V O L U M E 2 4 , NO. 1, J A N U A R Y 1 9 5 2 1 as

relative error was 1.0%. The standard deviation of a single value was 3 parts per thousand.

Advantages. The proposed method of analysis possesses the follou ing advantages over currently available procedures applicable to the same concentration range: freedom from co- precipitation or occlusion of boric acid; freedom from a blank correction; no requirement of boron-free glassware, because only

muin opportunity for loss of boron by volatilization because lengthy evaporation is not necessary; simple equipment; and

LITERATURE CITED

(1) Foote, F. J., IND. ESG. CHEM., * 4 ~ 4 ~ . ED., 4 , 3 9 (1932).

sis,” p. 179, New York, D. Van Yostrand Co., 1939.

70, New York, Interscience Publishers, 1947.

(2) Furman, N. H., “Scott’s Standard Methods of Chemical i ln : i lY-

(3) Kolthoff, I. M., and Stenger, V. A , , “Volumetric Analysis.’ p.

(4) Ib id . , p,114. (5) Mylius, W., Chenl.-Ztg , 57, 195 (19333.

(7) Ibid., 121, 170 (1941). (8) TschischeTVskl, s,, ~ ~ d .

acid solutions are boiled and then only for a fel\, minutes; mini- (6) Schafer, H., and Sleverts, A.9 2’. anal. Chenl., 121, 161 (1941).

cjLern,, 18, 607 (1926).

increased rapiditv and good accuracy. RECEIVED April 21, 1951.

Automatically Increasing Solvent Polarity in Chromatography

KENNETH 0. DONALDSON, VICTOR J. TULANE, AND LIWRENCE 11. 3IARSfIALL Howard L’niaersity, Washington, D. C.

The determination of organic acids separated on silica gel columns is limited in sensitivity by the re- duced resolution experienced for the more water- soluble acids. Although the literature contains procedures for improving resolution by manipula- tion of the solvent, the changes in polarity which accomplish this end arc abrupt and are obtained by frequent manual operations. The widespread use of

X SEPARATIOSS employing partition chromatography for I organic acids, the resolving power of silica gel systems de- crease8 with increasing water solubility of the solutes (2 , 4, 5) . Isherwood ( 2 ) proposed the use of several solvents, but was aware that no one, or even two, ITould suitably resolve an organic acid mixture of the complexity used in his study. In a procedure for the determination of furmaric acid ( 4 ) , this was again recognized

acetic + 5 4. 1 fumaric

P 7) x L

E 3 - 2

-0 si

0 62-

*

9

oconi t ic oxal ic

10 20 30 4050 60 70 80 90 f rac t ion r J r b e r f ract ion nJrnber

Figure 1. Chromatograms Typical curve (left) resulting when chromatographic solvent progressively increases in n-butyl alcohol content. Typical results ( r i g h t ) , when a eolvent of fixed ooinposition (10% n-butyl alcohol in chloroform, v /v ) is employed. The difference between the character of the curves as well BS the resolving power of the two systems with respect t o fumaric and ace- tic acids contrasts here the two types of chromatographic influent. Frac-

tion number X 2 equals ml. of effluent.

apd effluent fractions on the chromatogram were collected by geo- metrically increasing volumes. In effect, such a procedure grad- ually increased the volume of polar mobile phase in each effluent fraction. Marvel and Rands ( 5 ) successfully employed a series

mechanical apparatus for the collection of chromato- graphic fractions necessitates a procedure that requires no attention once the separation has be- gun. A simple device permits the delivery of a sol- vent automatically but gradually increasing in polarity. Recovery studies indicate that, with the exception of oxalacetic acid the method is suitable for the quantitative determination of organic acids.

of solvents progressively increasing in composition with respect t o n-butyl alcohol. Their solvents were added manually to the col- umn in order of increasing n-butyl alcohol concentration. This procedure, like the first, required constant attention and mas, therefore, more noticeably disadvantageous when mechanical ap- paratus was employed for the collection of effluent fractions. The above procedures reduced the number of effluent fractions required to collect a given acid by manually but not gradually in- creasing the polarity of the mobile phase. When the concentra- tion of each fraction n as plotted against its number, the curves on the chromatogram ivere sharper than those obtained Kith a sol- vent of fixed polarity.

