than 0.05 mmole per liter, which is the lower limit of the present method. This may be remedied either b y vacuum concentration of the liquor or by calibration. The data of Table I11 were obtained by placing 30 grams of 48 X 65 mesh sulfide mineral particles in a beaker containing 200 ml. of a n aqueous xanthate solution (0.625 m M per liter). The mineral slurry was agitated for 10 minutes, then centrifuged for 3 minutes. A 50-ml. aliquot of the supernatant liquor was removed and enough solid sodium hydroxide, potassium chloride, and eosin were added to yield the concentration of the normal stock solution. A polarogram was run to determine the concentration of xanthate. Xanthate in two other 50-ml. aliquots was determined in accordance with the iodometric method of Taylor and Knoll (16). The polarographic method may possibly be adapted for mill control. Mino (12) reported recently that a rugged portable polarograph has been developed and used in Japan for the purpose of mill control. He claimed
that this polarograph is simple and easy to operate, even in the hands of relatively unskilled technicians. ACKNOWLEDGMENT
The writers wish to thank Daniel L. Love for his technical advice. They also wish to thank Joseph Jordan and Richard Javick for their constructive suggestions. LITERATURE CITED
(1) Afanas’ev, B. X., Zavodskaya Lab. 15, 1271 (1949). (2) Berger, J., Acta Chem. Scand. 6 , 1564 (1952). (3) Brdieka, R., 2. Elektrochem. 48, 278 (1942). (4) D; Rei&, C. Hj., private communica-
tion, Bolidens Gruvaktiebolag Boliden, Sweden, 1952. (5) Fujii, Yujiro, J . Mining Inst. Japan
70, 99 (1954). (6) Hirschkind, W., Eng. &fin.-J.Press 119,968 (June 1925). (7) Kolthoff, I. M., Lingane, J. J., “Polarography,” 2nd ed., Vol. 1, p. 262, Interscience, Nec- York 1952.
(8) Kolthoff, I. M., Miller, G. S., J . Am. Chem. SOC.63, 1405 (1941). (9) Linch, A. L., ANAL. CHEM.23, 293 (1951). (10) Majumdar, K. K., J. Sci. Ind. Research (India) IIB, 260 (1952). (11) Meites, L., Meites, T., ANAL.CHEM. 20, 984 (1948). (12) Mino, H., Eng. Mining J . 156, 97 (September 1955). (13) Salamy, S. G., Nixon, J. C., Australian J . Chem. 7, No. 2, 146 (1954). (14) Shcherbakova, E. A., Zauodskaya Lab. 4 , 1053 (1935). (15) Taggart, A. F., “Handbook of Mineral Dressing,” pp. 1207-87, TViley, Kew York, 1945. (16) Taylor, T. C., Knoll, A. F., Trans. Am. Inst. Mining M e t . Engrs. 112, 384 (1934). (17) Wark, I. W.,“Principles of Flotation,’’ p. 84, Australasian Institute of Mining and Metallurgy, Melbourne, Australia, 1938. (18) Weiss, N., Trans. Am Inst. Mining M e t . Engrs. (Mining Sect.) 183, 145 (1949). (19) Wiesner, K., Collection Czechoslov. Chem. Communs. 12, 594 (1947)
RECEIVED for review December 23, 1955. Accepted April 17, 1957. College of Mineral Industries Contribution No. 55-35.
Quantitative Evaluation of Spots on Paper Chromatograms Light-Flux Measurement through Negative Printed Spots ROMAN MYKOLAJEWYCZ’
879 Elm St.,
New Haven, Conn.
b To determine quantitatively the amount of substance in paper chromatogram spots, a negative photocopy is made of the developed chromatogram. The spots appear more or less white on a black background and are translucent when illuminated from one side. The light flux transmitted through those spots is measured. The light flux is approximately linear in proportion to the spotted amount of substance, is in high degree independent of the size and form of the spot, and can be measured easily with a photometer.
I
the components of a mixture are separated and deposited as spots a t different distances from the starting point (7). After development with the proper reaN PAPER CHROMATOGRAPHY,
l Present address, Olin Mathieson Chemical Corp., Kew Haven, Conn.
