of acid-chloranilate ion. Figure 1 shows the effect of the solvent on sensitivity. The curve for ethyl alcohol-water was obtained b y the recommended procedure; t h a t for water alone was obtained similarly, except that 50 ml. of distilled water were used instead of 50 ml. of ethyl alcohol. The systems obey Beer’s law up t o a t least 400 y per ml. of sulfate. Measurements were made with a Beckman Model B spectrophotometer in 1cm. cells. K i t h 5-em. cells, the usable sensitivity is 1 y per ml. of sulfate in the final solution, or 2 p.p.m. in the original sample. Wave length of maximum absorption for acid-chloranilate ion in aqueous chloranilic acid solution and for that produced according to the colorimetric method is shown in Figure 2. Both curyes were obtained with a Beckman Model DU spectrophotometer and 1-cm. cells. Curve 1 represents the absorption of 0.017, aqueous chloranilic acid; the p H of this solution was 2.2. Curve 2 \\-as obtained by reaction of 0.3 gram of barium chloranilate with 100 y pctr nil. of sulfate at p H 4 in 50% ethyl alcohol. The broad absorption peak observed a t 530 mp has also been reported as occurring a t 535 m p (11) and at 550 mp (23). The pII of the solution governs the absorbance of chloranilic acid solutions at a particular n a v e length; chloranilic acid is yellow, acid chloranilate ion is dark purple, and chloranilate ion is light purple (19). However, the sohibility of barium chloranilate increases with decreasing pH, and blanks are too high a t p H 2 . At p H 4 the sensitivity is adequate, adjustment of p H is simple, and blanks are small. Color stability was reached 15 minutes after the addition of 0.3 gram of barium chloranilate to 100 ml. of an aqueous
solution containing 250 y per ml. of sulfate. The absorbance increased an additional 5% in 24 hours. Cations interfere in the colorimetric method by forming insoluble chloranilates (6, 6, W ) ; anions may interfere by forming insoluble barium compounds. In the determination of 250 y per ml. of sulfate, the per cent error introduced by 250 y per ml. of six cations was : cut+ K+
92 0.8
Mg;+
J
Na 4”
0.8 0
+
Zn++
96
illuminum, calcium, ferric. and plumbous ions completely precipitated the acid-chloranilate ion. However, any interfering cations are readily removed by ion exchange (3). Anions tested for possible interference were chloride, nitrate, bicarbonate, phosphate, and oxalate; a t the 100-p.p.m. level, none reacted with a suspension of barium chloranilate in 50% ethyl alcohol a t p H 4. CONCLUSION
The analysis for sulfate can be completed in 20 minutes. The method is being adapted to the determination of sulfur in the products of lamp combustion (1). It is useful for the determination of sulfur wherever i t can be converted to sulfate. LITERATURE CITED
(1) Am. Soc. Testing Materials, “ A S . T.hl. Standards on Petroleum Products and Lubricants,” Philadelphia, Pa., 1955, Method D 1266-55T. (2) Coutinho, A. B., Almeida, 14. D., Anais. assoc. quim. Brasil, 10, 83 (1951).
