Two-Dimensional Paper Chromatography Using One-Dimensional Apparatus Application to Sugars of Spent SulJite Liquor L. A. BOGGS Sulphite Pulp Manufacturers' Research League, Inc., ..lppleton, W'is. lJ\ 0
dimensional paper chromatography has been used extenively in studying the amino acid composition of protein hydrolyzates-e.g. (1 )-but the application of this technique to the investigation of sugar mixtures has been relatively rare (9). Most simple mixtures of monosaccharides are, with the proper choice of irrigating solvents (6, Q), relatively easy to resolve on paper chromatograms. Consequently, the usual laboratory doing this type of work is equipped with apparatus suitable only for irrigating the paper in one direction. Thus, when complex mixtures of sugars not readily resolved by one-dimensional paper chromatography are encountered, it would be convenient to have a method of running two-dimensional chromatograms without needing to resort to the use of the larger tanks conventionally eniployed. Procedures for running two-dimensional chromatograms using simple and readily available equipment have been described ( 7 , 11j. However, they involve irrigation of the paper in an ascending direction and cannot readily be applied when a satisfactory repolution of the components requires that the irrigating solvent be permitted t o flow over the paper for relatively long periods:. with the solvent front passing far beyond the end of the chromatogram, as is frequently true in the study of sugar mixtures. The sugar fraction of spent sulfite liquor, a by-product in the manufacture of pulp by the mlfite process, is a complex mixture. It has been established that its main components are xylose, arabinose, mannose, glucose, and galactose ( 4 ) . Qualitative and quantit'ative methods based upon the use of paper chromatography ( 3 , I O ) and starch column chromatography (8) have confirmed the presence of these sugars. However, none of the irrigating solvents so far trsted in the author's laboratory has given a completely satisfactory resolution on paper chromatograms of the five component,s. Accordingly, a method of studying thi.; misture of sugars by two-dimensional paper chromatography, using the available one-diinensiomtl apparatus, has been devcloped. EXPERIAIENT.4L
Sheets of filter paper (Khatmnn S o . I), 57 cm. long in the machine direction, were marked in pencil x i t h a starting line 9 cm. from the top of the paper. Vertical lines 2 cm. apart were drawn on the sheet as references to detect the extent of lateral migration during the irrigation. The prepared paper is illustrated in 9 of Figure 1. The following samples, dissolved in water or in aqueous ethyl alcohol were transferred with a small platinum loop to the starting line a t the points where this was intersected 1)y the vertical lines: mixture of standard sugars, mixture for analysis, blank, mixture for analysis, blank, mixture for analysis, mixture of standard sugars, and mixture of standard sugars. The papers were irrigated by the descending method, in the usual manner, for the period of time found necessary in previous experiments. The irrigated chromatograms were freed of solvent by evaporation a t room temperature and cut lengthwise into three strips, along the vertical lines to which no sugars had been added. The center strip was saved for subsequent irrigation with the second solvent and the components of the two outer, or indicator, strips of the chromatogram were located by spraying lightly with a solution of p-anisidine (0.2 gram) and trichloroacet,ic acid (1.0 gram) in water (50 ml.), They were then heated for the required period in an oven a t 100" to 150' C. (cf. 5 ) . This treatment resulted in the development of brown spots a t the positions where the aldoses were present and of yellow spots in the areas containing fructose. -4strip 2 cm. wide n-as cut from the unsprayed center portion of the chromatogram, a t the locations shown by the indicator strips to contain sugars (B, Figure 1). This unsprayed strip was woven into the starting line of a new chromatogram (B', Figure
1). The paper for the second chromatogram was prepared by drawing lines in pencil 7.9 and 10.1 cm. from the top of the filter paper shrxet. The space required for the unsprayed, sugar-containing strip from the first irrigation was marked off on these lines and within this area was cut a series of vertical slits (3 to 5 mm. apart) extending from the upper to the lower line. h piece of cardboard was placed under the filter paper and the cuts were made with a razor blade,
/
I'
r
.,
N
A
C
Figure 1. Papers Prepared for Running Two-Dimensional Chromatograms by Inweaving Technique Paper for the first-dimensional irrigation Unsprayed strip from first-dimensional irrigation, containing sugars to be woven into new starting line. Sugars are located by cutting off the two indicator strips (left and r i g h t ) and subjecting them to a color-producing reaction B'. Series of vertical slits (3 to 5 mm. apart) for receiving sugars from first-dimensional irrigation C. Paper for second-dimensional irrigation I ) . Vertical slits for receiving standard sugars for second-dimensional irrigation A. E.
