1645
V O L U M E 2 6 , NO, 1 0 , O C T O B E R 1 9 5 4 REAGENTS AND SOLUTIONS
All reagents are -4.C.S. grade. Fluoboric acid, 48 to 50’%,, purified ( B & A). Hydrogen peroxide, 30%. Iron powder, reduced by hydrogen. Antimony trichloride, 1%. Dissolve 5 grams of antimony trichloride in 100 ml. of hydrochloric acid and dilute to 500 ml. with wat,er. Standard tin solution, 1 ml. = 1.0 mg. Dissolve 1 gram of tin in 15 ml. of sulfuric acid and evaporate to strong fumes of sulfur trioxide. Transfer to a 1-liter volumetric flask and make to volume with 3Oy0hydrochloric acid. Standard potassium iodate solution. Keigh 0.1782 gram of pot,awium iodate, 15 grams of potassium iodide, and 1 gram of potassium hydroxide, and make to volume in a 1-liter volumetric flask. S h r c h solution. Dissolve 1 gram of soluble starch in 80 nil. of boiling WaTer. Cool and dilute to 100 ml. The Task Forre on Tin, Panel on JIethods of Analysis, Metallurgical hdviaory Board for Tit,nnium nnulyzed two titaniuni-
tin alloy samples according to the procedure presented by the author. I n addition, two laboratories determined the tin content of these two samples by other analytical procedures. These cooperative results for tin are presented in Table VIII. LITERATURE CITED
(1) Clennell, 3. C., Mining Mag., 55, 213-19, 278-84, 344-8 (1936). (21 Hillebrand, W.F., Lundell, G. E. F., Bright, H. A, and Hoffman, J. I., “Applied Inorganic Analysis,” Sew York, John X l e y &Sons, 1953 (3) Kolthofl, I X., and Furman, K, I3 “Volumetric .Inalysii,” Sew Tork. John Wilev 8: Sons, 1929. (4) S o r i v i t a , G., and Codell,-;\I., Frankford -1rsena1, Rept. MR-558
(June 1953). (5) Xogerman, K. D., J . Research S a t l . Bur. Standards, 33, 307
(1944). (6) Okell, F. L., A i d y s t , 60,803-11 (1935). RECEIVED for review April 3 , 1954. Accepted July 6, 1954. Presented a t the Pittsburgh Conference on Analytical Chemistry a n d Applied Spectroscopy, Pittsburgh, P a . , March 1954.
Quantitative Determination of Sugars on Paper Chromatograms by a Reflectance Method R. M. MCCREADY and E. A. MCCOMB Western Utilization Research Branch, Agricultural Research Service,
A general method for the direct photometric determination of sugars on developed paper chromatograms is proposed. Variables such as tl-pe of paper, volume and concentration range, and method of reflectance measurement have been evaluated.
UMEROUS methods for the direct quantitative determination of substances separated by paper chromatography have been described. Methods based upon visual comparison, weight of an excised spot, area, length, maximum transmittance density, and other measurements are summarized by Block, Le Strange, and Zweig (1). The first reports comparing transmittance with reflectance measurements ( I S ) reported that the reflectance and transniittance curves for a certain green compound formed by or-benzoin oxime and cupric ion resembled each other, but the transmittance va1ul.s followed Beer’s law while the reflectance values did not. Furthernioi e, for this compound greater sensitivity was obtained by measuring transmittance. Goodban, Stark, and 0 ens ( 3 ) measured the maximum reflection density (defined as log RO/RL,where I f 0 is reflectance of a white surface, such as the blank paper, and I?, is reflectance of the colored spot) of amino acidcj developed with ninhydrin for quantitative analysis. They obtained stiaight (or nearly straight) lines for plots of the log of the weight of amino acid against ieflection density for 2 to 10 y per .pot of the six amino acids tested. Vaeck ( I O , 2 1 ) determined nickel on chromatograms by direct measurement of the reflectance (reflectance of total area) of a colored spot and found that the values did not obey Beer’s law but followed the Kuhelka-Munk law (6). Direct ieflection density measurements on paper chroniatograms of the colored spots developed from reducing sugars and aniline-trichloroacetic acid, or fructose and derivatives with acid-resorcinol, have been found in the authors’ work to be reproducible and to follow a linear relationship between the logarithm of the sugar concentration and the reflection density. The method is particularly useful in the determination of the concentration of one or more pentoses in a mixture containing
U. S.
Department o f Agriculture, Albany 6, Calif.
