ANALYTICAL CHEMISTRY
1146 shape but with displaced maxima and minima. That there was
Consden, R., Gordon, A . H., and Martin, A. J. P., Biochem. J.,
a fixed order of the Ri values found in different solvent mixtures for some, but not all, of the amino acids of a group was deter-
Datta, S . P., Dent, C. E., and Harris, H., Science, 112, 621
mined by inspection of their superimposed curves drawn on transparent plastic sheeta. The importance of pH is emphapized by the relatively large differences in the R, values of the acidic and the basic amino acids with the solvent pairs No. 5 (methanolwater) and KO. 6 (phenol-water with hydrocyanic acid), No. 6 and KO.7 (phenol-water with ammonia), and No. 8 (m-cresolyater with ammonia) and No. 9 (m-cresol-water).
LITERATURE CITED
Arden, T. V., Burstall, F. H., Davies, G. R., Lewis, J. A,. and Linstead, R. P., N a t u r e , 162, 691 (1948). Bentley, H. R., and Whitehead, .J. K., Biochem. J . , 46, 341 (1 950).
Block, R. J., -4N.41.. CHEW,22, 1327 (1950). Bull, H. B., Hahn, J. R., and Baptist, T’. H., J . A m , Chem. SOC., 71, 550 (1949).
38, 224 (1944). (1950).
Goodall, R. R., and Levi, A. h.,Aiaalyst, 72, 277 (1947). Hanes, C. S., and Isherwood, F. A , .?‘atwe, 164, 1107 (1949). Helmer, 0. M.,Proc. SOC.E x p t l . Bid. Med., 74, 642 (1950). Karnozsky, hl. L., and Johnson, hI. J., AXAL.CHEM.,21, 1125 (1949).
Kowkabany, G. N., and Cassidy, H. G., Ibid., 22, 817 (1950). McFarren, E. F., Ibad., 23, 188 (1951). Moore, S., and Stein, W.H., J . Biol. Chem., 178, 53 (1949). Muller, R. H., and Clegg, D. L., A N ~ LCHEM., . 23, 408 (1951). Patton, A. R., and Foreman, E. AI., Food Technol., 4, 83 (1950). Rockland. L. B.. and Dunn. B.1. S.. J . Am. Chem. Soc.., 71., 4121 (1949). (17) Rockland, L. B., and D u m , AI. S., Science, 109, 539 (1949). (18) Ibid., 111, 332 (1950). (19) Rockland, L. B., and Miller, J., private communication. RECEIVED January 16, 1951. Paper 78, Chemical Laboratory,Cniversity of California, Los Angeles. For the preceding paper in this series see Rock..
land and Dunn ( 1 7 ) . This work was aided by grants from Institutes of Health (E. 9. Fublic Health Rervire) and the
the National University of
California.
Quantitative Determination of Sugars on Filter Paper Chromatograms by Direct Photometry EARL F. RZcFARREN, KATHLEEN BRAND,
AND
HENRY R. RUTKOWSKI N. Y .
National Dairy Research Laboratories, Znc., Oakdale, L. I.,
Neither conventional methods of sugar analysis nor paper chromatographic elution methods permit the quantitative determination of galactose and glucose in the presence of one another with any reasonable assurance of accuracy. Sufficient separation of galactose and glucose can be achieved on a paper chromatogram to permit the quantitative determination of each sugar by direct photometry. In this method the maximum densities of the developed sugar spots are determined by means of a densitometer and a standard curve is prepared by plotting
T
HE method presented here for quantitatively determining
the sugars is essentially the same as the methods published by Block (8) and Rockland and Dunn (10) for quantitatively determining the amino acids by direct photometry on filter paper chromatograms. The method of Rockland and Dunn requires determination of the density of the entire spot, while Block’s method, as in the present study, requires determination of only the maximum density of the spot. This study was begun before either of these methods was published and was developed as a result of a suggestion during a discussion with R. J. Block. Both Block (3) and Bull et al. (5) published separately a t about the same time another method for quantitatively determining the amino acids. In this method the density of consecutive 5-inm. segments of a strip chromatogram was determined using an electron transmission densitometer, The densities determined along the strip were plotted against the distance from the starting point and curves were drawn. Block pointed out that when such a curve was plotted it could be shown that the peaks of the curves varied in height with the concentration, the indication then being that there might be a simple relationship between the maximum density and the concentration. Fisher, Parsons, and Morrison (6) have shown experinientally khat a linear relation holds between the area of the spot of test
the logarithm of the concentration against the densities. The densities of the unknown sugar spots are similarly determined and their concentrations calculated from the standard curve. Data are presented which indicate that it is possible to determine the sugars present in a mixture with an error no greater than 5%. Quantitative analysis of samples by chromatography has revealed the presence of reducing substances not suspected of being present, which apparently have been calculated as lactose or total monoses in the usual sugar analysis.