Standard sugars were transferred M ith a platinum loop to the centers of pieces of filter paper 2 em. wide and similar slits were cut into the starting line of the nen- chromatogram to receive them. The weaving process was facilitated by cutting a pointed end, in a portion not containing sugars, on the strip to be woven into the starting line. After the weaving was completed, this end n-as cut off with a razor blade and the new chromatogram %-asirrigated with the second solvent. C, Figure 1, illustrates a paper prepared for rreaving in the samples for the second dimension of a t 11o-dimensional chromatogram. The two irrigating solvents used in the work reported here were phenol saturated with water, a t approximately 30" C., ( 9 ) , and the one-phase mixture ethyl acetate (9 volumes), acetic acid (2 volumes), and water ( 2 volumes) ( 2 , cf. 6). .2 fresh solution of the latter was prepared each n-eek. In addition to xylose, arabinose, mannose, glucose, and galactose the presence in certain samples of spent sulfite liquor of fructose has been reported (IO). Preliminary experiments showed that the centers of the spots corresponding to these six sugars were found in distinctly different positions on chromatograms which had been irrigated for periods of from 20 to 36 hours with the ethyl acetate-acetic acid-water solvent. Moreover, when a known mixture of the six sugars u as run on a chromatogram with this irrigating solvent the separation was sufficiently good so that the presence of each component could be detected unequivocally, although most of the spots overlapped and the separation of glucose and galactose was very slight. On the other hand, irrigation overnight with phenol saturated with water gave spots with R, values too close to each other for satisfactory characterization of the several sugars, and a mixture of all six gave a long smear Tithin which no individual components could be located. The sample of spent sulfite liquor sugars was prepared from a
1673
ANALYTICAL CHEMISTRY
1674
The ellipses show the relative positions of the spots obtained by two-diFirst irrigation, phenol saturated with water. Second irrigation, ethyl acetate (9 volumes), acetic acid mensional irrigation of a mixture of the ( 2 voluinesl. a a t e r (2 voluniesi. Sugars of spent suiti'te liquor (iirst irrigation) six sugars. Reading along the horizoiiR g h c o s e values." (1.85)1.73 (1.63)C 1.54 (1.46)/(1.36)1.23 (1.11)/(1.07)0.99 (0.93)C 0.88 (0.iY) tal axis from left to right these spots corE , values.6 (0.60)t o 10.35); smear, no distinct spots. Spent Sulfite Liquor respond to galactose and glucose (fused Spent Sulfite Liquor Sugars after Second toget.her), mannose, Fructose and arabiSpot CorSugars after Second Irrigation (by Inresponding Standard Sugars, Irrigation, Rglueose Standard Sugars, terpolation), R j nose (fused together), and xylose. The to R g l u e o s e Valuesa Values R/ Valuesb Values identity of the spot due to fructose ir Xylose (1.98)1.85(1.71) (1.94)1.81 (1.78) (0.53c)0.49( 0 . 4 4 ) (0.49) 0.44 (0.39) Arabinose (1.69)1.58(1.47) (1.72)1 62 (1 52) (0.61) 0.57 (0.53C) ( 0 . 5 6 ) 0 . 8 2 (0.49) readily apparent because of the characFructose (1.60)1.46(1.38) Sone ( 0 . 5 6 ) 0.52 (0.48C) Sone hlannose (1.36)1.24(1.14) (1.41)1 ZS(1.16) (0 49) 0.46 (0.43C) (0.47)0.42 (0.37) teristic lemon-yellow color developed Glucose (I .08)1 . O O (0.91) (1.08)1.02(0.96) (0.43c) 0.40(0.37) (0.39)0.36(0.34) with p-anisidine. The failure of the inGalactose (1.00)0.89(0.79) (1.00)0.91 (0.83) ( 0 . 