a uronic acid since orcinol methods (2) do not distinguish between these substances. IIATERIALS AND APPARATUS
Paper. Sheets of Whatman S o . 1, S o . 3MM, and No. 4 and Schleicher and Schuell (S&S) SO.507 and S o . 595 (not previously conditioned before chromatography) were used. The sheets were about 46.5 X 5 i em. in dimension and in the air-dry condition retained 3 to 47, moisture. Pipets. Kirk ( 5 ) micropipets of the self-filling type were used to apply a given volume of sugar solution to a sheet. Chromatographic Apparatus. Cylindrical glass jars, 25 em. in diameter and 45 em. in height, with tight-fitting ground-glass covers served as the chambers. The covers were lightly lubricated with petrolatum and weighted to ensure air-tight seals. Heuagonal glass racks supported the cylinders of filter paper as described by Stark et al. (9). Developing Solvents. Any of the solvents used according to the general technique of Block et al. ( 1 ) for the separation of sugars seems to he satisfactory. For separating mixtures of galacturonic acid, oligogalacturonic acids, and arabinose, a miscible solvent composed of 10 volumes of ethyl acetate, 6 volumes of water. and 5 volumes of acetic acid was used ( 7 ) . Xylose, arabinose, and galacturonic acid were separated by the organic layer of a solvent composed of 10 volumes of ethyl acetate, 6 volumes of n-ater, and 5 volumes of pyridine ( 4 ) . Indicator Dips for Color Development of the Separated Sugars. A dipping technique was used to apply the indicators to the paper chromatograms. T n o per cent aniline and 2% trichloroacetic acid in ethyl acetate, mixed immediately before use, were applied to the paper for analysis of the reducing sugars. The dipped paper wa? dried in air in a vertical position for 30 minutes and heated a t 85’ C. for 5 minutes to develop the colors. For fructose, sucrose, or raffinose, an indicator composed of 10 volumes of 85% phosphoric acid in 90 volumes of 0.1% resorcinol in ethyl acetate, mixed immediately before use, was applied. The dipped sheet mas suspended in a vertical position, dried in air for about 30 minutes, and heated at 90” C. for about 5 minutes. PROCEDURE
For quantitative TI ork one-dimensional paper chromatograms were prepared essentially by the technique given by Block et al. ( I ) with the solvents previously mentioned, and with sheets 46 X 57.5 cm. of Whatman S o . 1 paper. Exactly 5.0-pl. spots of sugar solution were placed in triplicate about 2.5 cm. apart on a starting line 5.0 em. above the edge of
ANALYTICAL CHEMISTRY
1646 the paper sheet. The concentration of each sugar was about 0.4 to 1.5% and the combined concentration of all of the sugars did not exceed about 6%. Two or three standard solutions containing all of the suspected sugars in a known amount, spanning the concentration of sugars in the unknown, were applied on the same sheet. The papers were dried in air for about an hour without heating. The paper sheets were then attached to the cylindrical racks and placed in the chromatographic jars. The depth of the developing solvent was adjusted so that the sheets were immersed to a depth of 2.5 cm. The chromatograms were developed in one dimension by the ascending method ( 1 2 ) . After the solvent had advanced about 40 em. (20 to 24 hours), the sheet and rack were removed from the developing tank and dried in air without heating for about 2 or more hours. The sheet was then removed from the rack and immersed rapidly and evenly in a shallow pan containing the indicator, suspended vertically with the starting line a t the bottom, and dried for 30 minutes in air without heating. The colored spots were developed in a circulating air-draft oven a t the desired temperature.
B Galacturonic Acid C Galactose D Arabinose
I 400
I
I
I
500
600
700
1
Wave Length in Millimicrons Figure 1. Spectrophotometric Reflectance Curves and Colors Produced on Paper Chromatograms from Galacturonic Acid, Galactose, and Arabinose With aniline-trichloroacetic acid indicator
Reflection densities were measured with the Photovolt reflectance and densitometer unit with light of 515 mp wave length preferably as soon as possible after development of the colored spots. The meter was set so that zero represented reflectance of the blank paper between the row of spots. Reflection densities (the maximum reading of absorbance of the darkest area of the spots) were averaged and results obtained from the standard applications of sugars were plotted on semilogarithm paper. A plot of the amount of sugar in the logarithm direction on semilogarithm paper against the reflection density was nearly linear over the range of 25 to 100 y of sugar per spot. The reflection density values obtained for the unknown sugars were applied to the standard curve prepared from the particular sheet in question and the amount of sugar was obtained by reference to the ordinate. A standard curve was usually required for each sheet. When extreme care was exercised t o duplicate all operations, standard curves from one particular variety of paper had the same slope for a particular sugar and they usually coincided. Variables in technique caused slight displacements of attempted replicate standard curves. The over-all accuracy of this procedure with about 50 y of a pure sugar was usually about &5%. EXPERIMENTAL
Measurement of Reflectance and Color of Spots. While the visual difference between colored spots developed from 5 pl. of 0.4 and 5.0% glucose solutions (20 to 250 y ) on Rhatman KO.1 paper is very great, the colors analyzed with the General Electric Hardy recording spectrophotometer showed that the wave
length of the color of the spots of 20 y was only 5 mp lower than that of color produced from 250 y of glucose. The color change was, therefore, mainly one of intensity and not of composition. Various reducing sugars react n4th the aniline-trichloroacetic acid indicator on the chromatograms to produce different colors. Galacturonic acid gives a flesh-tan spot, glucose a brown spot, galactose a dark chocolate bro\Yn spot, and the pentoses appear as red or pink spots. The reflectance curves obtained spectrophotometrically of the flesh, brown, and pink colors given by galacturonic acid, galactose, and arabinose, respectively, are shonn in Figure 1. No sharp reflectance maxima are shown here and it can be seen that filters from below 400 mp to 560 mp and above can be used in the reflectometer search unit to measure the colored spots. O w n s et al. (8) used a 365-mp filter to increase the sensitivity of the measurements of reflectance in determining small amounts of galactose by this procedure. The 515-mp filter used in the experiments reported here yielded reproducible and consistent results, but it is possible to extend the range by using filters passing light of lower wave length.
