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. ChromatoMg., hlg., % Av. Acids grams Av. Av. Recovery Deviation 0.32 Acetic 1.600 1.616 101.oo 0.62 Aconitic 0,612 100. BO 0.606 0.64 2.049 102.90 Fumaric 2.000 0.00 1.163 97.90 a-Ketoglutaric 1.193 71.850 0 Oxalacetic 1.155 0.826 0.04 1.536 Q8.84 Oxalic 1.164 0.10 1.538 QQ .84 Succinic 1.543 Lactic 0.00 2.100 100.00 2,100 Succinic)
.w
a S o a t t e m p t 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): p = Ae-ub Then, 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 it 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 eventdiffusion along the longitudinal axis of the column-the change of this event with respect t o time would follow from the well-known diffusion lams. Then the increase in polarity of the solvent in restricting “diffusion” is maximal when diffusion is maximal. Normal resolution by the column should be enhanced. The observed 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.,
10, 169 (1951). (2) I s h e r w o o d , 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., 179,1127 (1948). (5)
Slarvel, C. S., and Rands, R. D., Jr., J . Am. Chem. SOC.,7 2 , 2 6 4 2 (1958).
( 6 ) Steen, F. H., and BaUou, D. H., “Analytic Geometry,” p. 215, New York, Ginn and Co., 1946. RECEIVED J u n e 1, 1951. D a t a 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.
Separation of Organic Acids from Plant Tissues Chromatograp h ic Teehniq u e W. A. BULEN, J. E. VARNER, AND R. C. BURRELL D e p a r t m e n t of Agricultural Biochemistry, T h e Ohio S t a t e University, C o l u m b u s , Ohio Organic acids are of great significance in plant and animal metabolism. They ordinarily occur as complex mixtures, from which isolation and identification 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
C
URRENT biochemical studies have demonstrated a need 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 metabolism 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 unsatisfactory 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 techniques. It provides for separation and tentative identification of 16 biologically important organic acids. This method has been applied to the separation and identification of acids in tomato fruits and Bryop 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 organic 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 t o biological materials, as it fails t o separate some of the commonly 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.
ANALYTICAL CHEMISTRY
188 APPARATUS
Kitrogen tank and needle valve A chromatographic tube (Figure 1) 12 mm. in inside diameter, 24 cm. long, with attached stopcock A chromatographic tube 7 nim. in inside diameter] 75 cm. long, with attached stopcock Solvent reservoir and pressure system (Figure 1) Intermittent siphon RIicroburet, 10-nil. capacity REAGENTS
Silica gel was prepared from Rlallinckrodt's silicic acid (specially prepared for chromatographic analysis) by removal of the fine particles through repeated suspension in distilled water and decantation of the slower settling particles until approximately one third of the original material was removed. The remaining Fraction containing the coarse particles was filtered, dried in an oven at 100" C. for 24 hours, and stored in a closed container.
IOcm. H
Fipure:l.
Apparatus
K
solid with 5.7 ml. of 0.05 Ar sodium hydroxide and making up to 100 ml. Standard 0.01 Ar sodium hydroxide (carbonate-free). EXPERIMENTAL
The complete procedure for the isolation of the individual acid consists of a preliminary or survey separation] followed when necessary by additional separations. The survey separation is conducted by adding the mixture of organic acids to the top of a silica gel column and developing with a series of n-butyl alcohol-chloroform solvents. Fractions of the effluent are collected and titrated. Procedure. The standard survey column is prepared as follows: A glass-wool plug is placed in the bottom of the chromatographic tube (12 mm. X 25 em.) to support the column. Eight grams of the prepared silica gel are mixed with 5.5 ml. of 0.5 N sulfuric acid in a mortar. The resulting free-flowing powder is slurried in 60 to 70 ml. of chloroform and added to the chromatographic tube in successive portions. A gas pressure of 5 to 10 em. of mercury is applied to the top of the tube to speed the removal of excess solvent, care being evercised not to allow the solvent level to fall below the top of the column. This procedure gives a uniformly packed column 14.5 em. long. The mixture of free organic acids, containing a total of 10 to 100 mg , dissolved in 0.5 ml. of 0.5 N sulfuric acid is mixed thoroughly with 1 gram of dry silica gel and the resulting free-flowing powder is transferred quantitatively to the top of the column. This transfer is easily accomplished by pouring the mixture into the tube through a short-stemmed funnel which, along with the container, is rinsed m-ith 2 to 3 ml. of chloroform. If the acids are liberated from their sodium salts, a slight excess of