4 8 9 0.44 (0.40) (0.43)0.40(0.37) R g l u c o s e value. Distance from starting line t o point concerned divided by distance from starting dicated centers to fall exactly a t the h i e t o center of glucose spot, reported for ethyl acetate-acetic acid-water irrigations. Standards f o r centers of the areas marked is due printhis solvent were placed on t h e starting line in pairs, as follows: xylose plus fructose, arabinose plus glucose, a n d inannose plus galactose. Figures in parentheses indicate the leading a n d trailing edgea. ripally to the fact that the values were Figures not in parentheses indicate the point of greatest concentration of the component. The diagonal calculated to the nearest 0.01 unit. line ( / ) shows a definite separation between two components. b All Ri values reported in this article are for phenol saturated with water as the irrigating solvent. I n Tables I and 11, figures from pheStandards were plared on the starting line in pairs: arabinose plus xylose, fructose plus galactose, and mannose plus glucose. nol-saturated-with-water irrigations are Indicates a lack of complete separation between two components. given in conventional R, values. Those irom the ethyl acetate-acetic acid-water Table 11. Two-Dimensional Chromatogram of a RIixture of Xylose, Arabinose, irrigations are reported in Rsiuoosevalues Fructose, Mannose, Glucose, and Galactose (distance from starting line to point conFirst irrigation, phenol saturated with water. Second irrigation, ethyl acetate (9 volumes), acetic acid (2 volumes). water ( 2 volumes) cerned, divided by distance from starting Sugars of spent sulfite liquor (iirst irrigation) R g h c o s e values. (2.03)1.87 (1.74)c1.64 (l.?~2)~ 1.46 (1 39)c 1.31 (1,21)/(1.08)1.02 (0.99)C0.86(0.82) line to center of glucose spot), since the R j values. (0.60) to (0.38); smear, no distinct spots front had passed far beyond the eud of Sugar Mixture Sugar Mixture after Second Irthe paper during the irrigation. I n ortier Spot c o r a f t e r Second rigation (by responding Standard Sugars, Irrigation, Standard Sugars, Interpolation), to show the extent of spreading and overto H g h c o s e Values R g l u c o s e values R / Values R / Values lapping of the components, figures in par(1.95)1.81 (1.63) (2.13)1.97 (1.81) (0.53)C0.49 (0.44) Xylose Arabinose (1.76)1.60 (1.45) (1.90)1.76 (1.62) (0,60) 0.57 (0.53)C entheses, to show the locations of the Fruotose (1.51)1.39 (1.27) (1.69)1.57(1.47) (0.56) 0.53 (0.50) leading and trailing edges, have been inMannose (1 36) 1.23(1.09) (1.47)1.33(1.21) (0.50)0.48(0 46)C Glucose (1.09)1.00(0 91) (1.17)1.04(0.90) (0.46) 0.42 (0.40) cluded. Where incomplete separation Galactose (1.00)0.85 (0.71) (1.11)0.95 (0.76) (0.49) 0.45 (0.41) was found between two components in C Indicates a lack of complete separation between two coinponents a mixture, this fact is indicated. Zones of complete separation between two comDonents are marked with a diagonal calcium base spruce wood sulfite cook. The stripped spent line. The presence of xylose, arabinose, mannose, glucose, and liquor was subjected t o exhaustive dialysis through cellophane galactose and the absence of detectable amounts of fructose are against distilled water (8). The material passing through the indicated in spent sulfite liquor. membrane was evaporated under reduced pressure to approxiChromatograms irrigated with phenol saturated with water mately the original volume of the stripped liquor used. This solution was mixed with 6 volumes of acetone and the mixture were dried for 24 hours a t room temperature before spraying w m allowed to stand overnight (cf. I O ) . The supernatant layer with the rolor-producing reagent. Chromatograms irrigated was decanted and the sirupy precipitate was washed thoroughly with the ethyl acetate-acetic acid-water mixture were dried apwith small portions of acetone until it became granular. The washings were combined with the supernatant layer and the acetone was removed under reduced pressure. More water was added and the solution was passed successively through a column of the acid form of Amberlite I R 120 and a column of the basic form of Amberlite I R 4-B (cf. 8). The effluent was evaporated to a sirup and this was used for the preparation of the chromatograms. Paper chromatography indicated that none of the sugars was present in the other fractions. Table I.
