Small Scale Filter Pap1er Chromatography rs and 3oivents LOUIS B. ROCKLAND', JEREMIAH L. BLAW, AND MAX S. DUNN University of California, Los Angeles, Calif.
Investigations were undertaken hecause of the rapidly growing interest i n and importance of filter paper chromatography as a tool for the qualitative identifioation and quantitative determination of amino acids and other biological substances. Thirteen filter papers have been rated on the basis of seven physical and chemical oharaeteristics, the influence of the paper on the Rj values of eighteen amino aoids has been studied, the influence of water content of eight water-misoible solvents on Rj
MALL scale filter paper ehromatoBaphy, described in earlier papers (16-28)from the authors' laboratory, has heen'ap plied to the study of filter papers (2i) and solvents (8) and has been adapted to the rapid qualitative (9, 25) and quantitative (26) determination of amino acids. The authors' systematic investigations relating to filter and miscihle and im. papers .
values of amino acids has been investigated, and the R, values (ratio of lhe length of the chromatogram to the distance traveled by the aolvent boundary) of amino acids representative of the acidic, basic, neutral, and cyclic groups have been determined. The fixed and most common sequences of the amino acids, the solvents yielding inverted sequences, and the solvents most effective in separating individual amino acids, pairs of amino acids, and gronps of amino acids have been determined.
'lhe exact hnal positrons or tne 6oivenG uounamm were iocaceu in some instances under ultraviolet light. Chromstagritms developed with volatile solvents were dried in air, and those developed with phenol and other higher boiling solvents by heating them for 5 minutes in an oven a t 80". It was found convenient t o store paper strips spotted with standard solutions of amino acids in stoppered 25 X 200 mm. test tube8 and paper sheets spotted similarly in letter file folders. Tn shinine chromatoeram solvents were employed iu which ~.~~~~ the chromogenic reagen't, hut not the amino aEid,. waa appreciably soluble. The solutions found most useful were 0.5% ninhvA& in rlrv m d h v l C~llnsolven.nd 4 % ninhvdrin in Dvridine ~~
'lhe solvents were introduced conveniently into the test tubes without wetting the walls by means of a wooden ramp with a
a.
brated by &an8 of rulgd tapered brass strip, were seieeted which permitted the trzppeeoidal strip of filter paper to he held in position without touching the walls of the test tube except a t the upper end. A rectangular glass chamber (museum jar No. 4, outside dimensions 15 X 9 X 15 em.) with three 0.25-inch wooden dowels inserted in the grooves in the jar was found advantageous when it was desired t o determine simultaneously the R, values of a number of amino acids using a. single solvent mixture. Andogous types'of apparatus for macro chromatography have been desorihed recently hy Block (3) and Datta et al. (6). For tw-dimensiond chromatography i t is convenient to have available two museum jars, one for each solvent mixture. The test tube teehnique is particularly useful for solvent studies and preliminary separation of amino acids, while the museum jar method is more satisfactory for qualitative and quantitative analysis of amino acid mixtures. The Gilmont ultramicrohuret (0.01-ml. capacity) was entirely sat,isfactory for small-scale chromatogrt%phy of amino acids which requires that spots of minimum size he introduced on the filter paper. Uniform and relatively high precision in the delivery of solutions was accomplished by means of au aluminum (1/,Anch) platform (2.5 X 7.5 inches) attached to an auxiliary rod supporting the buret and provided with a, lever with which i t could he raised to make contact with the tlp of the buret a t any position of the filter paper. Using these devices ten aliquots of solution could he added t o the paper without increasing the initial spot size. A single spot (approximately lo-' ml. of solution) dries completely within 1 minute when exposed to a gentle stream of warm air. The initial position of an amino acid spot was marked accurately with apencil held in a 3-inch piece of aluminum tubing 0.375 inch in inside diameter, fixed t o the buret tip with Scotch tape.
Figure 1. Sample Holder for Photometric Determination of Ninhydrin-Stained Amino Add Chromatograms in Small-Scale Chromatography
.
?a Dot
.