1300
ANALYTICAL CHEMISTRY
gent, colored spots appear on the paper, each one corresponding to one of the separated components. This is a quick and sensitive method for separating components of a mixture that otherwise are difficult to separate, such as amino acids or sugars. QUANTITATIVE DETERMINATION
Paper chromatography was developed further to enable quantitative determination of the separated components. Various methods are used more or less successfully. Elution. This method seems to be most exact, b u t in practical use often gives unsatisfactory results. The spots have t o be eluted quantitatively and a relatively large amount of eluent must be used. The amounts of materials in t h e spots are small and therefore a very dilute sample is obtained. Other substances present, which may also be eluted, often inter-
fere and cause large variations in the results. For instance, small amounts of ammonia present in air and absorbed by the filter paper have caused large errors in the chromatographic determination of amino acids by the elution procedure (11, 15). Corrections due to the filter paper blank must be mdde. If several improvements are used, the average error of a single determination of amino acids by elution methods could be lowered in some cases to 5% (11, 13, 17, 19, 24). Area Measurement. As a rule, t h e area of t h e spot enlarges as t h e amount spotted is increased. This is shown graphically in Figure 1. The shaded bars in t h e upper line represent three spots of a substance in the starting position, spotted in a concentration ratio of 1 t o 2 t o 4. The height of the bars shows the concentration, and t h e base of t h e bars, t h e area. In t h e starting position t h e areas of the spots are the same; but
1
ORIGINAL POSITION
I FINAL POSITION
Figure 1.
Diffusion of spotted substance in chromatograms
Different amounts of spotted substance, same diffusion coefficient Maximum density value, shown by vertical arrows, decreases and area (diameter), shown by horizontal line, increases with distance traveled during chromatography, diffusion causes spreading of the substance over a larger area. As the amount spotted increases, the spreading proceeds more rapidly because of the higher concentration gradient on the edge of the spot, The spots flatten and their areas increase. It has been shown experimentally (10) and proved theoretically (4) that in the final position the areas of the spots are proportional to the logarithm of the amount spotted. LOW (STANDARD)
This relationship has been used for quantitative determination of the substances in the spots (2, 9). However, the areas of the spots are not well defined because of their diffuse and indistinct boundaries, and the color intensity of the spots disappears gradually into the surrounding background. Therefore, it is not possible to determine the area exactly. Color Density. An average color density (22) or maximum density
MEDIUM
HIGH
-I
ORIGINAL POSITION cc
c--c
I
1
INFLUENCE OF DIFFUSION RATE
r---
1 1
DIFFUSION
1 r--1
r--1
A
L
ITlmtIi II I I ! I I 1 1 ll\ll 1 1
FINAL POSITION
LIGHT FLUX TRANSMITTED THROUGH THE NEGATIVE PRINT
COLOR DENSITY OF NEGATIVE PRINT Figure 2.
(1, 16, 17, 10) has been used with success for quantitative determination of the spotted substance. I n Figure 1 the maximum color density of the spots is represented by the height of the bars and by the vertical arrow beside each bar. As the amount of the spotted substance increases, the density also increases. I n the final position the maximal color density is linearly proportional to the logarithm of the spotted amount within a certain range. This experimentally stated relationship is a basis for the optical determinations. A modified method, using measurement of light reflectance instead of color density, has also been developed (23) and used for quantitative chromatographic analysis of metals. Scanning the color density with a recording photometer along the run direction produces a curve in which each spot is represented by an elevation similar to bands or peaks in spectrograms. The area included by this elevation is proportional to the color density of the spot and its length in the run direction. This area, when planimetered, gives a value that is approximately linear in proportion to the amount spotted, and allows a quantitative determination ( 2 , 6, 6, 20, 21). This method was modified using light intensity of the negative photographed spots instead of color density and scanning along the run direction (12). The scanning method combines two factors of the spot: its color density and its length in the scanning direction, but not across this direction. It does not take into consideration all the spot area and, therefore, cannot be used in two - dimensional chromatograms. It requires rather complicated and tedious procedures, but gives good results.
