.06 8.92
~ L U T I O N OF MBOFLAYIN FROM ADSORBENTS
WITH PYRIDINE-ACETIC ACIDMIXTURE Galvanometer Readings on Riboflavin Solution* After elution After elution
ANALYTICAL EDITION
June 15, 1941
387
The combination of filter 511 for transmission of the inciCORNING GLASSFILTER COMBINATIONS FOR FLUORO- dent light with filter 351 for the fluorescent light gave the METRIC DETERMINATION OF RIBOFLAVIN highest reading with the lowest blank, and consequently Galvanometer Readings these were selected for use. For activating the riboflavin Incident Light Fluorescent Light Riboflavin Filter" Filtera solution Blank t o fluorescence, filter 511, transmitting light of approximately 511 + 038 72 368 18 445 mp, is superior to 584 which transmits ultraviolet light. 511 4- 038 62 12 038 f 368 Although filter 368 gives a high fluorescence reading, there 511 + 038 351 32 2 038 29 511 + 038 351 2 is also a correspondingly large blank, indicating transmission 17 511 f 038 440 + 401 6 37 440 f 401 511 7 of wave lengths other than those derived from the fluorescence 73 351 511 5 of the riboflavin. 351 + 428 511 40 3
TABLE I\-.
-
+
a
584 440 401 6 15 584 351 26 584 368 Filter numbers are those used b y Corning Glass Works.
T.4BLE
J'.
2 2
a
COMPARATIVE RESULTSOBTAINED BY CHEMICAL AXD BIOASSAY PROCEDURES
Slaterial Processed cereal KO.1 Frozen peas
Riboflavin Content Bioassay Chemical Mzcrograms/gram MMzcrograms/grain 2.50 1.55
2 29 1.45
tion proceeded a t a slower rate. At higher p H values the destruction is particularly rapid. S o loss occurred in any of the control solutions. ADSORPTIONSTUDIESWITH RIBOFLAVIN. An essential feature of this procedure is the use of a single extract of the food material for the combined determination of riboflavin and thiamin. This involves the assumption that none of the riboflavin is adsorbed on the Decalso column. To test 'this assumption, solutions containing 5 micrograms of thiamin and varying amounts of riboflavin were prepared at a pH value (4.5) corresponding to that of the food extract following enzymatic hydrolysis. Each solution was then passed through the activated Decalso column in the usual manner and the amount of riboflavin adsorption determined.
The results given in Table I1 show that no appreciable adsorption of riboflavin from solutions containing 3 to 10 micrograms occurred on the Decalso column. h study was also made of the adsorption of riboflavin on the two fuller's eart,h preparations (Floridin and Supersorb). Both adsorbents were reduced to 60- to 80-mesh and activated according to Ferrebee's directions (3). Solutions containing various amounts of riboflavin at pH 4.5 were passed through columns of each adsorbent at the rate of 1 cc. per minute. Each column was then washed thoroughly with hot distilled water and finally eluted with 25 cc. of the pyridine-acetic acid mixture. Fluorescence readings Tyere made on the eluates and on control solutions of riboflavin in pyridine-acetic acid containing the same amounts of the vitamin as originally passed through the columns. The results obtained are given in Table 111. Although the recovery of riboflavin from the Supersorb was excellent, the recovery from Floridin was incomplete. As it mas subsequently established that adsorption of riboflavin was quantit'ative on both adsorbents, the low recovery from Floridin is apparently due to failure t o obtain complete elution of the riboflavin from this column with 25 cc. of the pyridine-acetic acid mixture. Therefore, the Supersorb was selected for use in the method. SELECTIONOF LIGHT FILTERS FOR DETERMIXATION OF RIBOFLAVIN. Various Corning glass filters were tested in the fluorophotometer to determine their adaptability for transmitting the required incident light and fluorescent light. I n making the tests, 15 cc. of a n aqueous solution of riboflavin, containing 1 microgram of t,he vitamin, were placed in the cuvette of the fluorophotometer and its fluorescence was determined using various filter combinations. Readings were also made on a blank consisting of 15 cc. of distilled water. The results are shown in Table IV.