sulfuric acid may be used in the liberation with no undesirable effects. With the aid of a glass rod, the silica gel containing the sample is slurried in the chroma.tographic tube with the 2 to 3 ml. of chloroform used in rinsing, the ewess chloroform is drained through the column, and a glasswool plug is pressed down firmly on the surface of the sample. This plug serves to wipe down particles of silica gel adhering to the tube and also prevents disturbance of the column during addition of the eluting solvents. Development of this survey column proceeds by addition of a series of n-butyl alcohol-chloroform solvents experimentally selected to give maximum separation of the metabolically important acids. These solvents are added to the top of the chromatographic column through a reservoir which permits the application of pressure from a nitrogen tank and a t the same time allon s the addition of more solvent to the reservoir without interrupting the pressure a t the top of the column. The solvent schedule is 100 ml. of 5%, 135 ml. of 15%, 100 ml. of 25%, 300 ml. of 3 5 % ) and 150 ml. of 50% n-butyl alcohol-chloroform, A few m d l ~ l ~ t eof rs the 5% solvent are poured directly into the tube above the glasswool plug and the remainder is added to the reservoir. A gas pressure of 2 to 5 em. of mercury is applied and collection of the fractions is started. The pressure is adjusted so that the effluent
W
a
b
Figure 2. Results of Preliminary Separation
Elutin solvents were prepared from U.S.P. chloroform washed twice wit% water and C.P. n-butyl alcohol to contain 5, 15, 20, 25, 35, and 50% (v./v.) wbutyl alcohol in chloroform and from water-washed benzene and C.P. n-butyl alcohol to contain 25% (v./v.) n-butyl alcohol in benzene. Each solvent mixture was equilibrated against 0.5 N sulfuric acid by shaking the two phases in a separatory funnel and passing the solvent layer through dry filter paper to remove suspended water droplets. Phenol red indicator was prepared by grinding 100 mg. of the
is collected a t the rate of 80 ml. per hour. Subsequent solvents are added just before the last of the preceding solvent passes into the column. The effluent passes through an intermittent siphon adjusted arbitrarily to deliver about 3.3 ml. Individual fractions are collected for titration in 25-ml. Erlenmeyer flasks, to which 8 to 10 ml. of carbon dioxidefree distilled water and 2 drops of henol red indicator are then added, and the mixture is titrated y addition of 0.01 N sodium hydroxide from a microburet. Near the end point vigorous agitation is necessary to ensure intimate
E
V O L U M E 24, NO. 1, J A N U A R Y 1 9 5 2 contact between the two phases. tion requires about 9 hours.
The complete survey separa-
Figure 2 shows titration values obtained when a mixture of the acids indicated was chromatographed, and illustrates the relative positions of those acids completely separated by the survey Procedure and the position of those separating into groups that demand further attention. The order of elution of the acids was determined by chromatographing known acids singly and in pairs.
189 effected chromatographically by use of a modification of the method presented by Keish (6). The fractions which contain these acids are combined and the water layer is separated and evaporated to dryness. The free acids are liberated from their and added to the O f a survey The Procedure for the preparation of the column and addition of the acids is the Same as that for the survey separation. The column is developed by the addition of 25% (v./v.) n-butyl alcohol-benzene solvent. Figure 4 illustrates the separation achieved by this procedure.
2
0 3
0 0
z
GLUTARIC
5a
SUCCINIC
LL
FRACTION NUMBER
Figure 3.
Separation of Group I
Fumaric, glutaric, and formic acids are 'aJt separated from each other on the survey column, but can be readily. separated by a . second treatment on a longer column.
Figure 4. Separation of Succinic and Lactic Acids
* Compound that separates whenever commercial C.P. lactic acid is chromatographed
The fract,ions containing these acids are comiJined in a separatory funnel and the xvater layer is separated. The solvent layer is washed once wit,h water; if necessary, sufficient 0.01 N sodium hydroxide is added to make t8hesolution alkaline to phenol red. The w'at,ersolutions are combined and evaporated to dryness on a xater bath. The sodium salts obtained are dissolved in a volume not, exceeding 0.5 nil. of 0.5 S sulfuric acid. An amount of 4 .Ysulfuric acid equivalent to 'the organic acid salts present is added. This solution is mixed thoroughly with 1 gram of dry silica gel and transferred quantitatively to t,he top of a chromatographic column 60 c m . long and 7 nim. in diameter prepared, as dwcribed previously, from 12 grams of silica gel mixed nith 8.5 nil.of 0.5 N sulfuric acid. This column is developed by the addit,ion of 20y0 n-butyl alcohol-chloroform solvent. Fractions are collected at, the rate of 30 ml. per hour with the aid of an intermittent siphon delivering 0.9 nil.