substance and the logarithm of the concentration a t which it is originally applied. From Beer and Lambert’s law it is known that in a solution the concentration is proportional to the density. At first thought this relationship would seem to apply here. However, Brimley (4) in developing a theoretical derivation of the relationship of the area of a spot to the concentration supposed that the spot spread by diffusion as it moved along the chromatogram. Making this assumption, by analogy, it would seem to follow that the density of a spot on the chromatogram is linearly related to the log of the concentration. Block (1) has since shown that this relationship holds experimentally for the amino acids. This relationship is shown here also to hold experimentally in the case of sugars. Briefly, the method employed consists of separating the sugars in an ethyl acetate-pyridine-water solvent system containing silver nitrate, air drying, exposing the chromatograms to ammonia vapors, and developing the sugar spots by heating in an oven. The maximum densities of the developed spots are then determined by means of a densitometer, and a standard curve is prepared by plotting the log of the concentrations against the densities. The densities of the unknown sugar spots are determined and their concentrations are calculated from the standard curve.
V O L U M E 2 3 , N O . 8, A U G U S T 1 9 5 1
1147 ~-
in 2-microliter uantities by means of a G h ~ o n tultramicroburet. Seven spots were introduced along a line 7 . 5 cm. Standard Unknown from one end a t 2.5-em. inConcn., 31g./2 MI.__ Calculated Average. Theoretical. Sugar 5 7.5 10 1 2 . 5 111 1/3 112 Concn., -y,'p. G.1100 111. G . ?100 311 Error tervals' The four 'pots were the known sugar concen2.0 Lartose 0 53 0 9 1 I 17 1 . 3 1 0 . 6 1 0 8 1 1 . 1 6 1 0 23 1. 0 2 1.00 trations used to establish the 1. 0 0 2 0 0 52 0 92 1 . 1 7 1 . 2 1 0 . 5 8 0 87 1 13 9.83 0.98 standard curve, and the other Galactose 1 . 1 2 1 5 8 1 74 1 . 8 4 1 0 1 1 . 2 % 1 . 5 2 7 9.5 0.79 0 80 1 0 three spots were the unknown 0 9 2 I 4 1 1 64 . . 0 . 8 1 1 . 1 1 1 39 8 02 0.80 0 80 2.0 solutions applied a t appro7 42 Glucose 1 . 4 9 1 9 7 2 . 0 0 2 . 2 0 1 19 1 . 4 6 1 93 0 i 4 n0 ii t6i 3 .0 priate dilutions. 7 17 0 72 5 0 1.27 1.76 1.95 .. 1 . 1 3 1 . 4 1 1.69 After spotting the paper, ___ __ t h e chromatograms were placed in the .chambers and allowed to run for 16 to 20 EXPERIMENTAL PROCEDURE hours. In this period of time, the solvent front had Drogressed beyond the end of the paper and a better separation ofthesugars The solvent system employed was prepared by placing 2.5 resulted. Upon removal from the solvent chamber, the chromaparts of ethyl acetate, 1.0 part of pyridine, and 3.5 parts of distograms were air-dried for 1 hour and then placed in an ammonia tilled water in a separating funnel and shaking thoroughly. After chamber for 1 hour. The chambers used for the ammonia were separation, the water-rich layer was discarded and a portion of the same kind as those used for the chromatographic run. Conthe solvent-rich layer was placed in the bottom of a cylindrical centrated ammonia was placed in the bottom of a lined chamber, borosilicate glass chamber 24 inches (60 cm.) high and 12 inches and the chromatograms were hung on the glass rod of the (30 cm.) in diameter. The chamber was lined with two sheets chromatographic stand (without the trough) by means of a stainof Whatman No. 1 filter paper stapled together to form a cylinder less steel clip. On removal from the ammonia chamber, the which fits snugly against the