Comparison of Results by Bioassay and Chemical Procedures I n order to test the validity of the method for determining riboflavin, bioassays were run on certain samples using a rat-growth method, with the results given in Table V. I n conducting the bioassays, the following basal diet was employed : Labco vitamin-free casein, % Cornstarch, % Osborne-Mendel salt mixture, % Hydrogenated fat (Primex), % Cod liver oil. '-3 Laboo ricepoliih concentrate No. 2, 9; Thiamin chloride, 20 micrograms per r a t per day Pyridoxin, 10 micrograms per r a t per d a y
20 67 4 4 2
a
From Table V, it is apparent that the chemical values are in good agreement with those obtained by bioassay, and that the difference in results is well within the range of error inherent in the biological method. TABLE VI. Sample
Wheat germ Wheat \-ellow corn
PERCENTAGE
Riboflavin Content of Sample .Wicrograrns/ gram 5.56 1.74 1 1G
RECOVERY O F ADDED RIBOFLAVIS Riboflavin Added .Micrograms.'
Recovery of Added Riboflavin
gram
.Ificroyrams
2 50 4.00 2.00
2.49 3.85 1.93
Recovery cc
99 91 99
Recovery of Riboflavin _Addedto Foodstuffs In order to test the reliability of the method, various amounts of pure riboflavin were added t o food extracts and percentage recovery of the vitamin n a.; determined, with the results shown in Table T?. The recovery of added riboflavin was over 90 per cent in each case, which indicates that the procedure does not lead to appreciable 106s of the vitamin. TABLE VII. RIBOFL.IVIF; COSTENTOF FOODSTUFFS llaterial
Kihoflavin Content or Itange .IficruyrniliS/I"I.IL.II
Wheat Yellow corn White corn Rice polishinrz Rice bran Wheat germ Wheat bran Skim milk oudt.1 Whey p o d e r Frozen peas Frozen broccoli Fresh spinach Fresh lima beans Fresh broccoli Fresh string beans
0 89-2 0:I 02 1 27-2 3 2 .jL
(J Xd-2
2 41
3 78-5 5li
3 17 19 00-29 00 39 00 1 90 a 00 1 17-1.47 1 35-1 56 1.76 1.61
Riboflavin Content of Foodstuffs The method has been applied to various food materials with the results shown in Table YII. These values are in agreement with those reported by Hodson and Norris ( 5 ) for similar materials.
388
INDUSTRIAL AND ENGINEERING CHEMISTRY
Summary A rapid and accurate method has been developed for determination of thiamin and riboflavin on the same sample. The method is in close agreement with biological assays and has been applied to grains, milk products, and fresh and frozen vegetables. A study of the destruction of riboflavin by light in aqueous solutions a t pH values ranging from 2 to 8 showed that rapid destruction of the vitamin occurred, irrespective of the pH, when the solutions were exposed to the diffused light of the laboratory. The destruction from exposure to artificial illumination was slower and appeared to be dependent upon the pH. Ferrebee's procedure for the adsorption of riboflavin on Supersorb has been modified to use- a smaller extraction column. A study has been made of Corning glass filters suitable for the fluorometric determination of riboflavin. Filt'er 51 1 has been selected for transmitting the incident light and No. 351 for transmitt'ing the fluorescent light.
Vol. 13, No. 6
Literature Cited Cohen, R. H., Act. Brar. .veerland. Physiol. Pharmacal. Mierebid., 5, 18-19 (1935). Conner, R. T., and Straub, G. J., IND. ENG.CHEM.,Anal. Ed., 13, 38C-5 (1941).
Ferrebee, J. W., J . Clin. Investigation, 19, 251-6 (1940). Hand, D. D., ISD. ESG. CHEM.,Snal Ed., 11, 306-9 (1939). Hodson, A. Z., and Norris, L. C., J . Biol. Chem., 131, 621-30 (1939).
Karrer, P., and Fritasche, H., Helv. Chim. Acta, 18, 911-14 (1936).
Koschara, W., 2. physiol. Chem., 232, 101-16 (1935). Kuhn, R., and Moruzzi, G., Be,., 67, 888-91 (1934). Kuhn, R., and Wagner-Jauregg, T., Ibid., 66, 317-30 (1933). Sullivan, R. A., and Xorris, L. C., ISD. ENG.CHEM.,Anal. Ed., 11, 535-9 (1939).
Supplee, G. C., Bender, R . C., and Jensen, 0. G., Ibid., 11, 495-8 (1939). (12) Teisburg, S. M.,and Levin, J., Ihid., 9, 523 (1937). J . Am. (13) Whitnah, C. H., Kurnerth, B. L., and Kramer, M.M., Chem. Soc., 59, 1153 (1937). PRESENTED before the Division of Agricultural and Food Chemistry, Joint Program on Vitamins with the Division of Biological chemistry, at the lOlst Jfpeting of the American Chemical Society, St. Louis, 310.
