Determination of Some Components in Corn Sirups by Quantitative Paper Chromatography ROY
L. WHISTLER
and
JOHN L. HICKSON
Department o f Biochemistry, Purdue University, Lafayette, Ind.
Paper chromatography has been demonstrated as a practical procedure for the quantitative separation of nine corn sirup components, These components, in order of decreasing chromatographic mobility, have been tentatively identified as: glucose, maltose, isomaltose, maltotriose, isomaltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose. The nine components, separated chromatographically-, were quantitatively determined by absorptiometric techniques. A number of corn sirups differing in degree of hydrolysis were analyzed.
P
RODUCTIOS control in corn sirup manufacturing has been based primarily on determinations of moisture, ash, pH, color, and reducing sugars (8). Little has been known about the composition of the sugar mixture except its n-glucose content, determinable from Sichert-Bleyer reducing values (29),by various fermentation procedures ( 2 L . 30). or recently by the specific action of the enzyme glucose dehydrogenase (33). For the deteimination of the individual oligosaccharides there have been no such direct methods Maltose, maltotriose, and maltotetraose have been estimated in several sirups by fractional distillation of the methyl (15) or propionyl ( 1 6 ) derivatives, but the procedures were not adaptable to routine analysis. I n the present work, paper chromatographic methods ( 2 , 7 , 18, 23, 18, 36) for the separation of sugar mixtures have been applied BTith absorptiometric methods of determination to provide methodoloo for the analysis of corn sirups.
Table I. Comparison of Irrigating Solvents in Chromatography of Corn Sirup Components Glucose
bfove-
Irrigating Solvent n-BuOH, pyridine, Hz0 EtOAc. pyridine,
€120
(Epiphasej (One phase) (One phase) (One phase) n-BuOH. E t O H , H20
n-BuOH E t O H , 27% S H ~ O H
Volume Ratio 6:4:3
(4)
Rglucoae
Time, Hours 33
ment. Mm. 228
Maltose 0.60
Rlaltotriose 0.38
31 40 56 78 23
105 170 273 354 293
0.98 0.28 0.32 0.32 0.65
0.08 0.14 0.10 0.43 0.38 0.46 330 mni.
,..
8 : 2 : 1 (.W) 8 : 2 : 1 (38) 8 2 . 1 (36) 8 : 2 . 1 (36) 5 : 2 : 5 (19) 10:4:3 5:2:2 5:::2
23 23
10: 1 : 2 (36) 1 0 : 1 : 2 (36) 1 0 : 1 : 2 (36) 2:l:l
31 40 78 36
75 109 188 321
0.24 0.33 0.26 0.64
0:12 0.08 0.41
54
199 268
0.60 0.56
0.36 0.31
4:1:5 4:1:5
..
81
,, 0.65 256 0.68 Trisacch. moved
SELECTION OF CONDITIONS FOR CHROMATOGRAPHIC SEP4RATIONS
Irrigating Solvent. Several solvents were compared for separating corn sirup components on Whatman No. 1 filter paper (chromatographic grade) a t 2.5' to 30" by the descending technique (26') (Table I ) . Solvent selection was based on rate of movement of glucose and efficiency of separation of oligosaccharides. The epiphase of a mixture of ethyl acetate, pyridine, and water (5:2:5 volume per volume) seemed useful by these criteria. In practice it was
found that equivalent results were obtained with a monophase of solvent made up of 10 volumes of ethyl acetate, 4 volumes of pyridine, and 3 volumes of water. Also used with equal success was a mixture of n-butyl alcohol, ethyl alcohol, and water (2: 1: 1 volume per volume). This solvent was used for most of the analyses reported here. Indicator Spray Reagent. Aniline hydrogen phthalate (as), aniline hydrogen oxalate (H)and , ammoniacal silver nitrate were compared as chromatographic spray reagents. Aliquots of glucose or maltose solutions (0.1 y per p1.) on filter paper w-ere dried, sprayed with reagent, and heated a t 115' to 120" for 10 to 15 minutes. From observations recorded in Table 11, aniline hydrogen phthalate was chosen as the indicator spray reagent.