chamber wall. The remainder chromatograms were placed in a mechanical convection oven a t of the solvent-rich layer was made 0.15 N with respect to silver a temperature of 80" =t1' C. for 20 minutes. nitrate and placed in a stainless steel solvent trough which was The densities of the developed chromatogram (Figure 1) were held 22 inches from the bottom of the chamber by a stainless then determined with a standard Photovolt electron transmission steel stand. The chambers were covered with a 12-inch square of densitometer, using a 5-mm. diameter aperture and the No. 440 plate glass ground on one side. filter purchased with the instrument. This filter gives maximum The filter paper used was Schleicher and Schuell 589 JThite absorption and the highest density readings (Figure 2). Ribbon cut in 22 by 55 cm. rectangular strips. The paper was cut so that the long axis ran parallel with the watermarks. Four standard solutions containing, respectively, 5 , 7.5, 10, and 12.5 I I I I micrograms per microliter each of lactose monohydrate, glucose, and galactose, were prepared. These sugar solutions were applied 3 1 630 600 570 530 420
Table I.
Expected Error in Calculation of Concentration of Sugars from Density Readings Determined by Direct Photometry on Filter Paper
' I
-
. 2 -
Ki
K2 I
K3
K4
"I
I
I
"2
"3
LACTOSE 4
3 Figure 2.
GALACTOSE
GLUCOSE Figure 1. Photocopy of Quantitative Sugar Chromatogram Knowns applied a t concentrations of 15/2, 10/2, 7.512, and 57/2 pl., respectively, left t o right U's. Unknowns, i n this examp,le spotted repeatedly i n 2-pl. portions. A total of 20, 15, and 10 p l . were applied t o U I , Uz, and Ua, respectively
K's.
I 25
I
I
.50 .75 DENSITY
I
I
1.00
125
Variation of Density with Filter Used in Densitometer
This instrument was modified slightly by removing the opal glass from the receiving cone and by disconnecting the spring which normally holds the search head arm in a raised position. A cork was placed under the search head arm a t the middle of its length and high enough to leave a space between the receiving cone and the aperture disk of about the thickness of two sheets of paper. The shutter spring was also removed, so that it was not necessary to hold the shutter open while trying to determine the density of the spots. The instrument was first zeroed on a blank portion of the developed chromatogram, and then the masimum density of each sugar spot was determined by moving each spot slowly around underneath the receiving cone until masimum deflection was obtained on the density meter. The masimum density is easily found and can readily be reproduced for any one spot, if too long a time has not elapsed between readings. On exposure to light and volatile reducing substances in the atmosphere, over-all darkening of the chromatogram occurs with the passage of time. After the densities have been determined, the data are plotted on semilogarithmic paper with concentration on the log scale as ordinate and density as abscissa. A standard curve is typified by the data plotted for the 440 filter in Figure 2 . From this standard curve and the recorded densities of the unknown, the concentrations of the unknown are then calculated. For evaluating this procedure, a mixture containing 1% lactose, 0.80% galactose, and 0.76% glucose was prepared and run
1148
ANALYTICAL CHEMISTRY
as an unknown. In this case it was found by trial that it was necessary to run the unknown a t dilutions of one-half, one-third, and one-fourth. The data obtained and the calculations, including the per cent error, are recorded in Table I.