Ethylene Glycol Determination in and Removal from Cornmercial Alkyl Ethers of Diethylene Glycol MARGARET K. SEIKEL'. Massachusetts Institute of Technology, Cambridge, ;\lass.
I
N THE course of work in the Research Laboratory of Organic Chemistry, samples of commercial ethyl and methyl ethers of diethylene glycol have been found to contain 28 and 8.5 per cent ethylene glycol, respectively. No previous report of the presence of this impurity has been located. Two independent procedures for the determination of the amount of this ethylene glycol, the ditrityl ether method and the lead tetraacetate oxidation method (S), have now been developed and yield closely checking results. I n the first, the glycol is converted into its ditrityl ether which can be separated from the other monotrityl ethers and by-products because of its relative insolubility and weighed; this determination is rapid and yields excellent proximate results. I n the second, the glycol is oxidized with excess lead tetraacetate in acetic acid, the decrease in the oxidizing power of the solution serving as a direct measure of the glycol content for the alkyl ethers of diethylene glycol are substantially unaffected; this method requires a longer time and a more complex procedure, but gives more accurate results. The assumption that ethylene glycol is the only 1,2-glycol and the only easily oxidizable impurity present is supported by the close agreement between the two methods. Removal of the ethylene glycol from commercial alkyl ethers of diethylene glycol by ordinary fractionation, the method of purification reported previously by Veraguth and Diehl (6) and Whitmore and Lieber (7), is very inefficient (almost ineffective in the case of the ethyl ether), owing to the proximity of the boiling points of ethylene glycol (197"C.), diethylene glycol monomethyl ether (191' C.), and diethylene glycol monoethyl ether (198' C.). It can be removed readily, however, by a solvent partition method using benzene and water.
Ditritpl Ether Method Heat for 15 minutes on the steam bath in a test tube 0.25 ml. of diethylene glycol monomethyl or monoethyl ether with 1 to 2 ml. of pyridine and trityl chloride in excess over that amount 1 Present address, Chemistry Department, Kellesley College, Wellesley, Mass
calculated t o convert all the ethylene glycol to its ditrityl ether and the diethylene glycol alkyl ether t o its trityl ether. Leach with water, ice until the oil stiffens t o a partially crystalline ma&, and wash by decantation with several portions of fresh water. Extract the gum with 20 ml. of 95 per cent alcohol. The alcoholinsoluble (6) precipitate is the ditrityl ether of ethylene glycol and should melt around 170-180"; if it melts much lower, reextract. From the weight of this residue the per cent of glycol in the original can be calculated. In the present work the determinations shown in Table I were made. Identity of the impurity as ethylene glycol was attested by the following facts: The alcohol-insoluble precipitate obtained above melted a t 187' after two recrystallizations from acetone and did not lower the melting point of an authentic sample of ethylene glycol ditrityl ether (m. p. 187-187.5'). TABLE I. ETHYLENE GLYCOL CONTENT O F COMMERCI.4L ALKYL ETHERSOF DIETHYLENE GLYCOL Monomethyl Ether
Ethylene Glycol By ditrityl By lead tetraether method acetate method
7%
OI /O
Commercial material, 1937 Commercial material 1940 Artificial mixture, 1014% glycol Artificial mixture 9.8% glycol Artificial mixture: 1.95% glycol
9.2,8.6,8.4 9 . 2 , 8.4,7.9 8:7#'8.6 10.1,lO.l 10.1,ll.l 2 ,?, 2; 7", 1,7,
Highly purified (see below) C Highly purified, refractionated
Trace
... ...
1.YD
...
.. .
o :ii-o
.45c 0.42-0.45 Monoethyl Ether Commercjal materjal, 1937, discolored 24, 27 Commerc!al mater!al, 1937, colorless 28 2a:oa 2 8 . 0 26 26, 28 Commercial material 1940 28.2, 28.2 1937 material dehydr(ated and distilled 28: 29 Artificial mixture 27.270 g!ycol 25,24, 24 1937 material alltially purified 4.5,6.3 1937 material Eighly purified Less than 0.5 0 : i - 0 8. 0 7-0.W Monobutyl Ether Commercial material Not more than a trace a glvcol 8 Usinn Using enounh enough tritvl trityl chloride for 50% 50% glycol. b Using Using enough trityl chloride for 10% &col. glycol. C See paragraph, "Removal of Ethylene Glycol". Gly,col". d Curves of % glycol plotted against time did not flatten out but drifted slowly upward. However, between limits given there was a definitely sharp change of slope.
... ...
