V O L U M E 23, NO. 12, D E C E M B E R 1 9 5 1
1853
Table 11. Recovery Experiments Sample Size Grams
Sample Water I
500 500 Red clover I 4.123 4.125 4.008 8.134
Soil I
1.0
Soil I1
0.5 0.5
1.0
Iodine Content
Iodine Added
Iodine Found
Y
Y
%
1.50 1.50 1.00 1.00 1.00
2.90 2.85 2.02 2.02
1.00
3.05 8.61 8.53 3.50 3.60
101 98 10 1 101 105 106 98 96 95 100
9
1.38 1.38 1.01
1.01 0.98 1.99
3.80 3.80 1.60 1.60
4.90 4.90 2.00 2.00
2.03
Recovery
tube up to the point of constriction. Plug in the tube furnaces. Add to the tower of beads 5 ml. of the chromic acid solution and then 50 ml. of concentrated sulfuric acid. Connect the combustion tube to an oxygen line and introduce.oxygen slowly. As soon as the tube furnaces attain a dull red color, light the burner nearest the tube furnace. When the filter paper in the mouth of the sample tube catches &e, increase the flow of oxygen t o about 2 liters per minute. Light the burners in as rapid succession as possible without getting smoke over in the absorption tower. Volatile matter will burn a t the mouth of the sample tube until the wet filter pa er plug burns out. The charred residue will then burn, leaving onyy an inorganic ash in the sample tube. The time required for the combustion of a plant sample depends upon its nature and size. For a 20-gram sample of whole soybeans, the time required is 30 to 40 minutes. For a 20-gram sample of whole corn kernels, 20 to 30 minutes are usually required. Draw off the liquid in the absorption tower into a distillation flask. Fill the tower with water and draw it off. Then wash down the beads with two 50-ml. portions of water. Add the ash in the sample tube to the combined solution and washings by drawing the liquid up in the tube several times and washing out with a stream of water. Concentrate this solution and proceed as for water or soil.
Smooth curves and well defined waves were obtained even a t full sensitivity. Calibration curves were obtained by carrying known quantities of iodine through the entire procedure (Figure 5). Deviations of individual samples are within &lo% a t full sensitivity. The method has been used to determine the iodine content of a large number of soil, water, and plant samples. Some typical analytical results are shown in Table I. As little as 0.5 microgram of iodine can be determined with a maximum error of +10%. Determinations with a precision of within 5% of the amounts of iodine from 2 to 40 micrograms can be made. Experiments in which known amounts of potassium iodide were added to soil, water, and plant samples before combustion showed that recovery of iodine was quantitative within the limits of reproducibility of the method (Table 11). The sensitivity of this method permits the determination of iodine in the minute amounts present in plant materials without the necessity of handling large samples. LITERATURE CITED
Cizek, V., “Polarographie,” by Heyrovskg, p. 352, Ann Arbor, Mich., Edwards Brothers, Inc., 1944. Fraps, G. S., and Fudge, J. F., Texas .4gricultural Experiment Station, Bull. 595 (November 1940). Kolnitz, H. von, and Remington, R. E., IND.EKG. CHEM., ANAL.ED.,5, 38 (1933). h‘IcHarguc, J. S.,and Offutt, E. R., J . Assoc. Ojic. Agr. Chemista, 2 2 , 4 7 1 (1939).
Matthews, N. L., Curtis, G M., and Brode, W. R., IND. ENQ. C H E M .ANAL. , ED.,10, I312 (1938). Orlemann, E. F., and Kolthoff, I. M., J . Am. Chem. S O C 64, , 1044, 1970 (1942).
Rllich, A., Collection Csechoslov. Chem. Communs., 7, 288 (1935)
RESULTS
Typical polarograms are presented for solutions derived from wheat, water, and soil ( A , B, and C, respectively, Figure 4).
RECEIVED March 31, 1951. Journal Paper Number 517. Supported in part by funds furnished b y the Chilean Iodine Educational Bureau, Inc.. 120 Broadway, New York, N.Y.
