Fluorometric Determination of Malic Acid and 2-Naphthol ELMER LEININGER AND SIDNEY KATZ', Michigan S t a t e College, East Lansing, Mich. Q u a n t i t a t i v e fluorometric m e t h o d s f o r the d e t e r m i n a t i o n of m a l i c acid a n d 2naphthol are based u p o n t h e reaction of m a l i c acid a n d 2 - n a p h t h o l in s u l f u r i c acid. T h e effect of variations i n the o p e r a t i n g conditions for e a c h m e t h o d is discussed. The m e t h o d f o r malic acid is applicable to d e t e r m i n a t i o n s of 1 to 30 m i c r o g r a m s of m a l i c acid. Citric a n d succinic acids in large excess d o n o t interfere w i t h the m a l i c acid d e t e r m i n a t i o n , whereas tartaric acid in the ratio of 1 to 1 w i t h m a l i c acid causes a 3 to 5 % error. The d e t e r m i n a t i o n of malic acid i n a p p l e j u i c e is described. T h e m e t h o d for 2 - n a p h t h o l is applicable to determ i n a t i o n s of 1 to 14 m i c r o g r a m s of 2-naphthol. The presence of 1 - n a p h t h o l i n a 1 to 1 r a t i o with 2 - n a p h t h o l causes a n error of approximately 570.
T
HE method of the Association of Official Agricultural Chemists ( I ) for the volumetric determination of total malic
acid is long and tedious, and the results require an empirical correction. The fluorometric method of Barr (a), based upon the reaction of malic acid with resorcinol in concentrated sulfuric acid, cannot be used in the presence of citric acid. A fluorometric method which is relatively free of interferences, rapid, and simple in technique is described here. I t is based upon the qualitative test for malic acid employed by Eegrime ( 4 ) in which malic acid and 2-naphthol, when heated in concentrated sulfuric acid, produce a blue fluorescence. There is some evidence (3, 5 ) that 5,&benzocoumarin is the fluorescing product which is formed by condensation of the intermediate, formyl acetic wid. with 2-naphthol
One milliliter of reagent consisting of 12 mg. of 2-naphthol per 100 ml. of 92y0 sulfuric acid is added. The flask is tipped slightly to ensure complete wetting of the bottom surface and is then heated in an electric oven a t 90" to 95' C. for 30 minutes. The flask is removed and cooled, after which the solution is transferred with water to a 100-ml. volumetric flask and brought to volume. The temperature of the solution is adjusted to 25' =t 1.5"C. and the fluorescence intensity reading is taken. A straight-line calibration curve is prepared from instrument readings obtained from well-distributed points by carrying amounts of malic acid through the procedure described. The fluorescence meter is adjusted to give a reading of zero with a blank solution carried through the complete analytical procedure and a reading of 100 with the standard solution of sodium salicylate (2.000 grams per liter).
T a h l e I. APPARATUS
The fluorescence intensities are measured with a Lumetron fluorescence meter Model 402 EF with 25-ml. cells. The primary filter permits maximum transmittance in the spectral region of 365 mp. The secondary filters consist of a combination of a yellow filter, furnished by the Photovolt Corporation for use in vitamin B1 determinations, which does not transmit emission below 400 ms, and a Corning lantern blue filter S o . 5543. This combination of secondary filters permits maximum transmittance corresponding to the region of greatest fluorescence intensity of the final solution. REAGENTS
Malic Acid. Technical grade 2-malic acid was recrystallized from ethyl ether. 2-Naphthol. Technical grade 2-naphthol was purified by distillation. Sulfuric Acid, 91.5 to 92.5w0. One hundred milliliters of concentrated sulfuric acid are added to 7 ml. of water. The specific mavitv (20"/4" C.) of the solution must be within the range of 7.822 and 1.826. Lead Acetate Solution. Seventy-five grams of normal lead acetate are dissolved in water and 0.5 ml. of glacial acetic acid iQ added. The solution is diluted to 250 ml. Sodium Salicylate Solution. Exactly 2.000 grams of reagent grade sodium salicylate are dissolved in water and diluted to 1000 ml. It may be preserved by the addition of several drop? of toluene. Y
DETERMINATION OF MALIC ACID
Analytical Procedure for Solutions Free from .1 sample solution containing 1 to 30 micrograms preferably 1 ml. or less, is accurately measured Erlenmeyer flask and evaporated to dryness in a