Two-Dimensional Chromatogram of Sugars of Spent Sulfite Liquor
Q
C
Paper chromatograms of the spent sulfite liquor sugars showed the presence of xylose, arabinose, mannose, glucose, and galactose, but not of fructose, when the ethyl acetate-acetic acid-water mixture was used as the irrigating solvent. With phenol saturated with a a t e r the result was a long smear, identical p i t h that obtained from a known mixture of the six sugars. The data obtained by two-dimensional paper chromatography, carried out by the inweaving technique described above, of the sugars from a sample of spent sulfite liquor and of mixtures of known xylose, arabinose, fructose, mannose, glucose, and galactose are presented in Tables I and 11. I n order to explain clearly and concisely the method used for recording the data, the results obtained with a known mixture of the six sugars (given in Table 11) are presented in graphic form in Figure 2. D a t a from the first irrigation (phenol saturated with water) are plotted along the vertical axis, and those from the second (ethyl acetate, acetic acid, water) are given along the horizontal axis. Results of one-dimensional irrigations of mixtures of the six sugars are placed adjacent to the corresponding axis and next to them are given data for the individual sugars.
0.7
>
I
I OD
R (Ethyl
I 0.5 aucose
ACetalO- Acetic
I
I
I
I
1.0
1.5
2.0
25
VOIU~S
Acid
- W0t.r)
Figure 2. Two-Dimensional Chromatogram of a Jlixture of Xylose, .4rabinose, Fructose, Jlannose, Glucose, and Galactose First irrigation. Phenol saturated with water Second irrigation. Ethyl acetate-acetic acid-water
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V O L U M E 24, NO. 10, O C T O B E R 1 9 5 2 proxiniately 1 hour a t room temperature before spraying. Irrigations were performed in covered glass jars 12 inches in outside diameter and 24 inches in height. A commercially available type of rack (Berkeley Chromatography Division, University Apparatus Co.) holding two glass troughs was employed. The jars were kept in a constant temperature room a t 30' C. Indications are that spent sulfite liquor contains, in addition to the sugars reported here, other carbohydrate components. The investigation is being continued. LITERATURE CITED
(1) Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J . , 38, 224 (1944).
(2) Green, J.
iV.,personal communication.
(3) Gustafsson, C., Sundman, J., Pettersson, S., and Lindh, T., Paper and Timber ( F i n l a n d ) , 33, 300 (1951). (4 ) Hagglund, E., "Chemistry of Wood," New York, Academic Press, 1951. ( 5 ) Hough, L., Jones, J. K. N., and Wadman, W. H., J . Chein. Soc., 1950,1703. (6) Jermyn, M. A,, and Isherwood, F. ii., Biochem. J.. 44, 402 11949). \_._.,_
( 7 1) Ma, R. hl., and Fontaine, T. D., Science, 110, 232 (1949). (8)I Mulvany, P. K., Agar, H. D., Peniston, Q. P., and JIcCarthy, J. L., J . A m . Chem. Soc., 73, 1255 (1951). (9) Partridpe. S. hl.. with a note by Kestall, R. G., Biochern. J., 42, 238 (1948). (10) Sundman, *J., Paper and Timher ( F i n l a n d ) , 32B, 267 (1950) (11) Williams, R. J., and Kirby, H., Science, 107, 481 (1948). R E C E I V Efor D review February 6 , 1932. Accepted -4ugust 6 , 1932.