Indirect Colorimetric Micro-oxidimetry of Organic Compounds H. T. GORDON, Division of Entomology a n d Parasitology, University of California, Berkeley, Calif. The methods described in this paper are applicable to a great variety of organic compounds, in trace amounts (1 to 10 micrograms) and dilute solutions (0.001%). The analytical reaction is a stoichiometric oxidation with an excess of bromine, hypochlorite, permanganate, or ceric sulfate; the excess oxidant is then measured colorimetrically by reaction with an oxidizable dye. Each oxidation reaction is applicable to all compounds having certain reactive groups. Examples are given of reactions for cho-
V
ERY low concentrations of many oxidizing agents, of the order of 1 t o 10 micronormal, can be detected by using oxidizable dyes as reagents ( 7 , 8). The quantitative decolorization of a dye by various oxidizing agents is the basis of the analytical methods described in this paper. From 1 to 10 micrograms of many organic compounds can be determined oxidimetrically. The first step in an analysis is the reaction of the organic compound with an excess of oxidizing agent, and the second step is the reaction of the excess oxidant with a standard amount of oxidizable dye. The excess dye is then measured in a colorimeter.
lesterol (bromine addition to double bonds), amino acids and carbohydrates (hypochlorite oxidation of amino and aldehyde groups), citric acid (permanganate oxidation of alpha-hydroxy acids), glycerol and palmitic acid (ceric sulfate oxidation of C-C bond), and others. These micro reactions may be useful for identification of oxidizable structures in unknown compounds, for molecular weight determinations, and for analysis of very dilute solutions (distillates, extracts, chromatographic eluates).
All analytical results are expressed in terms of micrograms of dye protected from Oxidation by 1 microgram of organic compound. REAGENTS
Fast Green FCF (National Aniline and Chemical Co., Inc., New York, N. Y.), a water-soluble triphenylmethane dye (molecular weight as disodium salt 809), 0.1% solution in 1 M sulfuric acid. The dye is obtainable 94% pure and used without purification. The solution fades slightly in the first few days, but is then stable for months. Bromine, 1.2y0solution in carbon tetrachloride. This gradu-
1854 ally loses bromine, but can be kept several weeks at 5' C. A 0.15 to 10 dilution in 94.5y0 acetic acid is used in analysis. Sodium Hypochlorite, approximately 0.2 M in water. This is prepared by bubbling chlorine gas through a cold solution of 0.5 sodium hydroxide for 1 hour, and keeps indefinitely at 5" C. A 0.1 to 10 dilution in 0.1 M sodium hydroxide is used in analysis. Potassium Permanganate, approximately 0.2 M in water. A 0.08 to 10 dilution in 2 M sulfuric acid is used in analysis, and keeps for 2 to 3 hours. Ceric Sulfate, approximately 0.07 M in 1 M sulfuric acid. Two dilutions of this are used in analysis: a 1 to 10 dilution in 4 -If sulfuric acid, and a 1 to 10 dilution in approximately 17.5 X sulfuric acid, prepared by mixing 10 ml. of the stock reagent and 90 ml. of concentrated sulfuric acid, and heating until most of the water is driven off and crystals of ceric sulfate form (this heating destroys oxidizable impurities in the sulfuric acid). The solution is cooled and water is added, with cooling, to 100 ml. Potassium Bromate, approximately 0.2 M in water. A 0.05 to 10 dilution in 5 M sulfuric acid may be used in analysis. Potassium Bromide, approximately 0.2 hf in Jwter. Sulfuric Acid, reagent, approximately 10 M . Sodium Hydroxide, reagent, approximately 10 M. Solutions Used for Control of pH. I n each of the colorimetric analytical procedures described in succeeding sections, several aliquots of acidic solutions of varying molarity are added. In order to adjust the pH to 1.7 to 1.8 for the final colorimetric reading, special solutions of acid or alkaline sodium sulfate must be added. SULFATEBUFFER,prepared by mixing 150 ml. of 10 '$1sulfuric acid and 245 ml. of 10 Jf sodium hydroxide and diluting to 1liter. If 1ml. of this buffer is added to 9 ml. of water to which 0.15 