to ohtain maximum development of the spots. It is necessary to add 10 to 20 ml. of concentrated ammonia to the ammonia chambers every day when the ammonia chambers are in continual use. If this is not done, the concentration of the ammonia vapor is so reduced by opening and closing the chambers to add or remove DISCUSSION chromatograms that the intensity of the developed spots will diA number of factors influence the success of this method and minish. Immediately on removal from the ammonia chambers, cannot be ignored if satisfactory chromatograms are to result. the chromatograms are placed in a mechanical convection oven a t Solvent. The solvent system employed was that used by 80' C. for 20 minutes. This temperature and time are rather Jermyn and Isherwood ( 7 ) . However, the solvent proportions critical, as a higher temperature will cause greater background suggested did not always produce good separation of galactose coloration and a lower temperature will not develop the sugar and glucose, and irreproducible R, values were obtained. It was spots. absolutely essential to have airtight chambers, as even the smallBackground Coloration. In an effort to evaluate the suitability est break in the seal between the chamber and lid would cause variof various filter papers, the R, values for the sugars were deterations in R f values. Rf values varied with the size and number mined, using a number of grades and makesof filter paper. Litof strips or sheets placed in the chromatogram chamber a t one tle significant variation in the R, values was noted regardless of time, In order to obtain good separation of galactose and gluthe filter paper used. Schleicher and Schuell 589 White Ribbon cose, it was found necessary to change the solvent proportions of was finally chosen, for it gives a desirable rate of solvent flow ethyl acetate, pyridine, and water to 2.5: 1:3.5. Reproducible (about the same as Whatman No. 1) and it is an ash-free paper R / values were obtained by lining the chamber with two sheets which gives less background color with ammoniacal silver nitrate. of Whatman No. 1 filter paper stapled together to form a cylinder Reducing substances (such as formic acid) in the atmosphere durand by using the solvent-rich layer (same as in the trough) in the ing the air drying of chromatograms will also cause greater backbottom of the chamber. When this was done, reproducihle RJ ground color to develop and subsequently interfere in determinvalues resulted, regardless of the size of the sheet. ing the densities for quantitative purposes. In running a great number of chromatograms, it has been found desirable (after air drying for 1 hour) to hang Table 11. Variations of Rf Values of Sugars in Ethyl Acetate-Pyridine-Water with the chromatograms in an Vapor Content of Chamber and Size of Chromatogram empty air-tight chromatogram Chromatogram Size, 11 X 55 Cm. chamber until they can be dePhase in Bottom, Lined Chamber Phase in Bottom, Unlined Chamber veloped. This will prevent or Solvent Water Solvent Water rich rich Water rich rich Nothing considerably reduce darkening. O.lSfO.03 0.12f0.02 0.4OiO.20 Lactose 0.18f0.03 0.24f0.12 0.30i0.20 It is better, however, to stagger 0.32f0.04 0.22iO.03 0.47f0.18 Galactose 0.32f0.04 0.36i0.17 0.45fO.13 the chromatographic runs, so 0.37i0.04 0.27f0.04 0.5110.17 Glucose 0.3710.04 0.41iOo.16 0.483~0.16 0.62 fO.10 0.55 e 0 . 0 4 Ribose 0.43 f 0 . 0 3 0.55 f 0 . 0 4 0.56 f 0.11 0.55 i 0 . 1 7 that only one chromatogram Chromatogram Size, 22 X 55 Crn. is removed from the solvent Phase in Bottom, Lined Chamber Phase in Bottom, Unlined Chamber chambers every 30 minutes. So1,vent Water Solvent Water This procedure allows time for rich rich Water rich rich Nothing the chromatograms to be proc0.20 f 0.03 0.10 f0.01 Lactose 0.34 0 . 1 2 f 0.03 0 . 1 0 i 0.00 0.20 i0.02 0.20 f 0.01 0 . 2 0 i0 . 0 1 0.40 0.25 f 0 . 0 6 Galactose 0.21f0.01 0.34 i O . 0 1 essed one at a time without 0 . 2 4 f 0.00 0 . 2 4 f 0.00 Glucose 0.52 0.29 f 0 . 0 5 0.25 1 0 . 0 3 0.38 f O . 0 1 0 . 4 2 fO.O1 0.42 f O . 0 1 Ribose 0.70 0.49 f 0 . 0 6 0.46 i 0 . 0 2 0.59 i O . 