If the conditions of the initial estraction of the tissue are such that, cis-aconitic acid is not converted to tmns-aconitic acid ( 8 ) , &-aconitic acid is eluted from t,he survey column with glycolic and oxalic acids. The fractions containing these three acids are conibined, the \vater layer is separated, and 6he solution of the sodium salts is evaporated to about 10 ml. and transferred quant'itatively to a centrifuge tube. The solution is made just acid to phenol red Tyith dilute sulfuric acid and the oxalate is precipitated by adding a slight excess of solid calcium carbonate and digesting for 30 minutes in a boiling R-ater bath. The mixt,ure of calcium oxalate and calcium carbonate is centrifuged, the supernatant fluid is decanted, and the precipitate is washed tn-ice with 2 ml. of hot water. Oxalic acid may be recovered as such from this precipi-
X
Figure 3 shows hoiv fumaric, glutaric, and formic acids are separated with this longer column. I t can be seen from Figure 2 that lactic, succinic, a-ketoglutaric, and trans-aconitic acids are not completely separated in the preliminary separation. The degree of separation achieved does give information useful in the tentative identification of these acids. The second small peak in the trans-aconitic acid curve is a result of the solvent change occurring during its elution. For reasons discussed below, the separation of these acids on a longer silica gel column is not practical. If all of these acids occur together in amounts such that they are not separated from each other by the survey separation, the following procedure may be used:
tate by acidification and extraction, or it may be determined by the standard permanganate titration procedure. The supernatant solution and washings are combined and evaporated to dryness, and 1 ml. of 0.5 iV sulfuric acid is added. This acidified mixture is kept in a closed container in a water bath a t 65' C. for 1 hour. This treatment quantitatively transforms cis-aconitic to trans-aconitic acid (S). The resulting trans-aconitic acid and glycolic acid are separated by addition to the top of the survey column in silica gel as previously described and elution with 20% n-butyl alcohol-chloroform solvent.
The fractions containing these acids are combined and the water layer is separated. This solut.ion of the sodium salts of theorganic acids is evaporated to a volume of 10 ml. on a water bat'h. The e-ketoglutaric acid is separated as the 2,4-dinitrophenylhydrazone ( 2 ) by adding 2 ml. of a 1% solution of 2,4-dinitrophenylhydrazine in 10% sulfuric acid and allowing the mixture to stand for 30 minutes. The mixture is then extracted t r i c e with 2 to 3 ml. of ether. The combined ether extracts are extracted once with 10 ml. of water and this water solution in turn is extracted twice with 2 to 3 ml. of ether. The combined ether layers cont,ain the a-ket.oglutaric acid derivative, and the combined water layers cont,ain lactic, succinic, and trans-aconitic acid if all were initially present. I t is now possible to isolate trans-aconitic acid by rechromatographing this acid mixture according to the survey procedure. If both lactic and succinic acids are present, their separation is
Figure 5 shows that separation is effected by this procedure. Figure 2 shows that acetic, pyruvic, malic, citric, isocitric, and tartaric acids are separated completely during the survey separation. Any of the accepted extraction methods may be adapted for use with this separation procedure. If the isolation of volatile acids is desired, Isherwood's ( 1 ) procedure has significant advantages. With some tissues the extraction method of Pucher et al. ( 7 )is more convenient. Application. Table I lists the acids found in tomato fruit and Bryophyllum leaves. The tomatoes were extracted by the method of Isherwood and the Bryophyllum leaves by the method of Pucher et al. after freeze-drying.
FRACTION NUMBER
Figure 5.
Separation of Group
I11
ANALYTICAL CHEMISTRY
190
100” C. for 24 hours does not change the silicic acid in any way
Table I.
Acids of Tomato Fruit and Bryophyllum Leaves
Acid Acetic Formic Lactic Succinic trans-Aconitic Oxalic Malic Citric Isocitric
Bryophyllum calycinum Leaves
Tomato Fruit M e . X 102/10 g r a m f r e s h weight 2.1 2.6 0.9 .. 1 .a i.3 i:9
3:0
i:,
46
..
40
66
that is significant in this application. Flow Rate. For the survey column described the rate of flow may be any rate up to 100 ml. per hour. Rates greater than this tend to produce unsymmetrical curves and spreading of acid bands. Extremely s l o ~ rates are disadvantageous because of the increased time required and the increased losses due to esterification.