Table 11. Comparison of Developers on Corn Sirup Cornpon ent s (Spotted on paper) Component Developed ~Glucose, y Maltose,.-r_ Developer 0.5 1 1 10 Aminoniacal silver nitrate hniline hydrogen oxalate .hiline hydrogen phthalate T
-
++
-
-
SELECTION OF CONDITIOYS FOR DETERJIIN4TIONS
Recovery of Chromatographed Sugars. To recover sugars the paper \vas extracted Tvith condensate from a refluxing solvent (3). This technique gave rapid, complete recoveries n-ith minimum volumes and many units could be operated concurrently to provide efficiency. I n the later phases of this ivork it was found that complete extraction of sugar could be accomplished by simply stirring the paper in a small beaker with about 5 ml. of distilled water, filtering the liquid through a glass woo1 plug in a funnel, and rinsing the paper with a second 5-ml. portion of water To test the completeness of the extraction, 42 D.E. corn sirup was depleted of glucose and maltose by absorption on a carbon column (34) and elution with water and 3.5% ethyl alcohol. On analysis, 1463 y of this sirup were found to contain 32 y of residual glucose and 31 y of maltose. When 400 y of D-glucose were added to the same amount of sirup and the mixtlure was separated on paper chromatograms, 436 y of Dglucose (an average of 14 determinations) were recovered from the chromatogram. When 190 y of maltose were added, 224 y of maltose were recovered (an average of 11 determinations). Color Production. .Imong the reagents compared in the generation of a colored complex with the sugar Tyere anthronesulfuric acid ( 7 ) , 1-naphthol-sulfuric acid, phenol-sulfuric acid (21), and alkaline 3,5-dinitrosalicylic acid-phenol ( 3 ) . The latter two were found to be satisfactory and essentially equivalent. Both methods were employed in the analyses. The alkaline method, operating in the range of 100 to 600 y of D-glucose, was somewhat superior in regard to color stability but suffered from the disadvantage of a colored blank. At 543 mfi the method was more sensitive than a t the suggested (3) 500 mp. The phenol-sulfuric acid method, operating in the range 10 t o 125 y of D-glucose, had the advantage of low reagent requirement
.
1514
V O L U M E 27, NO. 10, O C T O B E R 1 9 5 5 but required strongly caustic reagents and had poor reprociucibility. Even blank determinations on carefully prepared paper n-ere large and poorly reproducible. To avoid the necessity of using individual standard curves for the oligosaccharides, each oligosaccharide was hydrolyzed by refluxing 0.01N hydrochloric acid in the extraction. ANALYTICAL METHOD
Separation. For the determination of components A through U (see section on identification of corn sirup components), prepare chromatographic papers (18 x 57 cm.) by tracing along the heavily outlined edges of a template cut from thin aluminum (Figure 1). Serrate ( I S ) the bottom edges-e.g., with pinking shears. Deposit replicate 10-pl. samples of sirup (diluted to about 25y0 solids) containing about 2.5 mg. of total sugars from a micropipet (Research Equipment Corp., Oakland, Calif.) along lines B . Put 5 - ~ l .spots of the same solution a t points A . Dry the papers, fold along line C. suspend in descending chromatographic equipment, and irrigate for 36 hours.
7 3 . 5CM.
+:
-6 C M - e
-18 CM.
1515 Table 111.
Determination of Components in Corn Sirup
Dry Solidsa, Sirup Sirup R D.E.6 AC B C D E F C H I 4 . 8 1.2 5.1 5 . 0 4.1 3 . 6 5 , 1 4.8 1.1 94.10 18.0 7 . 8 1.0 6 2 1.1 7 . 0 6 . 3 5 . 9 5 . 2 83.92 2 6 . 3 8 . 3 9.2 1.3 10.5 1.3 8.6 7 . 8 5 . 8 5 . 1 70.00 32.6 11.4 81.80 43.3 19.4 14.4 1 . 1 1 0 . 6 1 . 2 9 . 7 8 . 6 6 . 2 5 . 1 8 1 . 7 49.7 26.1 15.1 2 . 9 1 1 . 2 2 . 3 9 . 4 8 . 4 82.511 5 5 . 6 3 0 . 9 1 5 . 2 3 . 5 1 0 . 0 3 . 5 9 . 4 7 . 2 7.'0 5 . 0 8 2 . 6 0 59.9 3 4 . 6 l L 6 3 . 7 1 0 . 6 3 . 2 8 . 7 3 2.4 70.29 63.0 38.9 2 2 . 0 3 . 8 9 . 4 3 . 4 8 . 2 a Dry solids determined b y Filtercel method (6). D.E. determined by modified Lane-Eynon method ($2). c Glucose values A corroborated by glucose dehydrogenase a n d SichertBleyer determination (3s).