(3) Fritz, J. S., Yamamura, S. S., AKAL. CHEM.27, 1461 (1955). (4) Iokhel’son. D. B.. Ilkrain. Khein. Zhur. 9,‘25 (1934). ( 5 ) Iwasaki, I., Utsumi, S., Tarutani, T., J . Chem. SOC. Japan, Pure Chem. Sect. 74, 400 (1953). Johnson, IV. C., ed., “Organic Reagents for Metals,” p. 22, Chemical Publishing Co., Kew York, 1955, Jones, A. S., Letham, D. S., Analyst 81, 15 (1956). Kahn, B. S., Leiboff, S. L., J . Biol. Chem. 80.623 (1929). Klein. B.. IND.ENG.’ C m x . ASAL. ED.’16,’536 (1944). Kolthoff, I. XI., Sandell, E. B., “Textbook of Quantitative Inorganic Anal\-sisi” 3rd ed., p. 322 f f , .\lacmillan, New York, 1952. ’ Knbo, S., Tsutsunii, C., X e p t . Food Research Znst. (Tokuo) 2 , 145 (1949). La mbert, J. L., Tasuda, S. IC, Grotheei 11, P.. ASAL. CHEM. 27,800 (i955). ’ Lang, K., Biochenz. 2. 213, 469 e,
(1929). \ - - - - ,
hlahr, C., Krauss, K., 2. anal. Chem. 128, 477 (1948). Marenzi, A. D., Banfi, R. F., Anales farm. y bioquiin. (Buenos Aiies) 8, 62 (1937). Morgulis, S., Hemphill, I f . G., Biochem. Z. 249,409 (1932). Neuman, E. T., J . Am. Chem. SOC. 5 5 , 879 (1932). Rnbia Pacheco, J. de la, Blasco Lopez-Rubio, F , Informi. p i n . anal. (Madrid) 4, 119 (1950). Schwarzenbach, G., Suter, H., Helv. Chim. Acta 24, 617 (1941). Seifter, S., Novis, B., A N A L . CHIXI. 23,188 (1951). Snell, F. D., Snell, C. T., “Colorimetric hlethods of Analysis,” vol. 11, 3rd ed., pp. 767-8, Van h-ostrand, New York, 1949. Suito, E., Takiyama, K., Bull. Chem. SOC.Japan 28, 305 (1955). Tyner, E. H., . ~ N A L . CHEX. 20, 76 (1948). RECEIVEDfor review .iugust 3, 1956. Accepted Kovember 9, 1956.
Rapid Method for Determination of Malic Acid ALAN E. GOODBAN and J. BENJAMIN STARK Western Utilization Research Branch, United States Department of Agriculture, Albany 7 0, Calif.
,Because malic acid is one of the acids involved in the citric acid metabolic cycle, its determination in plant materials is often important. A method is presented which requires about 2 hours for the analysis of several samples, is specific for malic acid within the limits tested, and can be applied to an aqueous extract of plant materials. Recovery from standard malic acid solution was 100.3%, and standard deviation was 2.2%.
A crude separation of malic acid from other materials is effected by ion exchange resins, and malic acid is determined colorimetrically in an eluate after reaction with sulfuric acid and 2,7-naphthalenediol.
M
as well as other acids, can be determined b y fractionation of ion exchange resins in the acid form (5, 15, 20) or by partition ALIC ACID,
.
chromatography on silica gel columns (4, 8, I S , 14) and subsequent analysis of the fractions. These methods have the advantage of simultaneous analyses for a number of acids, but some require considerable time for each determination, and others lack the desired accuracy and precision. Chemical methods which have been used are time-consuming or low in specificity (1, 7, 11, 19, 18). Methods utilizing bacterial oxidation (9, 19) and VOL. 29,
NO. 2 , FEBRUARY 1957
283
paper chromatography (2) have also been proposed. The separation of succinic and malic acids from citric acid by the use of strongly basic anion exchange resin in the carbonate form has been noted ( 3 ) and the extension of this system to the analysis of lactic acid has been reported (21). [Strongly basic anion exchange resins in the hydroxyl form adsorb reducing sugars with concomitant production of hydroxy acids, which would interfere in the determination of lactic or malic acid (10, 1 7 ) . This difficulty is overcome b y the use of the carbonate foim.] Investigation showed that the method used for lactic acid is equally suited for malic acid by further elution of the column and use of 2.7-naphthalenediol, which