0 2 delay and the resulting darkening. Density Values. In general, density readings between 0.50 The Rr values obtained in chambers prepared in a number of and 1.70 have given the best results-namely, a linear relationways and on sheets of different sizes are recorded in Table 11. ship. Under the conditions specified for developing the chromatoReproducible R, values %-ereobtained only when the solvent-rich grams, glucose has greater reducing properties than galactose layer was used in the bottom of a lined chamber. Chambers prewhich in turn are greater than lactose. As a result, similar denpared in this manner could be used day after day with only the sity readings will not be obtained for equivalent concentrations of addition of more solvent to the trough prior to the introduction of each sugar. The standard curves are not reproducible from one the filter paper strips; it was not necessary to equilibrate chromatopaper strip to another, and the standards must be run each time grams overnight. The data in Table I1 indicate the necessity on the same chromatogram nith the unknowns. If the densities of assuring saturation of the chambers with both water and solof the unknown are not within the same range as the densities of vent. Lining the chambers appears to help in this respect, but the known, they should be discarded and the chromatograms run because of the volatility of this particular solvent, it is difficult to again after concentrating or diluting the unknown as indicated by keep the chamber saturated with solvent vapor unless the solthe initial run. Concentration can be done on the paper by revent-rich layer is also used in the bottom. peated applications of the same volume on the same spot after air It has not proved feasible for quantiDevelopment of Spots. drying or drying under an infrared lamp after each application. tative purposes to spray the chromatograms with ammoniacal silver nitrate, for the water in the spray caused diffusion and spreading of the spots in an unpredictable manner. This difficulty was eliminated by incorporating the silver nitrate in the chromatograming solvent, as had been done by Nicholson (9) with ninhydrin for chromatograming the amino acids. After the chromatograms have been removed from the chambers and thoroughly air-dried, it is necessary to expose the sugar chromatograms to ammonia vapor. This has been accomplished by hanging the strips in a lined chamber containing concentrated ammonia in the bottom of the chamber. A 1-hour exposure is required
APPLICATION TO MATERIALS OF BIOLOGICAL ORIGIN
In working with materials of biological origin, it is necessary to remove interfering substances such as proteins and salts. The removal of proteins may be effected in a number of ways. Of the many methods tested, the most effective method xyas the precipitation of the protein with barium hydroxide and zinc sulfate a s used by Somogyi (11) for blood clarification. In samples deproteinized the amount of reagents added varied with each sam-
V O L U M E 2 3 , NO. 8, A U G U S T 1 9 5 1 ple; however, it was necessary to add for each equivaleiit of zinc sulfate an equivalent of barium hydroxide.
1149 \. ILI E Oh' QUlVTITATIVE CHROMATOGRAPHY
The quantitative chromatographic analysis of natural materiknown or shown by chromatography to be composed of relatively simple mixtures of sugars, has given results comparable a i t h those obtained by the usual chemical or fermentation methods. In more complex mixtures, additional reduring substances have been found which r e r e apparentlv calculated as lactose or total monoses in the usual sugar analysis. Occasionally, other hugars havr been revealed ahich were not burpected of being prescnt :ind M ew not detrctrd hy conventional sugar analysis. ills
The barium hydroxide and zinc sulfate solutions were made approximately 0.3 i Y and the equivalents were determined by titrating the zinc sulfate solution with the barium hydroxide to a phenolphthalein end point. A sample of spray-dried skim milk was deproteinized by adding 20 ml. of zinc sulfate and 17.2 ml. of barium hydroxide (1.00 ml. of zinc sulfate is equivalent to 0.86 ml. of barium hydroxide) to 1 gram of sample dissolved in 50 ml. of water. The sample was then diluted to 100 ml. by adding distilled water. .ifter gently shaking, the solution was filtered through Whatman S o . 12 fluted paprr and a portion of the filtrate was used for chromatograming. Each sample under inveatigation is an individual caw, arid the amount of reagent used, and the extent of dilution, vary with the amount of sample to be deproteinized. To obtain a rapid filtration and a clear filtrate which marks an effective deprokinization, a pH of 7.2 to 7.6 is required in the sample-reagent mixture prior to filtration. This procedure has been applied to whole, dried, and skim milks, to egg whites, to cheese, and to tissue and body fluid extracts of animals. Satisfactory undiitorted chromatograms suitable for quantitative determinations have resulted in nearly all cases. Some interference from salts was occasionally encountered in the lactose region of the chromatogranio, but n hen this occurred, deionization TI ith pyridine (8) produced sati-factory