A Hunter Color and Color difference meter (Henry Gardner Laboratory, Inc., Bethesda, Md.) was used to measure the total reflection density (or reflection of the total area) and a Photovolt reflectance and densitometer unit (No. 5013) with a 515-mp filter in the search unit was used to measure the reflection density, or the darkest area within the spot. The Photovolt search unit for reflectance measurements is so constructed that an area of about 3 to 4 mm. in diameter is illuminated and the photocell is located 90" from the spot and not a t the angle of reflection. Only a small fraction of scattered light reached the photocell for a given position. -4reflection density reading of zero on the absorbance scale was obtained for a blank area of the paper, preferably between spots. The spot to be measured was searched for its area of maximum density by moving the paper across the illuminated area of the search unit. A maximum reading was recorded and the replicate spots were measured in like manner. The readings were averaged and the values obtained showed a linear relationship when plotted on semilogarithm paper as logarithm of the amount (or concentration) of sugar against the reflection density. Vaeck (10, 11) and Winslow and Liebhafsky ( I S ) have reported that total reflectance values (reflectance of the total area) of spots of paper chromatograms do not obey Beer's law (10, 11, I S ) . Vaeck ( 1 1 ) reported that the Kubelka-Munk law cannot be expected to hold true for measurements of the colored nickel spots of paper chromatograms. ilccording to this law
where R m = monochromatic reflectance of a material of infinite thickness (for reflectance purposes), K = absorption coefficient, and S = scattering coefficient. Vaeck, however, found that ratios of absorption coefficient to scattering coefficient, plotted against concentrations of nickel from 2 to 20 mg. per 100 ml., based on the original solution applied to the chromatograms, n-ere linear. Reflection densities, as measured in per cent reflectance with the Photovolt apparatus from sugar chromatograms, were plotted as absorption scattering ratios against the amount of sugar. A linear relationship was observed but over only a small part of the concentration range. The difference between these results and those of T'aeck may be due to the fact that Vaeck measured the reflectance of the total area (or nearly so) of the spot and the authors' measurements represent small fractions of the darkest area of the spot. Goodban et al. (3) observed a linear relationship for certain amino acids when the logarithm of the amount was plotted against the reflection density as measured with the Photovolt apparatus. Five-microliter spots of galacturonic acid, digalacturonic acid, arbinose, galactose, and glucose in concentration from 0.25 to 2% (12.5 to 100 y per spot) were applied to 'Vi'hatman No. 1
1647
V O L U M E 26, NO. 10, O C T O B E R 1 9 5 4 300
-
200
-
IO1 0
I
I
0.1
I
0.2
I
0.3
Reflection Figure 2.
I
I
0.4
I
I
0.5
I
I
0.6
C
Density
presented in Table I are typical of Photovolt reflection density readings of spots of galacturonic acid on paper chromatograms. Other Variables. It was not possible to apply the indicator uniformly by means other than dipping. The success of the quantitative method depended upon the uniform application of the indicator to the chromatogram, drying in the vertical position, and uniform development of the color upon heating. When fructose-containing sugars were analyzed, an indicator dip consisting of a mixture of resorcinol, phosphoric acid, and ethyl acetate was used followed by heating a t 90” C. for 5 minutes. Bscending development has been used here because the colored spots were round and compact, and the slower rate of movement of the developing solvent seems to increase the resolution of the sugars. Each sugar spot was placed on the paper sheet in a single application. When it was desirable to build up a given concentration of sugar in a single spot, the unit volume of application was kept as small as possible, since the sugars tend to concentrate a t the periphery of the spot. In such cases, it was advantageous to concentrate the solution and apply the sugar to the paper sheet in a single application.