has been used as a color reagent in analysis of glycolic and oxalic acids (6, 16). The method depends upon removal of cationic materials on a short column of cation exchange resin, then adsorption of acids, including malic, on a column of anion exchanger while neutral materials are allowed t o pass through. An initial elution of the anion column removes weakly held acids including lactic, glycolic, and glyceric, which r o u l d also develop colored compounds with the analytical reagent. Elution of malic acid from the column is speeded by increasing the strength of the eluting solution. An aliquot of the sample containing malic acid is heated with 2,i-naphthalenediol and sulfuric acid. The first reaction is the production of nialonaldehydic acid; the latter decomposes to carbon dioxide and water. The malonaldehydic acid probably condenses with the reagent to form hydrovynaphthalenea-pyrone according to the Pechmann reaction. Most other acids which might be expected to interfere in the color development are either removed during the resin step or produce colored products which do not have an appreciable absorbance a t 390 mp. MATERIALS A N D REAGENTS
Amberlite I R A 400, carbonate form, 60 t o 80 mesh (Rohm 8: Haas Co.). The resin is ground and sieved wet in the chloride form, then regenerated with 5% sodium hydroxide and washed with 1N sodium carbonate followed b y distilled water. The resin can be stored for considerable periods in a closed jar under water. Dowex 50, hydrogen form, 60 to 100 mesh, cross linkage 12% (Dow Chemical Co.). The resin should be stored in airdried condition. Ammonium carbonate, 0.255. Contains 14.25 grams of lump ammonium carbonate per liter. Ammonium carbonate, 1.OS. Contains 57.0 grams of lump ammonium carbonate per liter. Sulfuric acid, 96%, analytical grade, nitrate free. 284
ANALYTICAL CHEMISTRY
0.5,
,
,
I
,
I
I
I
W A V E LENBTH, MILLIMICRONS
Figure 1. Absorption curves produced with 2,7-naphthalenediol and carboxylic acids 0.50 mg. tartaric 0.50 mg. a-ketoglutaric 0.05 mg. malic
2,7-Saphthalenediol. 1 grain per 100 ml. in 96% sulfuric acid. Glass tubing, approximately 14 mm. in outside diameter. Sylon bolting cloth. PROCEDURE
The procedure in the ion exchange steps is similar t o that used in the determination of lactic acid (21). with the evception of further elution after removal of the lactic acid fraction. The columns are borosilicate glass, 14 mm. in outside diameter, stoppered at one end with a one-hole size 00 rubber stopper containing a short piece of glass tubing, A small circle of nylon bolting cloth retains the resin in the column. Care must be taken that no threads extend past the bottom of the column. One column, 20 em. long, is loaded with a mater slurry of IRA-400 (carbonate form) so that the volume after settling is 10 ml. A second column, 20 em. long. is loaded in the same way with 10 ml. of D o w x 50, hydrogen form, and supported above the first column so that the effluent drips directly into the anion column. The columns are loaded with a water slurry of the resins. No stopcocks or pinch clamps are necessary, as the columns will not drain dry if the resin is ground to 60 to 80 mesh. An aliquot of the sample to be analyzed, containing not more than 3 meq. of total acid nor less than 0.008 meq. of malic acid, is carefully added to the upper column and allowed to run freely through both columns. The upper column is washed with three 10-ml. portions of water, and each portion is allorr-ed to drain down to the top of the resin bed. The upper column is then removed and the lower column is washed with three 10-ml. portions of vater. This second washing may be omitted if no determination of lactic or glycolic acids is to be made.