E:;
DETERMINATION
Alkaline 3,5-Dinitrosalicylic Acid-Phenol Method ( 3 ) . To enough of the extract to contain 100 to 600 y of D-glucose, add 3.00 ml. of phenol-3,5-dinitrosalicylic acid reagent ( S ) , 2 ml. of 6 S sodium hydroxide, and 1 ml. of 50% potassium sodium tartrate. Dilute to 10.0 ml. with water and mix thoroughly. Heat the tube in boiling water for 10 minutes, cool to room temperature under tap, dilute with water to 10.0 ml., and measure the transmittance a t 543 mp. The amount of sugar present is determined by reference to a previously prepared standard curve. Phenol-Sulfuric Acid Method (21). To enough of the extract to contain 10 to 125 y of D-glucose, add 0.15 ml. of 80% aqueous phenol and 5.0 ml. of 96% sulfuric acid. Mix vigorously and let stand for 30 minutes, then measure the transmittance at 490 mg. Determine the D-glucose by reference to the corresponding standard curve and convert t o appropriate oligosaccharide values by applying the respective hydrolysis factors. Other Methods. I n the later phases of this work it was found that excellent quantitative results could be obtained with anthrone or the microferricyanide method of Hagedorn and Jensen (12). The ferricyanide method in particular gives excellent reproducibility and is not affected by the presence of filter paper fibers. For this reason it is now routinely used in the authors' laboratories. ANALYTICAL RESULTS
Eight corn sirups of various degrees of hydrolysis, as expressed by the dextrose equivalent (D.E.) m-ere analyzed by these methods. The first six components ( A through F ) vere determined by the alkaline 3,5-dinitrosalicylic acid-phenol procedure in all of the sirups. Values for components C through F in some of these sirups n-ere determined either by the phenolsulfuric acid method or the ferricyanide method and the analyses extended through spot I . Each value of the analytical results recorded in Table I11 is the average of four to eight replications the mean deviations of which were no more than &1.3%. Figure
1. Template for preparing paper chromatograms
.
Template length (28 cm.)-about half that of paper (57 em.)-is arbitrary
For the determination of the remainder of the oligosaccharides,
E through I , prepare chromatographic papers as explained above. but add a vertical line bisecting one line B. Put 5-pl. spots of eirup a t points A and a t either end of the bisected line, B. Deposit one 10-pl. aliquot of sirup along the remaining line, B . Dry, fold along line C, and sever the three adjacent locator strips except for a 1-em. band along the top edge. Suspend in descending chromatographic equipment and irrigate for 96 hours. Remove a locator strip each 24 hours to follow the progression of the sugars. Dry the resulting chromatogram. Cut into strips along the longitudinal lines and develop the locator strips with aniline h\ chogen phthalate reagent. Locate the respective sugars and excise appropriate sections of the chromatogram. Roll these sections into cylinders about 3 mi. in length by 5 mm. in diameter and suspend them from the hooks on the extractor condensers b r Sichrome n-ire clips. To 2.0-ml. aliquots of 0.OliV hydrochloric acid in test tubes, add small boiling chips. F i t the condensers with papers attached into the tubes and reflux in an oil bath a t 120" for 1 hour. All values are corrected for filter paper blanlie.
IDENTIFICATION OF CORN SIRUP COMPONENTS
By chromatographic comparisons with authentic samples *pots A , B , and C from representative sirups were identified as glucose, maltose, and isomaltose, respectively. From relative chromatographic positions and spot intensities, components D,F , G, H , and I were inferred to be nialtotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose, respectively. It is very likely that a t least components G, H , and I are not conPtituted wholly of pure mako homologs but are admixed with their isomers. Spot E was believed to be an isomaltotriose, but it did not correspond chromatographically to 0-or-D-glucopyranosyl- (1 6 ) -0-or-D-glucopyranos) 1- (1 -c 4)-~-glucopyranose (panose). To establish their identities, samples of components E , D ,F , and G v;ere isolated from representative sirups by cellulose column chromatograph\- (14) using ethyl acetate-pyridine-n a ter sol1 ent (10:4:3). Concentrates, so prepared, were rechroniatographed by the same method, decolorized in 25'37, aqueous ethyl alcohol n i t h activated carbon and dried for 16 to 30 hours a t '78" under reduced pressure over phosphorus pentoxide. These
-
ANALYTICAL CHEMISTRY
1516 Table IV.
Degree of Polymerization by Hypoiodite Oxidations Degree of Polymerization Inferred Found 2 1.98 3 2.83 4 3.74 5 5.08
Component
B
D F
G ~~
Cornponent
LITERhTURE CITED (1)
~~
Table V.