Interferences. of malic acid. into a 10-nil 105" C. oven
1 Present address, Carbide & Carhon Chemicals Corporation, Oak Ridge Tenn.
Reproducibility of F l u o r o m e t r i c Jfetborl fnr D e t e r m i n a t i o n o f Tlalic Arid
Xumber of samples, AT Malic acid present in each sample Average fluorometric reading, I Average deviation of fluorometric readings Standard deviation of fluorometric readings Standard deviation = [5-]"' v - 1
31 30.08 micrograrna 96.3 units I . 12 unite I , 28 unit,$
Discussion. The maximum fluorescence intensity is obtained the sulfuric acid solution of 2-naphthol is prepared from 92y0 sulfuric acid. With 90 and 95% sulfuric acid the apparent fluorescence intensities are approximately two thirds of those obtained with the 92% acid. The concentration of 2-naphthol is limited to 120 micrograms per sample because larger concentrations produce excessive fluorescence in the blank. The solution of 2-naphthol in 92% sulfuric acid is stable for a t least 2 weeks if stored in an amber bottle in a refrigerator. 4 calibration curve qhould be prepared for each batch of reagent. The maximum fluorescence intensity is reached in less than 30 minutes a t 90" C. and is not diminished by further heating. At 80' C. the maximum is reached in 28 minutes, whereas a t 100' the mavimum fluorescence is reached in 16 minutes. The maximum fluorescence readings obtained a t 80" C. are comparable to those obtained a t 90" C., whereas those obtained a t 100" C. are 2 to 4% lower. In the pH range of 0 to 5 and in any 3" temperature range betveen 20" and 30" C. the variation of fluorescence intensity of the final solution is qithin the experimental error. The fluorescence intensity is constant over long periods of standing. Table I demonstrates the reproducibility of the fluorescence intensity readings for 31 samples, each of which consisted of 30.08 micrograms of malic acid. if
1375
ANALYTICAL CHEMISTRY
1376 Table 11.
Separation of Pure Malic Acid
Malic Acid Present, M g . 50.0
40.0
Malic Acid Found, N g . 49.9 49.9 49.2 49.9 Av. 4 9 . 7 41,l 41.2 41.6 41.3 Av. 4 1 . 3
Error,
flo
0 6
3.3
Table 111. Separation and Determination of Malic .4cid in a Synthetic Mixture Containing Sucrose, Glucose, and Fructose Malic Acid Present, M g . 40 0
40.0
1Ialic Acid Found, Mg 38 9 38 9 39 7 38 1 I\ 38 9 39.5 38.9 41.2 39.7 AV. 3 9 . 8
aliquots were analvzed. These errors are mithin the same range as those found in the determination alone. Malic acid can be separated from sugars normally found in apple juice with the same accuracy as from solutions free of these sugars. Thus in Table I11 the error for separations from synthetic solutions of 40 mg. of malic acid containing 100 mg. of sucrose, 150 mg. of d-glucose, and 250 mg. of d-fructose is illustrated. In each separation four aliquots were determined fluorometrically. Results obtained by determining the amount of malic acid present in commercial apple juices before and after the addition of known amounts of malic acid are recorded in Table IV. Results are averages of four aliquots determined for each separation.
Error, 70
2 8
0 5
The total malic acid is determined by this method, inasmuch
as it was found that I-malic or dl-malic acid produces identical fluorescence intensities in this reaction. As much as 0.5 mg. of either citric or succinic acids has no influence on the fluorescence readings. Tartaric acid if present in a ratio to malic acid of 1 to 1 will produce an error of 3 to 5 % ; for a ratio of 3 to 1 the error produced is 20 t o 25%. The procedure requires 1 hour, during which time a large number of samples may be run simultaneously. Determination of Malic Acid in Apple Juice. The method of the ilssociation of Official Agricultural Chemists ( 1 ) for the separation of malic acid from fruit juices has been modified to use smaller volumes. In general, the protein matter is coagulated and removed from the juices by filtration along with other insoluble material. The malic acid is precipitated as the lead salt, washed, and put back into solution as malic acid by precipitating the lead as its sulfide. The elapsed time required for a complete determinat,ion by the fluorometric method is 2.5 hours compared to nearly 24 hours for the standard A.O.A.C. method. PROCEDURE FOR SEPARATIOli O F ;\l.%LIC -4CID FROM -4PPLE
A sample containing 20 to 75 mg. of malic acid is selected and the volume is brought to 15 ml. with water or by evaporation. The solution is transferred quantitatively to a 100-ml. volumetric flask and brought to volume with 95% ethyl alcohol. The solution is mixed, again brought to volume, and filtered through a folded filter paper covered with a watch glass. Seventy-five milliliters of the filtrate are pipeted into a 100-ml. centrifuge tube, and 10 mg. of citric acid and 1 ml. of lead acetate solution are added. The solution is thoroughly mixed, and centrifuged a t 1000 r.p.m. for 15 minutes. The supernatant liquid is tested with lead acetate solution for complete precipitation. If additional precipitate appears, more lead acetate solution is added and the centrifuging is repeated. The liquid is carefully decanted and discarded, leaving the precipitate in the centrifuge tube. The precipitate is washed with 75 ml. of 80% ethyl alcohol added in small portions. d stirring rod is used to ensure a homogeneous mixture and is rinsed with the last portion of 80y0 alcohol. The solution is again centrifuged and decanted as before. The precipitate is suspended by adding 50 nil. of water in small portions while using the stirring rod for thorough mixing. The solution is saturated with hydrogen sulfide by a rapid stream of the gas. The mixture is transferred quantitatively to a 100ml. volumetric flask and brought to volume with water. The contents are well mixed and filtered. Aliquot portions of the filtrate are taken for the determination of malic acid by the fluorometric method described. JUICE.