Present address. Fruit and Vegetable Cherniatry Laboratory, U. S. Deprtment of Agriculture. Pasadena, Calif L
1142
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..
. ....
V O L U M E 23, NO. 8, A U G U S T 1 9 5 1
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70.
in a glass baking dish. This technique conserves ninhydrin, as well as time, and permits more uniform application than spraying. The RJ values of the chromatograms were estimated with the aid of the partogrid (18). The transmittance characteristics (Table I ) of the filter papers were determined quantitatively using a sample holder (Figure 1) (constructed to hold a filter paper strip in the milled groove between Bakelite blocks) which was inserted in the sample compartment of a photoelectric colorimeter (Lumetron 402-E) (16).
(Eastman Kodak Co., white label). Rockland and Miller ( 1 9 ) have shown that use of the latter solvent facilitates the discernmenit of faint spots and, because the spots develop gradually a t room temperature, the differentiation of amino acids present in relatively high concentration or in close proximity in chromatograms. Uniform staining of the chromatograms was accomplished by immersing each paper strip in about 35 ml. of staining solution contained in a 20 X 150 mm. test tube and by drawing each paper sheet or series of paper strips through the staining solution
D1 SCUSSION
Table I.
Filter Paper Studies. Thirteen typcs of filter papers are listed in Table I apResolrSolvent Solrent ing proximately in the order of their suitTexBoundUniformWeight. Speede, Sinhydrin Powerg, Typea N o . tureb aryc ityd Mg./sq. om. hlin. Colorf Rp ability for use in amino acid chronuS. 8: S. 589 blue 1 B A A 11 260 Blue gray 9 tography as determined on the bmis '4 11 240 Blue gray 10 S. 8: S. 507 2 -4 4 11 180 Blue gray 11 of seven characteristics. These resiilts S.'&S 589red 3 B A 11 S. & S. 602 E. & D. 4 B A 270 Purple 12 are in good agreement with the less ~01x1Whatman 1 5 B A B 11 190 Purple 12 S. b- S. 602 '6 B A A 11 280 Pink purple 13 prehensive investigations of Kowkabany A 11 280 Blue gray 14 S. & S. 576 7 a B 8 60 Pink rose 14 and Cassidy (11) and Bull et al. (4). Rlunktdlls 0 % C A 13 100 Pink 15 S.&S.598YD '9 B A The latt,er authors have pointed out t h a t E. & D . 7 10 B B C 9 120 Pink 16 >Itinktells IF 11 D B B 9 80 Pale pink 15 slow solvent speed is one criterion of the E. & D. 248 12 D C B 11 240 Purple 13 most satisfactory papers. E. & D. 613 13 D C A 9 180 Pink 15 The R, values for 18 amino acids dea S. & s.,Schleicher and Schuell; E. & D., Eaton and Dikeman. b A, smooth; B. medium Tough; C. rough; D, very rough. termined on 13 filter papers using waterC A , even; B, uneven; C, very uneven. sat'urated phenol a t 26" as solvent are d Based on p w cent transmittances, each the average of four values determined with a photoelectric colorimeter (Lumetron 402 EF) and the sample holder (shown in Figure 1) over four different areas of a shown in ~ ~11. bIt is ]of interest ~ that trapezoidal strip of filter paper. Values found were S. & S. 598 YD 43, E. & D. 248 46,s. & S. 589 blue 47, E. & D. 613 5$, E. & D. 7 60, and all others 49-54. Mean deviation from mean values, 4 less than with only a few exceptions the order of 1%: B, l I t 0 2 7 ~ ;C, more t h a n 2%. arrangement O f the monoaminoniono6 Time requred at 26O for