Maize Co. for corn sirup samples; and to Edna Montgomery, Northern Utilization Branch, for a sample of isomaltose. They also wish to acknowledge the assistance of Wan Chen L. Ming, J. W. Armstrong, Ila G. Carroll, and C. K. Kolstadt in the course of the work.
Analysis of Sirup Components by Periodate Oxidation
Mole-Equivalents of Substance Periodate ( 1 7 ) Formic Acid ( 1 7 ) Formaldehyde ( 6 7 ) D.P. CalcuCalcuCalcuInferred lated Found lated Found lated Found
Bate-Smith, E. C., and Restall, R. G., Biochim. et Biophys. A c t a , 4, 427 (1950).
Blass, J., Nacheboeuf, AI., and Sunez, G., BuU. SOC. chim. b i d , 32, 130 (1950). (3) Borel, E., H o s t e t t l e r , F.. and Deuel, H., H e h . C h i m . A c t a , 35, (2)
115 (1952). (4)
Chargaff, E.,Levine. C., and Green, C., J . Biol. Chem., 175, 67 (1948).
Cleland, J. E., and Fetaer, W.R . , AN.AI..C H E h f . , 13, 858 (1941). (6) Consden, R . , Gordon, -4.H., and Ilartin, A. J. P., Biochem. J . , (5)
38, 224 (1944). ( 7 ) Dimler, R. J., Schaefor, W.C.. Wise, C. S..and R i s t , C. E.,
fractions were subjected to periodate (17, 27) and hypoiodite (34) oxidations; relative chromatographic mobilities (10) and specific optical rotations were compared with calculated values. Hypoiodite Oxidations. The degree of polymerization of an oligosaccharide is equivalent to the increase in reducing power (34) on hydrolysis. This information (Table IV) confirms the deduced degrees of polymerization. Periodate Oxidation. The analytical information obtained by periodate oxidation (Table V) confirms the inferred degrees of polymerization. Molecular Rotations. Specific optical rotations of the maltopolymer homologous series follow a regular order as expected (11) (Table IV). The information gained in the foregoing investigation demonstrates that components B, D , F , and G are maltose, maltotriose, maltotetraose, and maltopentaose, respectively.
Table VI. component
B D F G H I
Molecular Rotations of Corn Sirup Components
Oligosaccharide Naltose hIaltotriose Maltotetraose Maltopentaose llaltohexaose Maltoheptaose Jlaltodocosanose
M 01. Wt. 342 504 666 828 989 1152 3582
Chromatographic Mobilities.
[-“I1
++ 46,512 80,640 +117,482 + 148,543 +179,998
Observed [a1
h A L . CHEM., 24, 1411 (1952). Fetaer, W. R . , Ibid.,24, 1129 (1952). French, D., Levine, 11.L., and Paaur, J. H., J . Am. Chem. Soc., 71, 356 (1949). (10) French, D., and Wild, G. 11..Ibid., 75, 2612 (1953). (11) Freudenberg, K., Friedrich. K., and Bunian, I., Ann., 494, 41
(8) (9)
(1932). (12)
Hagedorn. H . C.. and Jensen, R . S . . Biochem. Z.,135, 46 (1923).
(13) Hanes, C. S.,and Isherwood, F. -I,, S a t u v e . 164, 1107 (1949). (14) Hough, L., Jones, J. K. S . ,and Kadman, W. H., J . Chem. Soc., 2611 (1949).
Table YII.