Diffusion of spotted substance in chromatograms
Same spotted amount, different diffusion coefficient In final position maximum density (height) decreases and area (diameter) increases with increasing diffusion. Light flux through negative prints keeps constant by changing diffusion
The amount of material in a chromatographic spot can be determined by the t n o factors, area and color density (or concentration), which represent something like two dimensions of a spot (the horizontal and the vertical). The area itself consists of two dimensions, length and width, but here and in the following discussion they are both combined as one “horizontal” dimension. Area and color density are functionally connected in a reciprocal way and behave as folloas: The larger the area, the lower the color density of the spots with the same amount of substance. (Compare the final position of the spots in Figure 2.) As the distance migrated increases, diffusion causes an increase of area and decrease of density. Both area and color density are greater as the amount spotted increases. By measurement of area or density, it VOL. 2 9 , NO. 9 , SEPTEMBER 1957
1301
possible to make quantitative determinations. Determinations based on these two methods are correct only in these cases where the E, value and the diffusion coefficient for all the spots are the same and the form of the spots is regular, as shown in Figure 1. But there are different factors influencing Rl value and the diffusion rate of the spots during chromatography, such as different rates of flow and irregularities in paper thickness and structure ( I d ) . The diffusion is affected also by some additional substances that are present mostly in the :tnalyzed sample. These substancese.g., salts or sugars in amino acid samples-usually increase the diffusion. causing irregular spreading or streaking. Thus, the sample spots are often irregular, diffuse, and streaky while the standard spots (made with pure subytances) on the same sheet are small. compact, and regular. T o make t h r diffusion conditions of the standard and sample solutions approximately the same, i t has been proposed to add some salts to the standard solution (S),but frequently this is not possible. A highly accurate comparison of standard and unknown spots is therefore not possible with the area or color density method. This is shonn in Figure 2. where three spots containing the same amount of substance have a different diffusion rate. The first spot, with the lowest diffusion rate, represents a standard solution, and the following two represent unknown solutions containing some additional substance that increases their diffusion rate. I n the starting position all the spots have the same area and density. B u t during the run, the higher diffusion rate of the second and third spots causes a more rapid spreading. Therefore, in the final position the spots have different areas and color densities. As the diffusion rate increases, the density becomes lower and the area larger. When the first spot is used as a standard for determining the amount6 in the other spots, the density comparison gives too low results and the area comparison, too high results. Thcl deviations of results made by the two methods are in opposite directions from correct value. These deviations can reach up to 30% in amino acid determinations in the presence of interfering substances such as sugars and salts Each of the two methods used alonc gives a relatively larger error, b u t when both area and color density are taken into consideration, the deviations compensate each other to some extent and give a more accurate average result. This effect was demonstrated h\ carrying out a run with serine solution. Spots of various sizes were made, from compact to long streaks, each containing 17 of serine. This created condi-
IS
1302
ANALYTICAL CHEMISTRY
Table I.
Effect of Diffusion Rate on Determination by Different Methods
[Results given in per cent of theoretical (correct) amount] Diffusion Rate of Sample Solution Same as Greater than Much greater Much greater Method Used standard standard than standard and streaking 132 166 123 lrea measurement 100 io 58 73 Max. color density 100 103 98 100 104 Light flux Only at exactly the same diffusion and spreading coiidition in both spotted solutionssample and standard-all methods give theoreticallg exact results (1007,). When the sample solution had higher spreading or streaking, area measurement gave too high results and the maximum color density method too lon-. The light flux method gave values that are close t o the exact amount and independent of the area or shape.
tions that resembled a successive increase in the diffusion of the spots. After chromatography the spots enlarged more, but their shape and area difference remained in their original relationship. The usefulness of applying both area measurement and maximum color density measurement simultaneously has been pointed out by Block and his associates (2, 3) in their studies on amino acids. They obtained good results using the relation that the determined amount is linearly proportional to the product of the area and color density of the spot. LIGHT FLUX MEASUREMENT THROUGH TRANSLUCENT SPOTS
An exact method must take into consideration both factors of the spot, area and concentration (or color density). Essentially, such a method divides the spot into small area elements, determines the concentration in each element, and integrates it over the whole area. K i t h this condition in vie\\, a new method was developed for evalnating the spots on chromatograms.