water-saturated phenol to ascend 120 mm. on trapezoidal filter paper strips. Each value is average of 5 t o 10 closely agreeing replicate determinations. acids is independent Of the f Most eommen colm observed for chromatograms of individual amino acids stained by spraying paper stripe wihh6.25% ninhydrin in water-saturated butanol and heating sprayed strips for 5 minutes type of filter paper; the values o b a t 80'. tained with the different papers were # Ratio of length of sfaimed amino acid chromatogram to distance traveled by solvent boundary. Each value is overage of values found for 12 different amino acids each spotted with 10-4 ml. of 0.03 M for the dicarboxylic somewhat solution on txapeseidal 61ter paper strip. and the basic amino acids; the sequence of arginine and histidine is reversed on Table II. Rf Values of Amino Acids on Different Filter Papers different papers; and the value for aspartic acid Filter Paper Number0 Amino Acid 1 2 3 4 5 6 7 8 9 10 11 12 13 Range differed widely from those for the other amino Aspartic acid 41 21 31 22 25 22 23 28 36 30 24 20 20 20-41 acids On each O f the filter papers except numbers 42 46 46 37 38 38 39 40 Glutamic acid 53 38 48 28 32 28-53 1,2, 3, and 7. Other amino acids for which sepa71 60 24-71 50 64 46 42 50 25 65 50 Lysine 24 24 30 78 64 61 68 58 58 52 34 Arginine 42 31 45 73 62 31-78 rations on certain papers (numbers given in par77 76 44-77 73 62 57 65 56 45 56 53 Histidine 44 50 50 48 45 43 45 38 38 30-48 entheses) appear to be possible include glutamic 36 36 34 30 Cystine 35 31 35 serine $: h j ji :!I6"$ acid (8, 11, 12, 13), lysine (I), histidine (5), Glycine Filter Paper Characteristics
i
i
Threonine Cysteine Alanine Tyrosine Valine Methionine Leucine Isoleucine
56 67 66 67 80 83 86
54 61 62 61 79 80 86
Proline
91
95. 72
a
:; it
60 64 66 64 82
84 88
56
:; :; :! $
58 8 5 64 66 66 67 68 83 82 84 83 90 91
ii :g t: 91
See Table I for filter paper types.
88
56
62
50 58
65 60 66 63 82 82 83 84 88 83
gz 2; [A 2: 2;
60 66 73 73 75 75 77 76 92 86 92 86 94 91
64 70 72 73 71 76 73 79 87 90 84 94 89
62 62 69 67 71 68 65 69 78 87 88 87 82 89
50-70 58-73 60-76 61-79 78-92 80-94 82-94
93
92
77
72-95
:; :: :: 91
87
97
i: E:
::
95
92
Solvent was water-saturated phenol.
cystine (1, 7, 12, 131, serine (11, 121, glycine (51, threonine (12), and proline (2, 3, 9). The shades of the ninhydrin colors for individual amino acids varied on different papers and the Rf values for Iysine'increased as the colors changed from blue gray. to light pink. As it was observed (data not shown) that the pII values of aqueous extracts of the filter papers varied from
ANALYTICAL CHEMISTRY
1144
S. &Y. 589 Blue IEihbon paper with phenol as ~olvent. The Rj values were 0.25 for untreated paper, 0.37 and 0.50 for Solvent Solvent Most Yielding Effective in paper treated with 0.005 S sodium hySequence Inverted Separating Amdroxide, and 0.42 for paper treated with Most common Sequence ino Acid Pair Group Fixed 0.005 iV hydrochloric acid. McFarren Acidic Glutamic acid > aspartic acid _6,-11 Basic Histidine > arginine 7,8,11 i.z,S,S,ll (fa) has shown recently that all the Histidine > lysine 7,8,11 5,S, 5 10,11 naturally occurring amino acids may be Arginine > lysine 5 6,g separated on a one-dimensional chroma3, 4._ .5,7 Neutral Serine and glycine > cystine togram by means of water-immiscible 1, 6 , 7, 8, 9 7,8,11 Serine > glycine 4, 5 , j Threonine > serine and glycine solvents and filter papers buffered to 7. 