Relative Mobilities of Corn Sirup Components
Component
Sugar R glucose Glucose 0.270 1.00 B lIaltose 0 . 1713 0.65 C Isomaltose 0.122 0.4;7 D llaltotriose 0.103 0.38 E Isornaltotriose 0.078 0.28 F llaltotetraose 0.057 0.21 G 0.041 Alaltopentaose 0.15 H llaltohexaose 0.030 0.11 I Maltoheptaose 0,022 0.08 a Solvent. E t h y l acetate, pyridine, water (10:4:3 v./v.). Temperature. 28-30’ C. Whatman Xo. 1 filter paper, descending technique.
d
+204,300 +691,300
The isolation of components
H and I by column chromatography was not attempted; hence there was no evidence obtained by chemical reactions that they belonged to the maltose homologous series. It has been observed (1,10,35) that, for members of a homologous series, there is a linear relationship between a function of the relative chromatographic mobility and the degree of polymerization. I n the solvent employed, the components were found to exhibit the relative mobilities expressed in Table VII. These data can be correlated by the a-function (e),a relationship between the areas of the mobile and stationary phases. French (IO) has simplified the estimation by employing the relationship a’-R,/(l -E,). This relationship, for the nine corn sirup roniponents determined in this work, is demonstrated in Figure 2. Hence, components H and I appear to be mnltohexaose and maltoheptaose, respectively. Component E is shown not to be a maltose homolog, and appears to belong to the “iso” series. ACKNOWLEDGMENT
The authors wish to express their appreciation to the Corn Industries Research Foundation for funds in support of this investigation; to the Corn Products Refining Co. and the American
Figure 2.
DEGREE OF POLYMERIZATION Relationship of chromatographic mobility to degree of polymerization
1517
V O L U M E 2 7 , NO. 10, O C T O B E R 1 9 5 5 Hurd, C. D., and Cantor, S. M., J . Am. Chem. SOC.,60, 2677 (1938).
Hurd, C. D., Liggett, R. W., and Gordon, K. K., I b i d . , 63, 2656, 2657, 2659 (1941).
Jackson, E. L., in Adams, R., et al., “Organic Reactions,” Val. 11, p. 361, TTiley, Kew York, 1946. Jeanes, A., Wise, C. S., and Dimler, R . L., d w . 4 ~ .CHEM.,2 3 , 415 (1951).
Jermyn, RI.
a.,and
Isherwood, F. A , , Bzochem. J . , 44, 402
(1949).
Kerr, R. W., “Chemistry and Industry of Starch,” 2nd ed., p. 174, Academic Press, New York, 1950. Koch, R. B., Geddes, W. F., and Smith, F., Cereal Chem., 28, 4 2 4 (1951).
Lane, J. H., and Eynon, L., J . SOC.Chem. Ind., 4 2 , 32T, 143T, 463T (1923); 44, 150T (1925); 46, 434T ( 1 9 2 i ) ; 50, 8 5 T (1931). Rfontreuil, J., Bull. SOC. chzm. btol., 32, 130 (1950).
Pan. S. C., Nicholson, L. W.,and Kolachor, P., A N ~ LCHEM., . 25, 231 (1953).
Partridge, S. M., Biochem. SOC.S u m p o s i a , S o . 3, “Chromatography,” Cambridge University Press, Cambridge, 1950. Partridge, S.hl., Nature, 164, 443 (1949). Reeves, R. E., J . Am. Chem. SOC.,63, 1476 (1941). Shu, P., Can. J . Research, 28B, 527 (1950). Sichert, K., and Bleyer, B., Z . anal. Chem., 107, 328 (1936). Stark, I. E., and Somogyi, M., J . Biol. Chem., 142, 579 (1942). Sugihara, J. M., and Wolfram, hl. L., J . Am. Chem. SOC.,71, 3357 (1949).
Whistler, R. L., and Durso, D., Ibid., 72, 677 (1950). Whistler. R. L.. Houah, L..and Hylin, J. W., ANAL.CHEM., 2 5 , 1215 (1953).
Whistler, R. L., and Tu, C. C., J . A m . Chem. Soc., 74, 3609 (1952).
White, L. bf.,and Secor, G. E., Arch. Bzochim. et Biophys., 43, 60 (1953).
Williams, K. T., and Bevenue, -4., Cereal Chem., 28, 416 (1951). RECEIVED for review October 12, 1953. Accepted June 9 , 1955. Journal Paper KO.758, Purdue University Bgricultural Experiment Station, Lafayette, Ind.
Determination of Alpha-Ketolic Substances in Urinary Extracts and Paper Chromatograms JOSEPH C. TOUCHSTON
and
CHIEN-TIEN HSU’
Endocrine Section, W i l l i a m Pepper laboratory o f Clinical Medicine, and Department o f Medicine, School o f Medicine, University of Pennsylvania, Philadelphia, Pa.