Procedure. After chromatography and color development, t h e sheets are printed as a negative photocopy. The lamp in t h e photocopying apparatus must produce a uniform illumination over t h e chromatogram area t o obtain t h e same copying conditions for all t h e spots t h a t have t o be measured and compared. I n these studies Apeco photocopy equipment and printing materials were used (American Photocopy Equipment Co., Chicago, Ill.). The exposure time and developing conditions must be adjusted to produce a negative with a dark background, but the spots must show a good gradation in their density as well as in size according to the amount spotted. This can be obtained best when the exposure time is properly estimated so that the weakest spot shows u p slightly on the dark background. The developing time must be long enough to let the developer affect the sensitive emulsion through all its thickness and not only a t the surface. The background then ap-
pears dark bj. reflected as well as transmitted light. Low contrast papers seem to produce plotting curves showing better linearity: but on the other hand, their backgrountl is not dark enough. Therefore, a high contrast paper was used although the plotting curves were more bent. Another advantage of the high contrast paper is that it makes weak spots on the chromatogram show u p more clearly, and increases small differences in density. Therefore, with high contrast paper it is possible to strengthen the spots and to increase their gradation. The spots too weak for evaluation by other photometric methods can be evaluated on these copies. The method can be made more sensitive and can be extended to smaller amounts or concentrations of the analyzed substance. The fixed, washed, and dried negatives sometimes show small white dots on the background caused by printing impurities or some dust particles. These dots must be covered with ink before photometering. From each chromatogram it is ttdvisable to make two or even three photocopies, changing the exposure time in the ratio of 1 to 2 to 3. If all these are used for photometering, i t is possible to obtain duplicate or triplicate results for each spot and then to calculate a more accurate average value. This eliniinates random irregularities in the density of the photocopy paper and the like. The negative is placed on a glass pane and illuminated from below. For this purpose the Photovolt Densitometer was used. The lens and the diaphragm of the illuminating lamp were removed and a glass pane was mounted on the box (Figure 3). To this glass pane was attached a sheet of dense black paper with a rectangular window cut out. The size of this window must be large enough to illuminate fully the largest spot of the series but still cover the adjacent spots. (It is possible to make a window with dimensions that can be changed by using two L-shaped sheets of strong black paper.) The dimensions of the window must be the same during measurement of one series of comparable spots. The negative photocopy is placed on the window and covered with another glass pane to keep it flat. The lamp must provide a homogeneous illumination of the entire Above the windon- a t all times.
window, at a distance not closer than five times the diameter of the spot, the photocell is supported by a holding arm. The photocopy is placed so that one spot a pears in the center of the window. l f t e r the light flux is measured by reading the photometer, the next spot of the series is shifted to the window.
PHOTOCELL
GLASS r
,
L
\
PANE
PnommY WINDOW GLASS PANE
ILLUMINATING
LAMP
Figure 3, Arrangement for measuring light flux
following spots transmit less intense light but over a larger area. The total light flux coming through is the same for each spot; it differs only in the distribution. To make a more detailed study of how the light transmittance is related to the spotted amount, the following assays were made.
in Figure 4. The transmittance curve is not a n exponential function-Le., it does not follow Beer's law. S t higher concentrations the transmittance is too high or the density too low. The reversal of the light values by making negative prints is shomm by the characteristic curve of the photographic material used. I n Figure 5,a. the usual form of the curve is plotted: the density-that is, the negative logarithm of the transmitted light-T emus the logarithm of the exposing light ( E ) . I n Figure 5,b, the same relation is shown but not plotted logxrithmically. These relations are all shown in Figure 6. I n section I the relation is the same as in Figure 4, but with reversed axes. I n section I1 the relation is the same as in Figure 5,b. The E axis, common for both, s h o w the amount of light that is transmitted through the colored portion of the paper and falls on the emulsion of the printing paper. This light produces on photoprints a transmittance shown in section 11. Many curves are possible here depending upon the exposing time, but only one is shown to avoid confusion. All of them have approximately the same shape.
The relationship of per cent light transmittance to the concentration of the spotted substance on the paper was studied. If the transmittance of the blank paper is assumed to be loo%, only the light absorption due to the spotted substance is taken into account. The concentration of the substance on paper is the amount per unit of area. Known amounts of tyrosine were spotted on paper, forming spots with an even and well-defined shape. The spots, without chromatographing. verc submitted to the usual color reaction by dipping into a solution of 0.4% ninhydrin in water-saturated butanol, heating to 60" C. for 15 minutes, and exposing to room temperature for 4 hours (ripening) to obtain a high color density. The light transmittance of these spots versus the concentration is shon7-n
W
/-x
+
" _1
0.1 CONCENTRATION.
02
03
7' NH2.N/cm'
b
Figure 4. Light transmittance of tyrosine spots developed with ninhydrin
The light transmitted tlirough the paper is dispersed in all dirrctions and only a part of all the flux comes to the photocell window. Only this part is measured, but it is exactly proportional to the whole transmitted light flux. The photometering must be done in a darkroom to prevent any outside light from coming into the photocell. The readings must be done using the light intensity scale, not color density. The readings of kno\ni standard spots on one sheet give a standard curve for the determination of unknowns. Usu:illy some five or 10 spots of tlic uilknown solution must be measured to obtain an average result with good :wwracy.