The present report, combining the principles underlying the de- termination of fumaric acid and those of Marvel and Rands, de- scribes a procedure for gradually and automatically increasing the alcohol concentration and thereby the polarity of the solvent entering the column. With this method no false chromato- graphic peaks or shoulders on the peaks occur, as they sometimes do when the solvent polarity is increased in discrete steps. The applicability of this approach for the separation of certain acids of physiological interest is examined. A typical chromatogram em- ploying the new procedure appears in Figure 1 (left).

APPARATUS

A device was arranged so that the stem of the solvent reservoir ( A , Figure 2 ) extended to xithin 3 em. of the bottom of the mix- ing vessel, B. When 4 was filled Kith 50% (v./v.) n-butyl alco- hol-chloroform and B with 175 ml of pure chloroform, the sol- vent flouing from column C created a fall in pressure within the system which was relieved by solvent delivered by the side arm. The 50y0 mixture from A flowed into B and because of the dif- ference between the density of n-butyl alcohol (0.804 gram per ml.) and chloroform (1.497 gram per ml.) mixing was obtained, as demonstrated below, by the time the ~olvent entered C. As a result, the solution entering the column gradual11 increased with respect to alcohol concentration. n-Amyl alcohol (density, 0.810 gram per ml.), where indicated be!ow, was substituted for n-butyl alcohol.

186

Table I . Observed Densities and Calculated Composition of Effluent Fractions

For a blank analysis to determine rate of mixing of two components of chromatographic solvent)

n-Butyl Alcohol-Chloroform n-Amyl Alcohol-Chloroform Density % composition Density % wmposition

Fractiona 23' n-butyl alcohol 23 n-amyl alcohol 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18

1.475 1.470 1.469 1.448 1.422 1.396 1.378 1.359 1.341 1.325 1 310 1.293 I , 280 1 266 1.258 1.243 1.234 1 226

0.00 0.00 1.00 3.00 5.50 9.30 12.20 15.00 18.00 2 0 , 5 0 22.50 25.00 27.00 29.30 31.00 32.50 34 00 35 50

a Fraction number X 10 = volume, ml.

1,47<i 1.467 1.470 1.451

l:i04

1:375

1:3i3

l'ii0

1 : 162

1 : i35 1 224

0.00 0.00 1.00 3.00

9.30 . . .

li'00

2 i 'io 25:50

34: 50

30:OO

36.50 . . .

NATURE OF iVfIXING OF COMPONENTS

Solvent irom the delivery apparatus was permitted to flow through a silica gel column prepared as described by Isherwood (2), and 10-ml. fractions of the solvent emerging from the column were collected. Densities de- termined at 23" are indicated in Table I. T h e d e n s i t y of n - b u t y l a lcohol o r n -amyl alcohol-chloro- form is a straight-line function of the alcohol concentration. The equa- tion for the n-butyl alcohol mixture is :

T = -151.4 p + 221.8

where x = '% n-butyl (v./v.) and p = density at 23".

APPLICABILITY FOR CHROMATO- GRAPHIC SEPARATION OF CERTAIN

ORGANIC ACIDS

Except for the progressively chang- i ng inf 1 u e n t , t h e chromatographic analyses were like those previously described ( 4 , 6). Three-gram columns were used with a 2 ml. 0.05 2\r su l fur ic ac id aqueous phase. The columns had a diameter of 10 mm. and a height of approximately 30 cm. Effluent fraction volume was 2 ml. The concentration of acid in each

Figure 2. Dis- penser for De- livering Solvent That Increases Automatically in

Polarity I t s ta r t of chromato- &ram A contains 50% chloroform-n-butyl al- cohol, B , pure chloro- form, and C, progres- sively changing mix- ture from side arm. Union of side a rm and column above C was by ground-glass joint, but can be by one-hole i topper.

fraction was measured by titration with stmdard alkali using thymol blue as the indicator.