8,Y Alanine > threonine particular pH values. 3 , 4 , 6 , 7 , 8,9.10 Valine a n d methionine > alanine Solvent Studies. In teats on solvents Methionine > valine 5 , 11 2,8 ; 4 i ~ ' containing 1-propanol or other types of Ieoleucine and leucine > methionine 3, E, y 2_. 8 , 9, 1 1 Leucine > isoleucine a ater-miscible alcohols and hydrochloric Cyclic Proline > tyrosine 1 , 2 , 1 0 , 1 17, ~ ,p. i" 10 acid utilized by Moore and Stein ( I S ) for Phenylalanine > tyrosine 10,11 1, 3, 6,7 , 32, 4 the separation of amino acids on starch Tryptophan > tyrosine 5 6 , z 99_ Phenylalanine > tryptophan 10,1 1 3 , s8 columns, the liquid phases formed two Phenylalanine > proline 5, 6 -1 . 5 3 , 4 , li,1 1 widely separated boundaries on filter paper strips and the amino acids did not Underlined figures indicate solvents which yield Kreater a See fGotnotes to Figure 6 for notations. (or greatest) differences in Hj values. migrate beyond the lower solvent boundary. On the other hand, there was only one solvent boundary and the amino acids separated satisfactorily when hy4.8to 6.5 and that there were marked differences in the nindr~chloll(~ ac*id was omitted from these solvents. Success in rmolving amino acids under these conditions depends, it would hydrin colors of chromatograms on the filter papers, these appear from the experiments of Consden et al. (5) on waterfactors may account for the variability of the RI values of the miscil~ksolvents, on the relatively Ion. water content of papers acidic and the basic amino acids. In general, the more acidic not subjected to preliminary equilibration with aqueous solvents. p H values were correlated with the deeper ninhydrin colors. The Kater logging of filter papers encountered in the use of watersignificance of pH was further denoted by tests of lysine ( i n saturated volatile organic liquids was prevented by Bentley and LVliitehead ( 2 ) and other n.orkers (1, 7 , 8, 10) through the use of air-tight chambers or reduced air space. The RI values of 19 amino acids with five solvent mixtures conLeucine 400 taining from 10 to 90% 1-propanol in water are shown in Figure 2. Cystine separated completely from all other amino acids with 50% I-propanol and tryptophan Kith 10% I-propanol. I t should be 80 possible, therefore, to separate cystine and tryptophan quantitatively under these conditions and to determine them quantita60 tively by the direct photometric method of Rockland and Dunn RF ' (16) or by some analogous procedure. 100 The Rf values of asparagine (tan colored spot with relatively 40 small RI value) and of leucine (purple spot with relatively high A. Ethanol o RI value) with solvent mixtures containing from 10 to 90% B. Methanol o water in various alcohols (methanol, ethanol, I-propanol, iso20 C. n - P r o p d o propyl alcohol, and twt-butyl alcohol) and Cellosolves (methyl, D. tert- Butanol 0
Table 111. Amino Acid Sequences Based on R / Values in Different Solvents"
-
-
1
1
IO0
90
80
70
60
50
40
30
20
IO
% Alcohol (Vv)
1
Leucine
100
25-
RP
250
-
3
80
I560
RF
-
'
00
/
40
520
I
I
100
't
4Oo 90
*
80
70
60
50
40
x)
20
10
YO Cellosolve (Vv)
Figure 3. Rf Values of Leucine and Asparagine in Aqueous Alcohols and Aqueous Cellosolves
I
I
80
I
60
I
I
40
I
I 20
I
I 0
% n-Propanol ("A) Figure 4.