LITERATURE CITED
CHEY.,22, 1327 (1950). ( l ) Block, R. J., -4x.t~. (2) Block, R. J., Proc. Soc. E x p t l . Biol. iMed., 7 2 , 3 8 7 (1949). Block, R. J., Science, 108, 608 (1948). (1) Rrimley, R. C., Saturc. 163, 215 (1949). ( 5 ) Bull, H. B., Hahn, J . W.. and Baptist, V. K., J . A m . Cham. Soc., 71, 550 (1949). R. B., Parsons, D. S., and Morrison, G . .i.,S n t u r e . 1 6 1 , 7 6 4 (1948). ( 7 ) ,Jerniyn, 11. .\.. and Ishwwood, F. .I..Rl'ochern. J.. 44, 402 (1949). Malgresc. 1:. H., arid AIorrison, A. R., S u t u r e , 164, 963 (1949). ') (9) Sirholson. 11. E., Ihid.. 163, 954 (1949). (1) Kockland, L. €3.. nrid D u m . 51. S.,J . An. Chern. Soc., 71, 4121 (1949). ( 1 1) Somogyi, RI., J . B i d . C h o n . , 160, 61 (1945). ((i)Fisher,
Fluorometric Determination of Zirconium in Minerals W. C. ALFORD', LEONARD SHAPIR02, AND CHARLES E. WHITE I.nirersity of MMnrylancI, College Purk, M r l .
The increasing use of zirconium in alloys and in the ceramics industry has created renewed interest in methods for its determination. It is a common constituent of many minerals, but is usually present in very small amounts. Published methods tend to be tedious, time-consuming, and uncertain as to accuracy. A new fluorometric procedure, which overcomes these objections to a large extent, is based on the blue fluorescencegiven by zirconium and flavonol in sulfuric acid solution. Hafnium is the only element that interferes. The sample is fused with borax
A
N O S G the more important methods for the determination of zirconium are the gravimetric procedures involving precipitation of zirconium with cupferron (9),phosphate ( 8 ) , mandelic acid ( 6 ) , m-dinitrobenzoic acid ( I I ) , and tannin (ti), and the colorimetric methods that make use of arsonic acids (4)or alizarin derivatives ( 3 , 7 ) . None of these reagents, except mandelic acid, is claimed to be specific for zirconium, laborious separations are required, and the results obtained are not always reliable, as evidenced by the fact that different analysts often obtain widely varying results on identical samples. Mandelic acid is thought by Kumins (6) to be a specific precipitant for zirconium, but the amount of zirconium required for a determination makes the method unsuitable for trace analysis. Thus the need for a rapid and reliable method for estimating small quantities of zirconium prompted the development of the method described herein. 1 2
Present address, National Institutes of Health, Bethesda, hld. Present address, V. S. Geological Survey. Kashington 25, n. C'.
glass and sodium carbonate and extracted with water. The residue is dissolved in sulfuric acid, made alkaline with sodium hydroxide to separate aluminum, and filtered. The precipitate is dissolved in sulfuric acid and electrolyzed in a Melaven cell to remove iron. Flavonol is then added and the fluorescence intensity is measured with a photofluorometer. Analysis of seven standard mineral samples shows excellent results. The method is especially useful for minerals containing less than 0.257' zirconium oxide.
The present method is based on the blue fluorescence given by zirconium and flavonol (3-hydroxyflavone) in moderately strong sulfuric acid solution when expowd to ultraviolet light. Under controlled conditions, the intensity of fluorescence is proportional to zirconium concentration. Measurements are made with a photoelectric fluorometer by comparing the intensity of fluorescence of an unknown solution with that of a known zirronium etandard. The method provides for the elimination of all known interferences except hafnium and overcomes most of the objections to previous methods. APP4RATUS AND REAGENTS
Aside from the usual laboratory glass and platinum ware, the only apparatus re uired is a Melaven (10) mercury cathode electrolysis cell a n 1 a photoelectric fluorometer. Fluorescence measurements were made with a fluorometer designed and built by Alford and Daniel (1). During the course of this work, a Corning glaps filter S o . 5871 was insrrted between