The procedure described was developed to fill a need for a reproducible method for determination of aketolic steroids in urinary extracts. The procedure has been extended to include determinations of aketolic substances on paper chromatograms. Paper strips containing the samples were sprayed with the blue tetrazolium color reagent. The color was eluted with a 7 to 3 mixture of ethyl acetate-methanol and the density determined. Recoveries of added a-ketolic steroids averaged 97%, and the duplicates showed an average deviation from the mean of 6’30. The blanks have not been more than 2% of the absorbance because of the a-ketolic content of the sample. A study has been made of normal, pregnancq, and cortical carcinoma urines. s
S 4,
EVERAL procedures have been reported for the determination of corticosteroids by the use of tetrazolium reagents (1, 2, 6 , 10). The results obtained with these methods when applied to urinary extracts are inconsistent because of the high blanks in some samples and the instability of the color. Hoffmann and Standinger ( 5 )have mentioned a procedure for eluting formazan spots from paper chromatograms and quantitation in a colorimeter, but gave no details and stated that the method mas not successful with urine extracts. I n experiments n ith paper chromatography, it was found that certain solvents used for elution of steroids from paper did not remove urinary pigments. Investigation was then instituted in an attempt to find a solvent mixture xhich would elute the color formed by various color reagents while leaving behind extraneous material, n hich would interfere with color density determinations. It was found that the blue colored formazan formed by reaction of a-ketole with blue tetrazolium (dianisole bisdiphenyltetrazolium chloride) was water-insoluble and could be washed on the paper with water. The blue formazan was eluted quantitatively from paper strips by a 7 to 3 mixture of ethyl acetate and methanol, after prior 1
Present address, Provincial Taipei Hospital, Taipei, Formosa.
washing on the strip with water, and was quantitated colormetrically. The procedure has been extended also to include 0-ketolic steroids on paper chromatograms. EXPERIMENTAL
Extraction of Urine. The urines were extracted with chloroform after incubation with glucuronidase (9). The values reported here are not maximum, as ideal conditions for extraction of urine have not been attained (8). It is probable that the values, in general, are low because 100 units per cc. of glucuronidase were used in hydrolysis of the urine specimens, and more recent experiments show slightly increased amounts when more glucuronidase is used. Procedure for Determination. For the determination of aketolic material, l/100 to 1 / 1 8 0 of the extract of a 24-hour sample, extracted as above, is sufficient. Using a minimum amount of 1 to 1 chloroform-methanol, the extract is placed directly on 0.5-inch strips of Whatman KO.1 filter paper and marked into sections of 3.5 inches; care is taken to keep the extract a t least i / 4 inch m-ithin the dividing line to allow for seepage when the strip is sprayed. The drying is aided by using a jet of dry nitrogen, or the strip is allowed to dry a t room temperature. A strip for the standard is set up in the same manner using 25 and 50 */ of desoxycorticosterone. Handling is facilitated when no more than three or four samples are placed on any one strip. The strips are sprayed Rith blue tetrazolium (two parts of 0.201, aqueous blue tetrazolium and one part of 10% sodium hydroxide solution, freshly prepared) until soaking wet (about 1 cc. of solution is required for each section) and allowed to dry a t room temperature. The strips are then washed 3 minutes by immeision in water and again allowed to dry partially a t room temperature. While still damp, each section is cut into small pieces, and thp formaaan eluted in a separate test tube containing 5 ml. of 7 to 3 (by volume) ethvl acetate-methanol mixture. The strips must not dry completely, or all the color will not be eluted. After 10 to 15 minutes with occasional shaking, the solvent is decanted into Evelyn tubes, another 5 ml. of elution mixture is added to the strips, and elution continued an additional 10 to 15 minutes. The second 5 ml. of solvent is decanted from the strips into the initial eluate, and the color density is read in an Evelyn photoelectric colorimeter using a 565 mp filter. Results are obtained by intrapolation against the curve obtained with the reference standards. For blanks, the following are used: a solvent blank consisting of the 7 to 3 ethyl acetate-methanol mixture, the reagent blank which is the eluate of a blank strip sprayed with the blue tetrazolium color reagent and eluted as described, and a urine color