ml. of 1 M sulfuric acid is added, the p H is adjusted to 1.75. Addition of 1 ml. of 94.5% acetic acid lowers the p H to 1.7, or addition of 0.1 ml. of 1 Jf sodium hydroxide raises the p H to 1.9. All pH measurements were made with a Beckman Model G glass electrode p H meter, standardized with p H 7.0 phosphate buffer. SoDxmi SULFATE, approximately 1 Jf in water. If 1.5 ml. of this solution are added to 8 ml. of water to which 0.15 ml. of 2 X sulfuric acid and 0.15 ml. of 1 ,?.I sulfuric acid have been added, the p H is adjusted to 1.75. ALKALINE SULFATE, prepared by mixing 100 ml. of 10 J4 sulfuric acid and 238 ml. of 10 M sodium hydroxide and diluting to 1 liter. If 1.5 ml. of this solution are added to 8 ml. of water to which 0.15 ml. of 4 -If sulfuric acid and 0.15 ml. of 1 M sulfuric acid have been added, the p H is adjusted to 1.75. SODIUM HYDROXIDE, approximately 4.1 .If in water. If 1 ml. of this solution is added to 9 ml. of water to which 0.15 ml. of 17.5 M sulfuric acid and 0.15 ml. of 1 M sulfuric acid have been added, the pH is adjusted to 1.7. Purified Organic Solvents. Most of the C.P. organic solvents obtainable contain several hundred parts per million of nonvolatile impurities, and are not suitable for use in oxidimetric microprocedures. If 2 drops of one of these solvents are evaporated to dryness in a small test tube, and 2 drops of fuming sulfuric acid are added, charring occurs on heating and the acid turns black. Of many solvents tested, only Baker's C.P. carbon tetrachloride passed the test. Even this solvent contains 10 to 20 p,p.m. of nonvolatile organic matter, and when tested by Method C5 the solvent blank causes complete decolorization of the ceric sulfate. The carbon tetrachloride is purified by standing over 10% fuming sulfuric acid for 1 week, with occasional shaking. It is decanted, shaken with 1% sodium hydroxide, separated, and slowly distilled. All glassware and receivers must be thoroughly cleaned xith chromic-sulfuric solution. The final product gives a very lorn blank in Method C5, and probably contains less than 1 p.p.m. of nonvolatile organic matter. C . P . petroleum ether can also be purified by this method, but two successive treatments with fuming sulfuric acid are needed. Acetic Acid, 94.5% (w./v.), prepared by diluting 90 ml. of glacial acetic acid nith water to 100 ml. COLORIMETRIC DETERMINATION OF FAST GREEN FCF
A Klett-Summerson photoelectric colorimeter, operated from a constant-voltage transformer, was used in all the work described in this paper. The standard No, 66 red filter was not satisfactory, because its transmittance is low and not selective enough and a red Lucite plastic filter was used instead. (The author has a limited supply of these filters available for distribution to other laboratories on request.) Two plates of this plastic '/le inch thick have a transmittance of less than 1yo from 400 to 600 mp, and above 80% from 650 to 990 mp.
ANALYTICAL CHEMISTRY Colorimeter readings are reproducible to 0.5 scale unit if the following precautions are taken:
A set of Klett test tubes is specially selected and matched to within 0.5 scale unit when filled with distilled water. Each tube is rotated in the holder until a position is found a t which a slight displacement to right or left causes less than 0.5 unit deviation from the standard. A vertical mark is engraved at the center of this position, near the rim of the tube, so that the tube can be replaced in this position every time it is used. Colorimeter tubes are cleaned with chromic-sulfuric solution and thoroughly rinsed with distilled water. Periodic checking of the matching of the tubes is desirable. The analytical procedures do not contaminate the tubes, and it is sufficient to rinse the tubes xith distilled water after use, and usually not necessary to allow them to drain dry. The 10-ml. calibration mark on the Klett tubes is accurate to about 2%; this may be checked by pipetting a given volume of dye solution into a series of tubes and diluting with water to 10 ml.; colorimeter readings in the 100 to 200 range should agree within 2 scale units. I
2401
210.