I
-I I
100
IO
I
0
LIGHT VALUE
Figure 5.
10 20 E LIGHT VALUE
30
Characteristic curves of photographic emulsion
t
CONCENTRATION 0.2 f NH,-N PER cmz AREA
--
- - - _ _ -- - -
THEORY
In the lower part of Figure 2 is sliown a cross section of the color density of negative printed spots, each containing the same amount of substance but having successively increasing diffusion rates. Light flux transmitted through these spots is represented by the bundles of arrows. The first spot transmits an intense light beam but covers a small areii, and the
E. LIGHT VALUE
\--{
OF THE POSITIVE
Figure 6. Relations of spotted concentration and light transmitted through positive original and negative copy VOL. 29, NO. 9 , SEPTEMBER 1957
1303
Section I11 shows the transmittance of the photoprint in relation to the concentration of the substance. This last relationship shows a n approximately straight line proportionality between the concentration (c)-Le., amount of substance spotted per area unit-and the transmitted light intensity ( I ) in a certain range a t not too high concentrations. Assuming the linear proportionality, this can be expressed as
+b = Io(ac + 6 )
Ill0 or
I
=
ac
(1)
where Io is the incoming light intensity, and a and b are constants. Here also many curves are possible depending upon the exposing time, but they show the same kind of approximate linear proportionality if the exposing time is in the proper range-i.e., the time usually needed to produce a good print. The amount of substance (dA) in a n area element ( d p ) is equal to the product of area and concentration (c) : d A = cdp
B u t as the concentration is different in different parts of the photometered area ( p ) , the total amount of the substance ( A ) in a spot is A
=
fpcdp
(2)
P is the total area taken into account by the integration. The light flux (dL) coming through a n area element ( d p ) is equal to the product of area and light intensity ( I ): d L = Idp
If the function of I defined by Equation
Equation 3 is the relation between the spotted amount of the substance ( A ) and the light flux (L)through a negative printed spot. This relation should be a linear function and the standard curve representing the spotted amount versus the photometer readings should be a straight line. But this linearity is based upon the assumption, expressed by the Equation 1, that the transmitted light is linearly proportional to the concentration. This proportionality is only approximate and therefore the real relationship of the spotted amounts and the photometer readings shows a deviation from a straight line. The standard curve is a more or less curved line. This deviation and the curvature of the standard line depend on different printing conditions, such as the characteristic curve of the negative material, exposing and developing time, and the validity of Beer’s laws on colored spots. Those conditjons are approximately the same for the spots located on the same sheet but different for different chromatograms. One standard curve is not valid for all chromatograms. Therefore, for each chromatogram a separate standard curve must be constructed based on measurement of standard spots on this sheet. This relation of the spotted amount to the light flux transmitted through the translucent spots is not dependent on streaking, diffusion, or other irregularities in the distribution of the substance. Therefore, it is much more accurate than the other relations of maximum color density or area. The experiments with serine, mentioned earlier, shon-ed this experimentally.
1 is substituted, this gives d L = Io(ac
+ b) dp
PRACTICAL APPLICATIONS
The total light flux ( L ) through the total area ( P ) photometered is L = I Of P
(UC
+ 6)dp
or
L
=
I o( a
f P cdp
+ bP)
and with Equation 2: L
=
Io (a24
+ bP)
and A = L
(&) - (F)
The photometered area ( P ) is equal to the window area of the illuminating box of the photometer. This area is the same through one series of measurements and is, therefore a constant. A11 the other figures in brackets in the above expression are constants, too. K h e n new symbols ( k and m) are substituted for the members in brackets, the following equation is obtained: A = k L - m
1304
ANALYTICAL CHEMISTRY
(3)
This method has been applied to the determination of amino acids and especially tyrosine, TI hich produces streaky spots and gives unsatisfactory results by the maximum color density method. Tyrosine determinations by the latter method give 13% average deviation of the mean value, nhile the light flux method gives 5% deviation under the same conditions. The same method has been applied with success for making quantitative determinstions of sugars and lactobionic acid, colored \T-ith silver nitrate and ammonia. The smallest measurable amount of substance could be decreased to one half or even one third of its previous value by using high contrast copying paper. Lactobionic acid did not produce spots of good color and shape and therefore the maximum color density method failed in this case. The light flux method showed 8% deviation when only one spot was measured. By measuring 10
spots and calculating an average result, the error could be lowered to 3%. In Figure 7, standard curves are shown for tyrosine chromatographed with m-cresol (pH 8.4, running time 40 hours) and colored with ninhydrin. The three curves were obtained from one chromatogram but a t three different exposure times. The sensitivity of the photometer was adjusted for each series of comparable spots so that the largest (or brightest) spot gave a scale reading of 100.