Standard Curves on Semilog Paper
Obtained by plotting amount of sugar (in the log direction) against Photovolt reflection density readings using light of 515mfi wave length
papers as described, developed, and treated with the anilinetrichloroacetic acid indicator. Both total reflection densities (reflection of the total area) of colored spots on paper chromatograms, obtained with the Hunter color and color-difference meter, and reflection densities (reflections of the darkest areas of spots measured by the Photovolt apparatus with light of 515mp wave length) were obtained. The data are not shown here but between the limits of 0.5 and 1.5% concentration (26 to 75 per spot) the total reflection density values plotted against the logarithm of the amount of sugar were found to fall on a straight line. The Photovolt reflection density values plotted against logarithm of the amount of sugsr were found to fall on straight lines between a concentration of below 0.5% to slightly above 2%, or 25 to 100 y per spot as shown in Figure 2. Measurements with the Photovolt apparatus viere reproducible and offered advantages in convenience and increased range over measments with the Hunter apparatus. Effect of Type of Paper. The type of paper does not appear to be critical, as far as measurements of reflection densities are concerned. Each paper offers certain advantages in its facility for separating sugars. For example, Whatman KO. 311hI is thick, and the volume placed on the chromatogram can be doubled as compared with Whatman KO. 1 and No. 4. Schleicher and Schuell S o . 507 is a thin hardened paper that permits resolution of sugars with minimum diffusion. The background color with aniline-trichloroacetic acid indicator is a uniform but much darker yellow than that produced with Whatman Xo. 1 and S o . 4 filter papers. The choice of paper, therefore, can be made on the basis of usefulness in separating substances under particular experimental conditions. Effect of Volume Applied. One to 5.0 pl. of solutions of sugars ITere applied to papers. Variability of results with 1.0 pl. was probably due to the difficulty of applying small volumes to papers in a uniform manner. Most consistent results x5ere obtained when 2.0 to 5.0 pl. of solution were applied. Reproducibility of Measurements. The reproducibility of the reflection density measurements of a qiven colored spot with the Photovolt apparatus by one or more operators was almost invariable. Variations in density readings occurred from spot to spot and i t was therefore necessary to obtain measurements from duplicate or preferably triplicate spots. Measurements
Table 1.
Typical Photovolt Reflection Density Readings with 515-Mp Filter
Chromatograms developed from 5-fiI. spots of solutions of galacturonic acid Amount of Galacturonic Reflection Density Acid per Spot, y Whatman No. 1 S. & S. No.507 12.5 0.158 0.09 0.156 0.145
25
50 125
0.205 0.210 0.210 0.290 0.300 0.305 0.370 0.370 0.350
0.09 0.10 0.155 0.145
0.150 0.230 0.230 0.225
... ... ..,
ACKNOWLEDGMENT
The authors wish to thank H. C. Lukens for measuring the total reflection densities and analyzing the color of some of the spots, and H. S. Owens for many helpful suggestions. LITERATURE CITED
(1) Block, R. J., Le Strange, R., and Zweig, G., “Paper Chroma. tography,” Ken, York, Academic Press, Inc., 1952. (2) Drury, H. F., Arch. Biochem., 19, 455 (1948). (3) Goodban, A. E., Stark, J. B., and Owens, H. S., J . Agr. Food Chem., 1,261 (1953). (4) Jermyn, AI. A., and Isherwood, F. A,, Biochem. J., 44, 402 (1949). (5) Kiik, P.’ L., “QuantitativeUltramicroanalysis,” p. 22, New York, John Wdey 8: Sons, 1950. (6) Kubelka, P., and Munk, F., 2. tech. Phys., 12, 593 (1931). (7) McCready, R. M., and NcComb, E. A., J . Agr. Food Chem., 1. 1165 (1953). (8) Owens, H. S., McComb, E. A, and Deming, G. W., Proc. Am.
SOC.Suoar Beet Technol.. in Dress. (9) Stark, J. B . , Goodban, A . E., and Owens, H. S., ANAL.CHEM., 23.413 (1951). \ - - - - ,
(10) (11) (12) (13)
Vaiyk, S. V.,Anal. Chim. Acta, 10, 48 (1964). Vaeck, S.V., A‘ature, 172,213 (1953). Williams, R. J., and Kirby, H., Science, 107, 481 (1948). Winslow, E. H., and Liebhafsky, H. 9., ANAL.CHEM.,21, 1338 (1949).
RECEIVED for review March 8, 1954. Accepted J u n e 18, 1954. Mention of manufacturers a n d commercial products does not imply t h a t they a r e endorsed or recommended by t h e Department of Agriculture over others of a similar nature not mentioned.