-0 z
180.
W
150.
30
90
60
MICROGRAMS
DYE
IN
120
IO
Ix)
I
ML.
Figure 1. Standard Curves for Fast Green FCF Circles, experimental data. Solid line plotted from uncorrected, dotted line from corrected equation
The standard calibration curve for Fast Green FCF is shown in Figure 1. Colorimeter readings againEt a distilled water blank are plotted for 20 to 150 micrograms of dye, plus 0.15 ml. of 1 M sulfuric acid, plus 1 ml. of sulfate buffer, diluted with water to 10 ml. The pH is buffered at 1.75 by the SO*---HSOd- system (pK about 1.7), because the absorption curve of Fast Green changes slightly with pH in the range from 1.5 to 7. The absorption maximum of Fast Green is a t 628 mp. The absorbancy of a water solution containing 2 micrograms per ml., measured with a Beckman Nodel D U spectrophotometer a t 628 m p with a 1-em. light path, is 0.32. There is marked deviation from Beer's law a t concentrations above 4 micrograms per ml. All volumetric measurements were made with Kahn and other Mohr-type graduated pipets. The procedures differ from standard microchemical practice as described by Kirk (6) and Benedetti-Pichler ( I ) , and are more like conventional macroprocedures. The tips of the pipets were drawn out in a flame to a fine point, with a relatively thick wall and an inner diameter of a few tenths of a millimeter. This facilitates slow delivery using finger control, and makes it possible to pipet a dense liquid into a lighter liquid without mixing inside the pipet. After filling to the zero mark, a pipet is held nearly horizontal and the tip is wiped dry with a clean paper toFel. Delivery is made with the fine tip immersed in the surface of the liquid to which an addition is being made, and is from one mark t o a lower mark on the pipet stem. If the tip is not immersed too deeply, upward creepage and retention on the tip after withdrawal are negligible for deliveries of theorderof0.l ml.
1855
V O L U M E 2 3 , NO. 1 2 , D E C E M B E R 1 9 5 1 Most of the liquid volumes delivered in the analytical procedures described below do not need to be absolutely precise, but should be reproducible to about 2y0. I n any series of analyses, one pipet is used exclusively for each reagent solution, and the volume of reagent delivered is the same. Only in the preparation of the standard calibration curve is high absolute precision desirable; for this, the standard 0.1% Fast Green solution was prepared with great care, and h h c o micropipets (Microchemical Specialties Co., Berkeley, Calif.) were used for measuring the solution, with the standard rinsing procedure, and absolute precision of the order of 1%. Because of the deviation from Beer’s law, the standard curve was fitted \\itti an equation of the form y = Az/(z B ) , where y is colorimeter reading and z is micrograms of dye per 10 ml. The best-fitting constants are il = 420 and B = 104. As oxidation of Fast Green by bromine, hypochlorite, and ceric sulfate causes a color change from blue-green t o yelloir, the equation for the standard curve used t o estimate the residual dye in reactions with these oxidants must be corrected for the residual reading of the yellow product. The corrected equation is y = 420z/(z 104) (150 - s)21/150. Permanganate Oxidation causes nearly complete decolorization of Fast Green, and so the uncorrected standard curve can be used to estimate residual dye in a permanganate reaction. Notes on Analytical Procedures. The techniques described in the procedures in the following sections represent the optimal conditions of volume, reagent composition and concentration, time of reaction, and temperature, for each of the several types of reaction studied. Each method given is the most satisfactory of an extensive series of trials, when judged from the standpoint of speed, convenience, accuracy, and approach to precise stoichiometry. For each such method, one or more examples are given of its application to a pure, known compound. The observed “protection ratio,” P (micrograms of Fast Green protected from oxidation by 1 microgram of the compound), is compared t o the P value calculated for the stoichiometric reaction. The per cent variation among duplicates or triplicates in a single test is given, and also the variation in the observed P value in replications, using freshly prepared reagent solutions, solutions of compounds, etc.