Absorption Characteristics of the Dithizone Mixed Color System A Theoretical Treatment ROBERT G.MILKEY Geological SurGey, Lnited States Department of the Interior, Washington, D. C.
T IS common procedure to determine trace amounts of lead by means of dithizone extraction. A solution containing the lead is adjusted to a pH of 9 to 10.5, and various complexing agents are added to prevent the extraction of other reacting element? that may be present. The lead is then estracted with a dilute solution of dithizone in chloroform, and the optical density of the dithizone solution is determined with a spectrophotometer a t an appropriate wave length of light. T h r lead content of the solution is determined from a standard cmve, expressed in terms of the optical density of solution versus micrograms of lead. The question arises whether the standard curve has a constant or a varying slope. Conformance to Beer's law in one part of the curve (dilute solutions) does not ensure a straight-line function throughout the range of the curve. Orice the standard curve has been established, the analyst will discover that it does not necessarily remain dependable for an?. appreciable length of time. Alteration of the various components within the analysis can result in an almost daily change of position of the curve. This lack of dependability of the curve is a matter of some importance, as thr qtandard curve is the basis of the analysis. The explanation for these, and for other characteristics that affect the slope and shape of the curve, lies in an analysis of the factor- that enter into the derivation of the standard curve.
If one analyzes the system mathematical1~-,the resulting equations make for an understanding of the disturbances introduced by these factors and of their relative importance. DERIVATION
ilssume a divalent metal in aqueous solution, estracted with a dilute solution of dithizone in chloroform. Me++
+ 2Dz --+;Ile(Dz)? + 2H'
The optical density of the resulting organic solution is equal to density of dithizone present plus density of metal-dithizonate formed. Assuming conformance to Beer's law: Density = kiclL
where k is the extinction coefficient, c is the concentration of the absorbing constituent, and L is the lengt,h of light path in the solution; subscript 1 refers to dithizone and subscript 2 refers to dithizonate. Let T' = unit weight metal per milliliter of organic solution Let C = original concentration of dithizonr solution in milligrams per milliliter Add 1 unit weight of metal. Dithizone equivalent of metal = KIT' Dithizonate equivalent of metal = K2T' Density of solution = density of dithizone density of dithizonate
+
=
klClL
FirPt of all, it is necessary to define some of the factors that determine the standard curve: 1. The presence of a mixed-color system. The density of the dithizone solution is the result of two absorbing systems-the absorption due to the lead dithizonate formed in the extraction and, in addition, the absorption due to the dithizone left in the solution after the reaction. 2. The characteristics of the dithizone solution used. (a) Changes in concentration from one dithizone solution to the next. I t is difficult to prepare dithizone solutions that are identical in concentration. (P) Degree of instability of the solution, whirh is especially significant in sunlight and warm weather. (c) The presence of impurities in the dithizone used. 3. ('ontamination from apparatus, reagents, and the atmosphere. T h r questions then arise: To what degree does a change in any of these factors affect the usability of the curve? If these factors are susceptible to appreciable change, will it be necessary to make almost day-to-day rechecks to be sure that the curve still applies?
+ k*c*L
+ k*K,T.'L = k,(C - 2K1V)L + 2kZKgVL
= ki(C DISCUSSION
+ k2c2L
-
K1V)L
Add 2 unit weights of metal.
Density of solution n unit weights of metal. Density of solution = kl(C - nKIV)L nk2KzVL = klCL - nklKIVL nk2K2VL = nVL(k2Kz - klK1) klCL which is, thus, the equation of the standard curve. When n = zero units: Density = (0 units) X [VL(k2K2- k1Kl)I klC' I, =k,CL The slope of the density curve-that is, the rate of change of the density with change in unit weights of metal-is the differential of t'he density expression: Slope = d(density)/dn = L (k2K*- kl K,) Add
++ +
+
v
IYTERPRETATIOR-
The curve of density versus weight of metal estracted is a straight line, as t,he slope is a constant and the original density. or concentration of dithizone, C, appears only in the expression that defines the zero inkrcept. Then the concentration of the dithizone solution used in the extraction has no effect on the dope