The lower column is noJv eluted with 50 ml. of 0.25N ammonium carbonate in 10-ml. portions. This eluate contains all of the lactic, glycolic, and glyceric acids which would otherwise interfere in the color step. A 50-ml. volumetric flask is no\y placed under the column, which is washed nith 49 ml. of 1N ammonium carbonate in 10ml. portions. The solution is made to volume with water. This second eluate contains the malic acid. The color development is carried out b y placing a 1-ml. aliquot of the second eluate, containing 5 to 80 y of malic acid, in a culture tube (25 X 200 mm.) and very carefully adding 6.0 ml. of sulfuric acid by allowing it to run down the sides of the tube. If the first drop of the acid is allowed to run slo~vlydown the side, the evolution of carbon dioxide is not rapid enough to cause spattering of the sample on the upper portions of the tube. It is generally more convenient to choose the sample size to allow a dilution of four-fold or greater prior to this step to reduce the concentration of carbonate. The remainder of the acid mag be added more rapidly with gentle swirling of the tube. The concentrated sulfuric acid is conveniently added from a 50-ml. buret attached to a 2-liter resemoir in an allglass system. Xon- 0.1 ml. of 2 , i naphthalenediol solution is added to the tube and the tube is heated in a boiling water bath for 20 minutes. After cooling, the contents of each tube are transferred to colorimeter tubes and read in a spectrophotometer at 390 mp. The curve of concentration os. absorbance is a straight line from 5 to 80 y of malic acid, using a Beckman Xodel B spectrophotometer. EXPERIMENTAL
Factors Affecting Color Development. Malic acid heated with sul-
IO YEATING
20
30
IO HEATING
T I M E , MIN
Figure 2. Malic acid, 407; water, 0.5 ml.; sulfuric acid, 6.35 ml.
Figure 3.
1 .O ml.;
TIME,
20 YIN
10 HEATING TIME,
30
Malic acid, 407; water, sulfuric acid, 6.0 ml.
20
30
YIN
Figure 4. Malic acid, 407; water, 1.5 ml.; sulfuric acid, 5.6 ml.
Effect of heating time and reagent concentration on color produced
fiiric acitl in tlic presence of 2 , i naphthalenediol pioduces a pale yelloir color which has a strong absorption peak a t 385 nip and practically no absoihance above 470 mp (Figure 1). The effect of reagent concentration. sulfuric acid concentration, and time of heating on the absorbance pioduced hy malic acid a t 390 mp were investigated. The thrce variables were changed independently a t ieagent levels of 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 mg.; heating periods of 5> 15, 30, and 60 minutes at 100" C.; and water (sample) volume of 0.5, 1.0, 1.5, and 3.0 ml. n i t h enough sulfuric acid to make a final volume of 6.717 ml. Some of the results are slioivn in Figures 2 to 4 for a malic acid concentration of 0.04 mg. per tube. The optimum water concentration is close to a ratio of 1 ml. of water to 6 nil. of 96% sulfuric acid. With water volumes between 0.1 and 1.0 ml.. color production increases with increasing water concentration. Color production also increases with time of heating and n i t h increasing ieagent concentration, but the maximum color is produced in 15 minutes with 1 nig. of reagent or in 30 minutes with 0.5 nig. of reagent. Increasing reagent concentration to 5 mg. or the heating period t o 1 hour did not result in further increase in color. Figure 5 shows the isochromes for varying reagent concentrations and heating periods with 1 nil. of water, 6 nil. of sulfuric acid, and 0.04 mg. of malic acid. Essentially the same iesults were obtained for 0.02 and 0.08 mg. of malic. The reagent blank increases with increasing concentration and n i t h heating time. The conditions chosen were 20 minutes' heating time with 1 nig. of reagent, using a sample volume of 1 nil. and 6.0 nil. of snlfuiic acid. One