+
+
+
ANALYTICAL PROCEDURE USING BROMINE
Reaction of Fast Green with Bromine. If 0.1 ml. of the approximately 180 micrograms per ml. of bromine in 94.5% acetic acid is pipetted into about 1 ml. of 94.5% acetic acid in a Klett tube, and 0.15 ml. of the 0.1% Fast Green in 1 JI sulfuric acid (150 micrograms of dye) is added, there is a quantitative reaction, two bromine atoms per molecule of dye, complete in 30 seconds. On adding 1 ml. of sulfate buffer and diluting with water to 10 ml., and reading in the colorimeter against a comparable blank (no dye added), the residual dye can be estimated from the corrected standard curve. If the bromine is allowed t o stand for 10 minutes after addition, before the dye is added, a loss of about 570 is noted. The reaction is quantitative in the range of 0 t o 25 micrograms of bromine; an excess of 20 micrograms or more of dye must be present. Analytical Procedure for Cholesterol and other BromineReactive Compounds. Cholesterol adds two bromine atoms and will therefore protect one Fast Green molecule from oxidation. The theoretical “protection ratio,’’ P , micrograms of dye protected per microgram of cholesterol, is 2.1. This is the ratio of the equivalent weights of the dye and of cholesterol, 809/386.
A solution of 200 micrograms of cholesterol per ml. in 94.570 acetic acid was prepared. (Either water or carbon tetrachloride can also be used as solvent for the compound to be oxidized.) Aliquots of 0, 0.05, 0.1, and 0.15 ml. were pipetted into 1 ml. of 94.5% acetic acid in a series of Klett tubes. To each tube 0.125 ml. of the bromine reagent was added, and each tube was gently shaken to mix, and allowed to stand 10 minutes at room
temperature. To each tube, 0.15 ml. of the dye reagent mas added, and the tubes were gently shaken to mix. After 1 to 2 minutes, 1 ml. of sulfate buffer was added to each tube, and water to the 10-ml. mark. After mixing, the tubes were read against a comparable blank. Readings for duplicate tubes agreed within 2%, duplicate readings were averaged, and the residual dye was estimated from the corrected standard curve. For 0, 10, 20, and 30 micrograms of cholesterol, there were 50, 70, 90, and 112 micrograms of residual dye and observed P values were 2.0, 2.0, and 2.1 micrograms of dye protected per microgram of cholesterol, all within 5y0of the theoretical. In a replicate, P values were 1.96, 2.0, and 2.05. ANALYTICAL PROCEDURES USING HYPOCHLORITE
Method A l . Reaction of Fast Green with Hypochlorite. The approximately 0.002 1M hypochlorite in 0.1 M sodium hydroxide does not react with Fast Green unless the solution is acidified to p H 1. If 0.1 ml. of hypochlorite is pipetted into about 1 ml. of water in a Klett tube, and 0.15 ml. of the dye reagent is added, the reaction, 1 hypochlorite molecule per dye molecule, is complete in 30 seconds. On adding 1 ml. of sulfate buffer and diluting to 10 ml., and reading against a water blank, the residual dye can be estimated from the corrected standard curve. The reaction is quantitative from 0 to 0.2 micromole of sodium hypochlorite; an excess of 20 or more micrograms of dye must be present. 2. Analytical Procedure for Glycine and Other Amino Acids. The a-amino acids, R-CH(KH2)-COOH, are oxidized by 2 hypochlorite ions to carboxylic acids, R-COOH. Two dye molecules will be Drotected bv 1 amino acid molecule. The theoretical P for glycine is 21.6 micrograms of dye per microgram of glycine. X solution of 40 micrograms of glycine per ml. in water was prepared. Aliquots of 0,0.05, and 0.1 ml. were pipetted into about 1 ml. of water in a series of Klett tubes. To each tube, 0.095 ml. of hypochlorite reagent was added, and the tubes were gently shaken to mix and allowed to stand 10 minutes at room temperature. To each tube, 0.15 ml. of dye reagent was