89
z
’,FORMIC AND FUMARIC
0 2I-
2 a LL
DISCUSSION
FORMIC AND FUMARIC I -
K W
The reproducibility of a chromatographic procedure of this type is such that the acids present in a given tissue may be tentatively identified from knowledge of the threshold volumes of their elution. Each individual apparatus, however, should be standardized by chromatographing a mixture of several known acids before running the unknown mixture. In this way fractions of the effluent can be coordinated with each acid without the tedious task of exactly duplicating the apparatus described here. Column Length. For a given column diameter and solvent mixture, an increase in the column length results in a directly proportionate increase in the separation of the threshold volumes of the individual acids. If the column length is increased when a series of solvent mixtures is being used, a corresponding increase in the volume of each solvent mixture is required. Attempts to adapt this relationship to the separation of those acids not separating on a short column often fail because of an increase in the volume of solvent required to elute individual acids. The “spreading” effect is most pronounced n4th acids having low R values-that is, a low ratio of movement of solute to movement of developing phase (4). For example, the separation of the acids in gronp I1 is not complete even on a 60-cm. column. Column Diameter. For a given column length and solvent mixture, the relationship between the cross-sectional areas of the column and the amount of solvent required to elute the acids is one of direct proportionality. An increase in column diameter may be desirable when relatively large amounts of the acids are to be separated. Solvent Composition. Changes in the n-butyl alcoholchloroform ratios produce different effects on the individual acids. The order in which the acids are eluted changes with solvent composition. For example, in the 5% solvent, acetic acid immediately precedes pyruvic acid. If 10% solvent is used, the order of emergence is acetic, fumaric, pyruvic, and glutaric acid. In 20% solvent, the sequence is fumaric, acetic, glutaric, and pyruvic acid. An association effect between fumaric and formic acids was noted. These acids fail to separate in the 5% solvent but separate completely when 10% solvent is used (Figure 6 ) . Variation in the molar ratios of formic to fumaric acids from 0.75 to 3.7 does not alter this association effect. Degree of Hydration of Silica Gel. Within certain limits this does not seem to be a critical factor. Amounts of water varying from 30 to 70% of the weight of the dry silica gel produce minor changes in the properties of a column. As the degree of hydration of the silica gel increases, more solvent is required to elute the acids and the separation of the threshold volumes of some of the acids is increased. If 0.5 ‘V sulfuric acid in excess of the weight of silica gel is used, excessive spreading of the acids with low? R values occurs. Preparation of Silica Gel. Removal of the finer particles from Mallinckrodt’s silicic acid facilitates packing of a uniform column and reduces the pressure required to obtain a satisfactory flow rate. A column prepared from the larger particles has the same characteristics with respect to variables other than rate as a column prepared from the finer particles. Oven drying a t
p.
L 2
* p
- FORMIC
I,’L.
ILL
2
0
FUMARIC I I
FRACTION NUMBER
Figure 6. Association Effect
Quantitative Considerations. Recoveries of the acids vary according to the individual acid but, with the exception of formic and oxalic acids, are 95 to 100%. The recovery of formic acid after elution from the two columns is 85%; the recovery of oxalic acid is also 85%. The threshold volume of each acid is characteristic and after standardization of the apparatus may be used as a means of tentative identification. The sensitivity of the method permits detection and tentative identification of quantities as small as 2 to 10 pe. of the individual acids. If one acid is present in a relatively large amount, the presence of the acid immediately following it may be obscured. In this instance the use of a smaller sample or a coli mn of larger diameter is recommended. Sample Addition and Suggested Uses. The method of adding the sample to the column permits the ready addition of watersoluble acids and eliminates the difficulties encountered when a sample is introduced in a small volume of one of the solvents. The simplicity of this method of sample addition suggests its usefulness in the direct determination and separation of organic acids from such biological materials as expressed plant sap, fruit juices, milk, and certain milk products. If, as in the case of dairy products, the material has a high fat content, this can be separated from the acids by elution with chloroform before beginning the development of the column. LITERATURE CITED
Isherwood, F. A, Biochem. J . , 40, 688 (1946). Krebs, H. A., Ibid., 32, 108 (1938). (3) Malachowski, R., and Maslowski, XI., Ber., 61B, 2521 (1928). I., J . , 35, 1358 (4) Martin, A. J. P., and Synge. R. L. &Biochem. (1) (2)
(1941). (5)
Marvel, C. S.,and Rands, R. D., Jr., J . Am. Chem. Soc., 72, 2642
(1950). (6) Neish, A. C., Can. J . Research, B 27, 6 (1949). (7) Pucher, G. W., Wakeman, A. J., and Vickery, H. B., IND.END. CHEY.,ANAL.ED.,13,244 (1941). (8) Thimann, K. U.,and Bonner, W. D., Jr., Ann. Rev.Plant Phwiol., 1 , 75 (1950).
RECEIVED May 14, 1951. Work supported in part by a contract between the Charles F. Kettering Foundation and The Ohio State University Research Foundation.