The pobitions on the chromatogram and the degree to which these were reproducible are indicated in Table 11. The location of all acids on the chromatogram for the mixtures was supported by evidence beyond that supplied by the repeated appearance of the acids in their first observed chromatographic positions. Ef- fluent fumaric and aconitic acids showed, rcspectively, molar ex- tinction coefficients a t 230 mm. of 3.9 X lo3 and 6.1 X lo3 as compared with 4.1 X lo3 and 6.3 X lo3 of the reagent acids. The 2,4-dinitrophenylhydrazones of oxalacetic and a-ketoglutaric acids showed extinction values (3) identical to those for these de- rivatives of the reagent acids. Acetate, oxalate, and lactate were

A N A L Y T I C A L C H E M I S T R Y

identified by known chemical procedures. Effluent succinate was mixed with an equal quantity of reagent succinate and re- chromatographed. The rechromatographed succinate appeared at a position on the chromatogram like that of the original, and recovery of the total succinate admitted to the column, expressed as per cent, 'Ivas 101. Recovery studies from the organic acid mixtures (Table 111) indicate that, with the exception of oxala- cetic acid, the method is suitable for the quantitative determina- tion of the acids employed. KO other arids were used in the study, and the purity of the acids employed was greater than 94% in every case

DISCUSSION

The suitability of the dispensing device as an aid in controlling resolution by the chromatographic system was tested by changing from one polar solvent to another. n-Amyl alcohol, employed in vessel A, permitted (Figure 3) the separation of two hitherto un- resolvable acids (1 ) by this change in the nature of the polar sol- vent in this procedure.

The difference between the character of the two curves (Figure 1) illustrates that the total volume of solvent required for the re- lease of the acids from the column is decreased when the influent progressively increases with respect to alcohol content. As the rates of f l o ~ through the column for the influent of fixed compo-

2 .o

W G $ 1.6 cz n > I I I .2 2 n 0 a 0.8 i t 0 80.4

I

f

(50% N.AMYL ALCOHOL-

CHLOROFORM $)

LAFTIC

SUCCINIC

100 120

i \ i l i / . '. ....,'

100 120 FRACTION NUMBER

Figure 3. Chromatographic Separation of Mixture of Lactic, Succinic, and Aconitic

Acids Employing 50% n-amyl alcohol in vessel A of Figure 2

Fraction number = ml. of effluent

Table 11. Observed Positions of Acids on Chromatogram [As described by fraction of highest concentration (peak) for each acid]

s o . of AV. Peak Standard Acid.: Chromatograms Effluent" Deviation

Acetic Aconitic Fumaric a-Ketoglriravic Oxalacetic Oxalic Succinic Lactic,

a Peak effluent X 2 = volume, ml b Range.

20.20 42 40 29 oc) 42 00 47 00 49 40 37 00 37 00

0 30 0.50 0 0 O b 0 0 O b 1 O O b 0 50 0 00 0 00 I

V O L U M E 24, NO. 1, J A N U A R Y 1 9 5 2 187

Table 111. Recovery Data for Determination of Organic Acids from Mixtures

(When chromatographic solvent employed automatically increases in polarity)

No. of Added, Found, Av. Chromato- M g . , hlg., % Av.

Acids grams Av. Av. Recovery Deviation Acetic Aconitic Fumaric a-Ketoglutaric Oxalacetic Oxalic Succinic Lactic Succinic)

1.600 0.606 2.000 1.193 1.155 1.164 1.543 2,100

1.616 0 ,612 2.049 1.163 0.826 1.536 1.538 2.100

101 .oo 100. BO 102.90 97.90 71.850 Q8.84 QQ .84

100.00

0.32 0.62 0.64 0.00 0 .w 0.04 0.10 0 . 0 0

a S o a t tempt was made t o account for low recoveries of oxalacetate. Decomposition probably accounted for 1045.

sition and that which is changing do not differ widely, the release of the acids by the procedure described above would require a shorter time.

The observed data of Table I, where density is plotted against fraction number, would describe a curve for which the equation would be (6):

Then, p = Ae-ub

C Ke-bl

On differentiation this becomes d C l d t = - K C b t where p = density, A, b = constants, y = fraction number, t = time, and C = alcohol concentration (and thereby polarity).