DISCUSSION. The error incurred by passing known amounts of malic acid through the above separation and the fluorometric determination is illustrated in Table 11. I n each separation four
DETERMIW4TIOW OF 2-NAPHTHOL
Analytical Procedure. A solution containing 1 to 15 micrograms of 2-naphthol dissolved in benzene is measured volunietricnlly into a 10-nil. Erlenmeyer flask and evacuated to drliiess by means of a vacuum desiccator and a mater pump. One milliliter of reagent, consisting of 1 gram of malic acid dissolved in 100 ml. of 92% sulfuric acid and allowed to age at room temperature for 24 hours, is added. The flask is tipped slightly to ensure complete wetting of the sample, and is then placed in a 35’ to 40” C. oven for 10 minutes. The contents are transferred quantitatively to a 100-ml. volumetric flask with water and brought to volume. The temperature should be 25 ’ * 1.5’ C. when the fluorescence reading is taken. A linear calibration curve is prepared from 2-naphthol samples which are tieated as in the standardized procedure. Zero on the fluorescence meter may be adjusted with distilled Tvater and the 100 setting obtained with the sodium salicylate solution (2.000 grams per liter).
DISCU~SIOS. Benzene was used as the solvent for the 2naphthol in this investigation. The “aging” of the malic acid in the 92% sulfuric acid solution increases the fluorescence intensity approximately sixfold ThiP reagent is stable for 10 days if refrigerated. The same calibration curve may be used for successive batches of the solution of malic acid in 92% sulfuric acid if the 92y0 sulfuric acid is from the same source. The concentration of the malic acid is not critical. The reaction goes to completion rapidly and after 3 or 4 minutes no change in the fluorescence intensity is noted. Above 25’ C. the fluorescence intensities produced from the reaction are a t a maximum. The fluorescence intensity is not increased a t reaction temperatures above 40” C. but the possibility of interference due to decomposition of otherwise noninterfering substances is increased. The fluorescence intensity of the blank is comparable to that
Table I\’.
Determination of Malic :icid in Commercial -4pple Juice
Sample Taken
Malic .4cid ddded
MZ.
MQ.
10
Kone
5
34.7
5
57.8
10
None
6
10.0
6
20.0
Malic Malic Acid Acid Found per Found 5 111. Juice Mg. MQ. .4pple Juice A 34.2 17.1 34.4 17.2 36.4 18.2 54.8 54.8 51.7 51.7 73.6 73.6 72.4 72.4
Malic Acid Found Less Malic Acid Added per 5 M1. Juice
.Wg. 17.1 17.2 18.2 20.1 17.0 15.8 14.6
Apple Juice B 57.6 57.8 57.4 38.5 39.2 39.7 40.8
28.8 28.9 28.7 38.5 39.2 39.7 40.8
47.4 48.6 49.2 48.3
47.4 48.6 49.2 48.3
28.8 28.9 28.7 28.5 29.2 29.7 30.8 27.4 28.6 29.2 28.3
V O L U M E 21, N O . 11, N O V E M B E R 1 9 4 9 Table
V. Reproducibility of Fluorometric Method for Determination of 2-Naphthol
Number of samples, N 2-Naphthol present in each sample Average fluorometric reading P Average deviation of tluorom’etric readings Standard deviation of fluorometric readings
48
14,OO micrograms 8 7 . 1 units 1 . 2 0 units 1 . 3 5 units
1377 1-naphthol is not greater than that of %naphthol. If the amount of the 1-naphthol is equal to that of %naphthol, the error is approximately 5% positive. If the ratio of 1-naphthol to 2naphthol is 5 t o 1, the error is approximately 15% positive. LITERATURE CITED
Assoc. Offic.d g i . Chemists, “Official and Tentarlve Methods of
produced by distilled water and therefore the latter may conveniently be substituted for the zero adjustment of the fluorescence meter. Table IT illustrates the reproducibility of the fluorescence intensity readings for 48 samples. Each sample consisted of 14.00 micrograms of 2-naphthol. The fluorometric method may be used for the determination of 2-naphthol in the presence of I-naphthol, if the concentration of
hnalysis,” 6th ed., pp. 395-6, 1946. Barr, C. G., Plant Phvsiol., 23, 443-54 ( 1 9 4 8 , . Dey, B.. Rao, R., and Ssnkaranarayan, Y., J . Indian Chem. SOC., 9,7177 (1932). (4) Eegriwe, E., Z . anal. Chem., 89, 121 (1932). (5) Peckman, H., and Welsh, W., Ber., 17, 1651 (18541. RECEIVEDJanuary 19, 1949. Presented before the southeastern Regional SOCIETY, Oak Ridge, Tenn., J u n e 10, Meeting of the . 4 M E R I C A N CHEMICAL 1949. -4bstracted from a portion of a thesis submitted by Sidney Katz in partial fulfillment of the requirements for t h e P h . D . degree.