100
50 PHOTOMETER
READINGS
Figure 7. Standard curves for tyrosine for different exposure times
The curves for amino acids can be approximated in their middle range by a straight line; this range is best for making a determination. Figure 7 shows that this straight line range shifts to lower amounts of substance )Then the exposing time of the pliotocopy is shorter. Lactose and carbohydrates, colored with silver nitrate and ammonia, produced standard curves which also can be approximated by a straight line. Some ultraviolet light absorbing substances (purines, pyrimidines) can be detected on chromatograms by making a contact print with ultraiiolet light, as suggested by RIarkham and Smith ( I S ) , without using any color reagent. The same prints can be used for making quantitative determination of these substances by measuring light flux. DISCUSSION
The light flux method combines the measuring of the two factors of the spot -color density and area. Therefore it gives more accurate results, especially when the substance separates irregularly and not in well formed spots and when the area or density measuring method fails.
The method is simple and requires relatively simple equipment. This method requires a n additional step in the procedure-making negative photocopies of the chromatograms-but this is the only disadvantage. On the other hand, by copying on a high contrast paper it is possible to increase the transmittance gradation of weak spots. Thus, the method can be made more sensitive and useful for smaller amounts or concentrations of the substance to be determined. The original chromatograms usually lose their color intensity in a short time, while the photocopies remain as a permanent record and allow measurements to be repeated or checked a t any time. ACKNOWLEDGMENT
The author expresses his thanks to Gotfred Haugaard for his helpful discussions and to Helen Brezicki, Chester Hargreaves, Leon Marker, and Eve
Marker for their advices in the preparation and correction of the manuscript. LITERATURE CITED
(1) Block, R. J., ANAL. CHEM.22, 1327 (1950). (2) Block, R. J., Science 108,608 (1948). (3) Bolling, D., Sober, H. A., Block, R. J., Federation Proc. 8, 185 (1949). (4) Brimley, R. C., Nature 163, 215 I 1 949 ). (5) Brown, J. A , , Marsh, M. M., ANAL. CHEM.25, 1865 (1953). (6) Bull, H. B., Hahn, J. W.,Baptist, V. H.. J . Am. Chem. SOC.71. 550 (1949 j. (7) Consden, R., Gordon, A. H., Martin, A. J. P., Biochem. J . 38, 224 (1944). \ - - - ~ I -
(8) Decker, P., Riffart, IT.,Wagner, H., Klin. Wochschr. 29, 418 (1951). (9) Fisher, R. B., Parsons, D. S., Holmes, R., Nature 164, 183
11949). (10) Fisher, R. B., Parsons, D. S., Morrison, G. A., Zbid., 161,764 (1948). (11) Fowden, L., Biochem. J . 48, 327 (1951).
(12) Gustafsson, C., Sundman, J., Lindh, T., Paper and Timber (Finland) 1, 1 (1951). (13) Klatzkin, C., Nature 169, 422 (1952). (14) Kowkabany, G., Cassidy, H. G., ANAL.CHEM.24, 643 (1952). (15) Landua, A. J., Awapara, J., Science 109,385 (1949). (16) McFarren, E. F., Brand, K., Rutkowski, H., ANAL.CHEM.23, 1146 (1951).
McFarren, E. F., Mills, J., Zbid., 24, 650 (1952).
Markham, R., Smith, J. D., Biochem. J . 45, 294 (1949). Martin, A. J. P., Mittelmann, R., Zbid., 43, 353 (1948).