lot of analytical grade sulfuric acid gave a visible color when heated with 2.7-naphthalenediol. with consequent interference in the determination of malic acid. The blank should be colorless after heating. T o test stability of color, malic acid a t thiee concentrations was made to react 11ith 2.7-naphthalenediol and the absorbance was determined. The tubes were then covered with Parafilni and allowed to stand. The absorbance was determined at intervals u p to 90 hours. Results are shown in Table I. The color is stable for several days if the tubes are covered to prevent absorption of water. The reagent solution is stable for several months if stored in a refrigerator. If only a limited number of analyses are to be made. the reagent may be incorporated in the sulfuric acid (170 mg. per liter) and 6.0 ml. added to 1 ml. of the malic acid sample. Although the concentrated reagent stored in a refrigerator discolors after a few days, this has no effect on the determinations, because the color disappears after heating. Elution of Malic Acid from Anion Resin. T h e elution positions of glycolic and malic acids were determined b y loading a 10-ml. column of I R 4 400 (carbonate form) with 5 mg. of each acid and eluting with six 10-ml. portions of 0 . 2 5 s a n d five 10-nil. portions of 1.V ammonium carbonate. Fractions of 10 ml. were collected and 1-ml. aliquots taken for color development with 2,i-naphthalenediol. Glycolic acid v a s estimated from the absorbance at 545 nip and malic acid from the absorbance a t 390 mp. The results (Table 11) sho~vthat glycolic acid is removed by 50 ml. of 0.25N ammonium carbonate, and that malic acid begins to bieak through with the
next 10 ml. of 0.25h7 eluant. Lactic acid comes off slightly faster than glycolic acid, and the conditions of elution were chosen to remove lactic and glycolic acids without appreciable loss of malic acid. All of the work in this laboratory has been done with a single lot of IRA 400; if the capacity of other lots varies, it would be necessary to determine the elution position of malic acid and to adjust the resin volume accordingly. There is a slow production of material from the resin. presumably formaldehyde, which reacts with 2 , i naphthalenediol and sulfuric acid to produce a red color. For this reason the operation of the column should not be interrupted during the time the malic acid fraction is being collected. I n 1 hour formaldehyde equivalent to 0.05 mg. of malic acid will be produced. Interference of Other Acids. Because cationic materials are removed on t h e cation exchange resin and neutral materials are presumably washed on through. t h e only interfering substances n-ill be those adsorbed on t h e anion evchanger and eluted n i t h t h e malic acid fraction. T h e
Table I. Stability of Color Produced from Malic Acid and 2,7-Naphthalenediol
Hours 26 90 Absorbance, 300 nip-
Malic acid,
0
20 20 40 40 80 80
0 348 0 338 0 640 0 656 1 230 1 220
,
0 348 0 328 0 644 0 656 1 250 1 228
VOL. 29, NO. 2, FEBRUARY 1957
0 0 0 0 1 1
338 325 630 6.50 238 228
285
Table II. Elution of Acids from IRA 400 with Ammonium Carbonate
(5 mg. each of glycolic and malic acids,
10-ml. fractions)
Fraction 1 2
3 4 5 6 7 8 9 10 11
Eluent Concn., N 0.25 0.25 0.25 0.25 0.25 0.25 1 1 1 1 1
Acid, Mg. Glycolic Malic 0 1.1
2.6(about) 1.4 0.1 0 0 0 0 0 0
0 0 0 0
0.03 0.16 1.4
>2 0.53 0.04 0
absorbance of malic acid and 2,7naphthalenediol in the presence of a number of acids which might be expected to interfere is shown in Table 111. Those acids which interfered to the extent of more than 6% were then carried through the column separation in the presence of malic acid. The results are shown in Table IV. The only acids showing significant interference a t this level are tartaric, a-ketoglutaric. and the sugar phosphates. I n each
Table 111. Reaction of 2,7-Naphthalenediol with Various Acids in Presence of Malic Acid
(40 y malic acid, 250 y other ac,id)
Other Acid None Tricarballylic Aconitic Citraconic Citric Mesaconic Succinic Pyrrolidone carboxylic Quinic Fumaric Isocitric Oxalacetic Pyruvic Dih droxytartaric HyJoxymalonic Tartaric a-Ketoglutaric Glucose-6-phosphate Glucose-l-phosphate Fructose-6-phosphate Glyceric Fructose-I ,6-diphosphate Lactic Glycolic
Interference by AbsorbSixfold ance, excess, 390 mfi % 0.592 .. 0.575 -3 0.581 -2 0.588 -1 0.591 0 0.596 1 0,598 1 0.608 0.622 0.623 0.630 0.630 0.648 0.675 0.685 0.761 0,815
are shown in Table V. As might be expected from the absorption curves, the use of this factor does not completely eliminate the interference due t o a-ketoglutaric acid, but the recovery of malic acid in the presence of excess tartaric and other acids is satisfactory. The apparent recovery of malic acid from solutions containing only the interfering acids shows that the suggested correction factor does not compensate completely for the presence of these acids, and sets a lower limit on the concentration of malic acid which may be determined in such a mixture.