HYDROGARBON TYPE ANALYSIS Estimation of Six-Membered Ring Naphthenes H. C. RAMPTON, Anglo-Zranian Oil Co., L t d . , Sunbury-on-Thumes, England A method of hydrocarbon type analysis of petroleum naphthas is described, based on examination of cuts of specified boiling ranges, prepared by fractionation of original and dearomatized samples. The cut points selected ensure the segregation of particular groups of hydrocarbons and contents of aromatics, paraffins, and total naphthenes are obtained in detail throughout the boiling range initial boiling point to 225” C. The “reactable” six-membered ring naphthene contents are determined by a dehydrogenation procedure and do not embrace gemin i l cyclohexane derivatives. The latter are included in the “unreactable” naphthene content, together with the cyclopentane derivatives.
F
R O N the commencement of the petroleum industry many
investigations have been carried out with the object of correlating the physical properties and chemical constitution of naturally occurring hydrocarbon mixtures, but, until recent years, little progress had’ been made in the development of reliable analytical methods for the accurate assessment of hydrocarbon type composition. The analytical difficulties encountered in such work are extremely great and are due to the involved and complex chemical nature of the raw materials. Nevertheless, considerable effort has been directed toward the ultimate goal of analytical petroleum chemistry-i.e., the resolution of petroleum into individual hydrocarbons and other constituents-and much progress has been accomplished. The hydrocarbons present in petroleum may be conventionally and broadly classified as paraffins, naphthenes, and aromatics, a classification that is sharp for the gasoline boiling range but becomes ambiguous for compounds of high molecular weight, which may contain the characteristic structure of all three type. combined in the one molecule. For the purposes of the present paper, the simple classification is tenable, since the investigation has been restricted to materials boiling within the gasoline 1 ange. The paraffins represent the well-defined saturated open-chain structure hydrocarbons. The aromatics may be specified as cyclic hydrocarbons, as a predominant feature of their structure is the benzene ring with its characteristic resonating double bond unsaturation. The naphthenes have fully saturated carbon ring structures. In the absence of evidence t o the contrary, straightrun petroleum fractions are considered to contain five- and sixmembered ring naphthenes only and it is with the estimation of
these two classes of compound that this investigation is concerned. ESTI&IATION OF HYDROCARBON TYPES
Fractional Distillation. The primary step in the estimation of hydrocarbon type is that of fractional distillation, and progress in recent years in this connection has been stupendous. Various forms of ultraefficient column packing have been designed and used with great success in the analytical laboratory, notably the glass and stainless steel helices developed by Fenske et al. (3,4,10, 11), the spiral packings due to Podbielniak (9).and the spiral screen packing described by Lecky and Ewe11 (1I . Using columns packed with such materials it has become normal practice to fractionate a gasoline sample into close boiling cuts, with subsequent analysis of these for aromatic, naphthene, and paraffin contents (6, 8). By these means a hydrocarbon type analysis throughout the hoiling range can be obtained. I t has been realized that the removal of aromatic hydrocarbons, prior to the fractionation, facilitated estimation of naphthene and paraffin hydrocarbons. This fact is due partly to abnormal vapor pressure relationshipi existing hetween certain of these compounds and the aromatic hydrocarbons, and also to the reduction in the number of individual compounds present in the distillat,ion charge. The removal of aromatics, without affecting the other hydrocarbons present, is accomplished by the convenient process of selective adsorption. The sample is percolated through a column packed with silica gel adsorbent (each 100 grams of gel absorb 8 grams of aromatics) and the aromatic-free percolate of constant refractive index is segregated, equivalent t,o the naphthene-