Patton, A. R., Chism, P., ANAL. CHEII.23, 1683 (1951). Redfield, R. R., Barron, E. S. G., Arch. Biochem. and Biophys. 35, 443 (1952).
Rockland, L. B., Dunn, M. S..
J . Am. Chem. Soc. 71, 4121 (1949). (23) Vaeck, S. V., Nature 172, 213 (1953). (24) Woivod, A. J., Biochem. J . 45, 412 (1949).
RECEIVEDfor review June 21, 1956. Accepted April 22, 1957.
Chromatographic Separation of 2,4-Dini trop henylhydrazones E. L. PIPPEN, E. J. EYRING, and MASAHIDE NONAKA Western Utilization Research Branch, Agricultural Research Service,
b Chromatography
of 2,4-dinitrophenylhydrazones of aliphatic aldehydes and ketones on silicic acid-Celite columns was studied. Columns packed to a height of 75 cm. permitted separation of hydrazone mixtures of adjacent members of the homologous series of saturated normal aldehydes as high as C11. In addition, the feasibility of separating various combinations of the derivatives of 34 aliphatic aldehydes and ketones is described.
I
CONNECTION with flavor studies under way a t this laboratory it was necessary to separate a complex mixture of aliphatic 2,4-dinitrophenylhydrazones. Existing reports (1, 2, 6, 6) concerning chromatographic separation of aliphatic 2.4 - dinitrophenylhydrazones, while helpful, were limited primarily to studies of somewhat simple mixtures of carbonyl compounds having one to six carbon atoms. The possibility of separating known mixtures, particularly of some higher aldehydes and ketones, and certain other combinations of 2,4dinitrophenylhydrazones not previously reported was therefore studied. The results not only describe N
U. S. Department o f Agriculfure, Albany 7 0, Calif.
the relative chromatographic behavior of many new combinations of 2,4dinitrophenylhydrazones but also show that it is possible, by using longer columns, to separate the 2,4-dinitrophenylhydrazone derivatives of the homologous series of normal, saturated aldehydes from Cg through C1,. EXPERIMENTAL WORK
'Reparation of 2,4-Dinitrophenylhydrazones. Most of the hydrazones used in this study n-ere prepared from commercially available carbonyl compounds. The carbonyl compound was added to a solution containing a 10% excess of 2,4-dinitrophenylhydrazine (2 grams per liter in 2-l' hydrochloric acid). After the hydrazone precipitated it n a s filtered off and washed thoroughly with water, followed by one or more recrystallizations from alcohol. The 2,4-dinitrophenylhydrazones of glycolaldehyde, methyl cyclopropyl ketone, 2-methylbutanal, 2-methyl-2butenal (tiglaldehyde), n-pentanal, n-heptanal, and 2-hexenal were authentic samples available within the laboratory. 3-iVethylmercaptopropional was prepared as described by Patton (4). Melting points of all hydrazones were checked to ensure their authenticity. I n some instances chromatog-
raphy followed by recrystallization was necessary to remove impurities. Diacetyl mono-2,4-dinitrophenylhydrazone was obtained by adding the hydrazine solution slowly to an excess of diacetyl dissolved in water. Ordinarily when diacetyl-2,4-dinitrophenylhydrazone is prepared, both carbonyl groups in the diacetyl molecule react to form the bis-2,4-dinitrophenylhydrazone. If only one carbonyl group reacts, a hydrazone is obtained n hich is referred to here as the mono derivative. The yellow precipitate was filtered off, washed with mater, taken up in hot alcohol, and filtered to remove any insoluble his derivative. The mono derivative in the filtrate was crystallized several times from alcohol. It melted a t 178" C. Analvsis showed C. 45.1%: H I 3.79%; and f, 21.1%. Calculateb for C1,HIoO5S4:C, 45.117,; H, 3.79%; N. 21.05%. 'Chromatography of 2,4-Dinitrophenylhydrazones. Chromatography was carried out on glass columns having an outside diameter of 35 mm. The procedure, solvent mixtures, adsorbent (silicic acid-Celite, 2 to 1 weight ratio), etc., used were those of Gordon and coworkers ( 2 ) . Solvents were redistilled before use; the petroleum ether used boiled a t 40' to 50" C. TWOcolumn lengths, Fvhich permitted packing the adsorbent to maximum VOL. 2 9 , NO. 9 , SEPTEMBER 1957
1305