R, Values of Representative Amino Acids in 1-Propanol-Water Mixtures
. . . . . . . . . . . .Running
time (minutes) of solvents on S. & S. 589 Blue Ribbon filter paper
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[Since this work was completed and the R ~ ~ I I U script submitted for publication, Muller and Clegg Min. I I I I I I I I (14) have reported that the density, as well a s the I I viscosity, of the solvent is a significant factor in retarding solvent trawl. If the curve (Figure 5 ) 90 for viscositv-surface tension is modified t o viscosity x 'density , it more closely approxi70 21 surface tenpion mates the shape of the solvent velocity curve in 70 agreement with the findings of Muller and Clegg. 1 It is of practical importance that there n a s no significant change in the Rj values of alaiiiiie 17 50 or glycine determined from four chromatogrune of each amino arid prepared successively with 50 the same aliquot of water-saturated phenol (111 trapezoidal strips of Whatman No. 1 paper. The 13 30 R j values were also identical when deterniiiied similarly with a fresh aliquot of this solvent in the 30 unuashed test tubes. The R/ values for each of 19 amino acids were identical when determined o n IO 9 trapezoidal strips in test tubes and a museum jar, 0 20 40 60 80 100 but differed significantly from those obtained on+ rectangular sheets of paper in a museum jar. In$ % Ethanol general, the Rf values were higher and the running Figure 5. Correlation of Running Time of Aqueous Ethanol Solutimes longer for the trapezoidal strips than for the tions on Filter Paper with viscosity and Surface Tension of Solvents rectangular sheets of filter paper, especially for the acidic and the basic amino acids. The fixed and most common sequences of amino acids, the ethyl, and n-butyl Cellosolves) are shown in Figure 3. The values solvents yielding inverted sequences, and the solvents most e€increased regularly with increasing percentage of water in the fective in separating individual amino acids, pairs of amino solvent mixture and decreased with increasing number of carbon acids, and groups of amino acids are shown in Tables 111 and IV. atoms in the molecule of organic substance. The greatest As shown in Figure 6, curves drawn from plots of the RJ values in spread in the K/ values occurred over the 10 to 30% range of eleven solvent mixtures for amino aridp representative of t h e water concentration. Methanol was found to give the greatest acidic, basic, neutral, arid ryclic gri~upsare ,strikingly similar in spread in R , values and butyl Cellosolve the least. Combinations of alcohols or Cellosolves in water yielded RJ values intermrdiate between those for the individual solvent-water pairs.
VIS ST
Time
("/VI
l a b l e 1V. Solvents Most Effective in Separating Groups of Amino Acids and Individual Amino Acids in a Group ~
Group Feutrai Cyclic Basic Bcidic
-4cidic 7 4,6,7,8,9
5 , 6, 7,_S
Group Basic 5,12 3,4,5,9,10,ll3 7 ,5 10
..
Cyclic 6,7. 8 4,8,9
___Neutrai
5 i ,S.Y_
6. 11
Underlined figures indicate solvents which yield greater (or greatest) differences in Rf values. Spe footnotes to Figure 6 for notations.
The R, values (see footnote g, Table I) of the amino acids varied with the percentage of water in the solvent mixture. r2s shown in Figure 4 for four groups of amino acids (only one representative of each group listed), the R, values of arginine (also histidine and lysine) decreased linearly with increasing propanol in the solvent mixture, the R, values of aspartic arid (also glutamic acid) increased linearly in the solvent mixtures containing more than 50% 1-propanol, the R, values of leucine (also cysteine, isoleucine, valine, phenylalanine, and tryptophan) were minimal a t about 50% propanol, and the R, values of alanine (also glycine, serine, and threonine) did not vary oignificantly with changing solvent Composition. The lowest values for leucine were found with the alcohols and Cellosolves of highest molecular ueights and for the solvent mixtures of the percentage composition which gave the longest running times, I t was also determined (Figure 5 ) that the solvent running time for solvent mixtures of different ethanol concentrations nearly paralleled the ratios of the viscosity and surface tension of these solvent mixtures.
Figure 6. R / Values of Representative Acidic, Basic, Neutral, and Cyclic Amino Acids in Different Solvents Whatman S o . 1 filter paper Solvents and methods. 1. Water-saturated benzyl alcohol. Descendlng, large scale (6) 2. tert-Amyl alcohol ( 5 ) 3. tert-Butyl alcohol, 70%. Ascending, small acale sheets, museum jar technioup ?. l-Propanol, 70% as,in 3 J. Xethanol, 6 7 % as in 3 6. Water-saturated phenol with HCN ( 5 ) 7. Water-saturated phenol with 3% ammonia ( 5 ) 8. Water-saturated n-cresol with cupron and 0.1% ammonia 9. Water-saturated m-oresol with cupron ( 5 ) 10. Collidine-lutidine. Ascending, niuscurn jar 11. Tetrahydrofurfury1 alcohol. is';&
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 Dum, 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.