case the interference is less than expected from the results in Table 111, showing that some separation of these materials from malic acid is achieved on the resin column. The ratio of phosphate esters to malic acid in plant extracts is ordinarily so low that significant interference will not be encountered] as the color produced even by: fructose diphosphate is only about 15% that of malic acid. The presence of the phosphate esters of fructose can be detected by the immediate appearance of a golden yellow color upon addition of sulfuric acid and 2,7-naphthalenediol. The two glucose esters do not give an immediate color, b u t turn red-brown after heating. Because the absorption due to malic acid is low at 490 mp (Figure 1) while tartaric acid shows a maximum at this wave length, it would seem that a correction could easily be made for the presence of this acid. I n view of the fact that tartaric acid is much more likely to be present in plant extracts in excess of malic acid than a-ketoglutaric acid, a factor was chosen which would correct for a11 of the interference of tartaric and approximately one fourth of the interference due to a-ketoglutaric. assuming that the absorbance due to interfering acids a t 390 nip is one half the total nbsorhance nt 490 inp
Table IV. Recovery of Malic Acid in Presence of Interfering Acid after Fractionation on IRA 400
(2.01 mg. malic acid, 10 mg. other acid)
Other Acid Added Kone None None Glyceric Glycolic Hydroxymalonic Lactic Pyruvic Dihydroxytartaric a-Ketodutaric Tartarii Glucose-I-phosphate Glucose-6-phosphate Fructose-6-phosphate Fructose-1.6-diohosphate ’ For first 9 columns Av. Std. dev.
hlistures containing the eight carbosylic acids listed in Table IV plus malic acid were made containing 2 or 4 mg. of each of the other acids per milligram of malic acid. 1 M i c acid was estimated after fractionation on the IRA 400 column by use of Equation 1. The results
A
%
1.96 2.01 2.05 1.97 1.96 1.94 2.02 1.96 1.92 2.23 2.26 2.30 2 37 2.56
97.5 100 102 98 97.5 96.5 100.5 97.5 95.5
127
3.29
163.5
1.98 0 042
98.3 2 1
111 ___
112.5 114.5 118
14
16 28 38
1.017
72
1.130
91
1.225 1.29
118
1.333 1.70 3.0
125 190 500
107
I
I
I
5
I
I
I
Figure 5.
I
I
I
15
10 HEATING
ANALYTICAL CHEMISTRY
hlg.
3 5 5 6 6 9
I
20 TIME, MIN.
Absorptivity isochromes
Data taken from Figure 3
286
Found as Malic Acid, Recovery,
I
25
I
I
30
Table V.
Estimation of Malic Acid in Presence of Several Interfering Acids by Use of Correction Factor Io
a
32 64
..
..
2.00
..
28 56
..
2.00
..
0
32 .. ..
Recovery of Malic Acid
Recovered
i Ar.
Std. dev.
.
Recovered as AIalic Bcid Mg. % 2.06 2.08 2.10 2.24 1.98 1.93 0.17 0.18 0.08
.. 28 56
1
..
..
28
..
..
28
103 104 105 112 99 96.5
Equal parts of glycolic, glyceric, citric, pyruvic, hydroxymalonic, lactic, tartaric, and e-ketoglutaric acids. Same as I without tartaric acid. Same as I without a-ketoglutaric acid.