Thus i t follows that dC/dt or the change in polarity is decreas-

ing. If the sum of the physicochemical processes accounting for the broadening of a chromatographic zone during its descent on the column were considered as a single but hypothetical event- diffusion along the longitudinal axis of the column-the change of this event with respect to time would follow from the well-known diffusion lams. Then the increase in polarity of the solvent in re- stricting “diffusion” is maximal when diffusion is maximal. Nor- mal resolution by the column should be enhanced. The ob- served data (Figure 3) illustrate the resolution when the system described above is applied t o succinic and lactic acids, a mixture previously unresolvable (1 ) on silica gel.

ACKNOWLEDGMENT

This investigation was supported in part by a research grant from the Sational Cancer Institute of the National Institutes of Health, U. S. Public Health Service.

LITERATURE CITED

(1) Busch, H., H u r l b e r t , R. B., and Potter, V. R., Federation Proc.,

(2) I sherwood, F. A., Biochem. J . , 40, 688 (1946). (3) Lepage, G. A., Cancer Research, 6 , 393 (1950). (4) Marshall, L. M., Orten, J. M., and Smith, 8. H., J. Bid. Chem.,

(5) Slarvel, C. S., and Rands, R. D., Jr., J . Am. Chem. SOC., 72,2642

( 6 ) Steen, F. H., and BaUou, D. H., “Analytic Geometry,” p. 215,

RECEIVED June 1, 1951. Data taken from a thesis presented by Kenneth 0. Donaldson in partial fulfillment of the requirements for the degree of master of science, Howard University, 1951.

10, 169 (1951).

179,1127 (1948).

(1958).

New York, Ginn and Co., 1946.

Separation of Organic Acids from Plant Tissues Chroma tograp h ic Tee hniq ue

W. A. BULEN, J. E. VARNER, AND R. C. BURRELL Department of Agricultural Biochemistry, T h e Ohio State University, Columbus, Ohio

Organic acids are of great significance in plant and animal metabolism. They ordinarily occur as com- plex mixtures, from which isolation and identifica- tion are difficult or impossible by methods currently available. Several recent papers have pointed toward the possibility of chromatographic separations. However, these have been of limited usefulness either because accurate identification was difficult or because they failed to include many important naturally occurring acids. The method reported

URRENT biochemical studies have demonstrated a need C for a complete and fairly rapid separation of small amounts of organic acids from biological materials. Extensive studies of the functions of organic acids as intermediates in plant metabo- lism are dependent upon a satisfactory method for their isolation. Most of the currently available methods for the determination of organic acids are restricted to individual acids and prove unsatis- factory when several acids are to be determined in the same sample. These methods often require treatments that destroy or alter the acids involved. Some of the limitations of existing methods have been discussed by Thimann and Bonner in a recent review (8).

The most satisfactory proof of the occurrence of a specific acid in plant tissues is given by its actual isolation and identification. Isherwood ( 1 ) has published a chromatographic method employ-

uses an initial separation on a silica gel column followed, when necessary, by additional separations using both chromatographic and chemical tech- niques. It provides for separation and tentative iden- tification of 16 biologically important organic acids. This method has been applied to the separation and identification of acids in tomato fruits and Bryo- p h y l l u m leaves. It should have applications in metabolic studies as well as in identification of acids present in a wide variety of biological materials.

ing a silica gel column for the isolation and determination of or- ganic acids from fruit, by which several specific acids can be s e p arated without severe or destructive treatment. When previous knowledge of the acids present is not available, this method offers little aid in their tentative identification. Marvel and Rands (6) published a similar chromatographic method suitable for the separation of various organic acids but not especially well adapted to biological materials, as i t fails to separate some of the com- monly occurring acids. It was felt that the real potentialities of this chromatographic technique for the separation of organic acids of metabolic importance had not yet been realized.

By the use of mixtures of known organic acids, this method has been successfully extended and many separations not previously reported have been achieved. The improved technique has been applied to separation of organic acids from typical plant tissues.