(15.18 mg. added to each column)
Column
IIIC
2.00
Recovery of Malic Acid. Eight columns of I R A 400 (carbonate form) were prepared. E a c h was loaded with 13.18 mg. of malic acid. T h e columns were eluted and t h e malic acid was determined as described above. Average recovery was 15.24 mg., standard deviation was 0.33 mg. and t h e range was kO.5 mg. (Table VI). The recovery of malic acid added to a n aqueous extract of sugar beet root is shown in Table VII. No special preparation of the sample was made before addition to the resin columns. This extract contained about 12% sucrose in addition to amino acids, proteins, and organic acids. The results in Table 1-11 n-ere obtained b y use of the correction factor and duplicate columns. Under the described conditions, the leakage of formaldehyde from the resin does not interfere n-ith determination of malic acid.
Table VI.
Acid Added, Mg. II*
AIg.
15.5 14.7 l5,24 0.33
%
101.9 96.6 100 3 2 2
columns, undue disturbance of t h e resin surface during addition of liquid t o t h e column should be avoided and resin particles which have been left on t h e walls of t h e column should be washed down with succeeding additions of wash water or ammonium carbonate. This has been found to be simple enough so that no pad or glass float over the resin is necessary. T o ensure efficient and reproducible washing and elution of the column, the liquid level should be allowed to drain down to the resin surface before addition of the next portion. During addition of ammonium carbonate to the anion exchanger there is a shrinkage of about 10% in resin volume, so that addition of 49 ml. of 1N ammonium carbonate results in almost 50 ml. of eluate.
LITERATURE CITED
(1) Barr, C. G., Plant Physiol. 23, 443
11948).
B&nt,'F., Overell, B. T., Biochim. et Biophys. Acta 10,471 (1953). Bryant, F., Overell, B. T., Nature 167, 361 (1951).
Bulen W.A , , Varner, J. E., Burrell, R. d., ANAL.CHEW24,187 (1952). Busch, H., Hurlbert, R. B., Potter, V. R., J . Biol. Chem. 196, 717 (1952).
Calkins, T'. P., ANAL. CHEW 15, 762 (1943).
Ferris, L. W., J . Assoc. Ofic.Agr. Chemists 36, 266 (1953).
Frohman, C. E., Orten, J. M,, J . Biol. Chem. 205,717 (1953).
Gachot, H., Journe'e vinicole-ezport 22, KO.6313, l(1948).
Hulme, A. C., Nature 171,610 (1953). Hummel. J. P.. J . Biol. Chem. 180. 1225 (1949). '
latz, S.,
Table VII. Recovery of Malic Acid Added to 5 MI. of Sugar Beet Diffusion Juice
Added, Mg.
Found, Mg.
0 0.47 0.94 2.35
1.46 1.94 2.34 3.91
CHEW
Recovered Mg. %
...
0.48 0.88 2.45
102' 93.5 104
Lactic acid produces about 30% as much color as malic acid with 2,7naphthalenediol; therefore, a small plug of cotton should be placed in the top of the pipets to prevent contamination a i t h lactic acid from saliva. ACKNOWLEDGMENT
Technique. Aside from t h e usual attention t o analytical techniques, certain precautions should be noted. I n t h e operation of t h e ion exchange
for some of the analyses, and t o Rosie Jang for the samples of sugar phosphates.
The authors are indebted to the late
H. S. Owens for his encouragement and advice during the course of this work, to Taysir Jaouni and Harold Barrett
PuchLr, G'. W.; Wakeman, A. J., 1-ickery, H. B., ISD.ENG.CHEM., h A L . ED. 13,244 (1941). Rentechler, H., Mitt. Gebiete Lebensm. u. Hyg. 39,30 (1948).
Schenker. H. H.. Rieman. W.. ANAL.CHEM.25.'1637 (1953). ' Stark, J. B., Goodban, A.'E., Owens, H. S., J . Agr. Food Chem. 1 , 564 (1953).
RECEIVEDfor reviea- May 31, 1955. Accepted November 13, 1956. Division of Analytical Chemistry, 128th meeting, ACS, Minneapolis, Minn., September 1955. Mention of specific products does not constitute endorsement by the Department of Agriculture over others of a similar nature not mentioned. VOL. 29, N O . 2, FEBRUARY 1957
287