added, and then 1 ml. of sulfate buffer and water to 10 ml. After mixing, the tubes were read against a water blank. Readings for duplicate tubes agreed lrithin 2%, duplicate readings were averaged, and the residual dye was estimated from the corrected standard curve. For 0, 2, and 4 micrograms of glycine, there were 33, 78, and 120 micrograms of residual dye, and observed P values were 22.5 and 21.8, within 4% of the theoretical. In a replicate, P values were 23.0 and 22.5. Groups other than the a-aminocarboxylic group, such as the e-amino group of lysine and the SH group of cysteine, are also oxidized by hypochlorite. 3. Analytical Procedure for Glucose and Other Carbohydrates. The procedure used for amino acids is not directly applicable t o glucose, because the cyclic lactal form of glucose is not oxidized and isomerizes only slowly to the oxidizable aldehyde form. It is necessary to preheat an alkaline solution of glucose to 100” C. for several minutes to convert it to the aldehyde; the solution, when cool, can be analyzed by the procedure described for glycine. The theoretical P for oxidation of glucose to gluconic acid by 1 sodium hypochlorite is 4.5. A solution of 100 micrograms of glucose per ml. in water was prepared. To a series of Klett tubes, 0.5 ml. of water and 0.1 ml. of 0.02 M sodium hydroxide were added. Into these, aliquots of 0,0.04,0.08, and 0.12 ml. of glucose solution were pipetted. The tubes were gently shaken, heated in a boiling water bath for 4 minutes, and cooled in Rater, and the solutions were analyzed by the 10-ml. final volume procedure described for glycine. Observed P values for 4, 8, and 12 micrograms of glucose \?-ere0.8, 0.7, and 0.7, about 15y0of the theoretical. The above procedure for glucose was exactly repeated, using 0.1 ml. of 0.2 ;II sodium hydroxide in each tube for the preheating reaction. Observed P values \yere 2.9, 2.5, and 2.6, about 58y0 of the theoretical. The above procedure m-as exactly repeated, using 0.1 ml. of 2 M sodium hydroxide in each tube for the preheating reaction. Because of the excess of sodium hydroxide present, a 0.1% dxe
1856
ANALYTICAL CHEMISTRY
reagent in 2 M sulfuric acid was used; the pH, after addition of 1 ml. of sulfate buffer and dilution to 10 ml., was 1.73. Observed P values were 3.10,3.15, and 3.05, about 66%of the theoretical. The above procedure was exactly repeated, using 20-minute heating a t 100'. Observed P values were 3.10, 3.0, and 2.9, about 64% of the theoretical. These results indicate that complete conversion of glucose to the aldehyde form is not practicable. Under the optimum conditions, a t a sodium hydroxide concentration of 0.1 to 0.3 ill, and 4 to 8 minutes' heating a t lOO", only 66% of the glucose becomes oxidizable, and so an empirical P of 3.10 must be used. ANALYTICAL PROCEDURES USING PERMANGANATE
Method B1. Reaction of Fast Green with Permanganate. This reaction in acid is a complex reduction of manganese(VI1) to manganese(II), with more extensive destruction of the Fast Green molecule than occurs with other oxidants.
If 0.15 ml. of the approximately 0.002 M permanganate in 2 Af sulfuric acid is added to 0.5 ml. of water in a Klett tube, and 0.15 ml. of dye reagent is added, a transient red colorappears, changing to a stable green within 2 d n u t e s . On addition of 1.5 ml. of 1 M sodium sulfate and water to 10 ml., and reading against a water blank, residual dye can be estimated from the standard curve Approximately 105 micrograms of dye are decolorized by 37.5 micrograms of potassium permanganate; but 55 micrograms are decolorized by 18.75 micrograms of potassium permanganate. The reaction is therefore not stoichiometric, but the deviation from linearity is not great when the excess of dye is about 50 micrograms. Approximately 1.8 potassium permanganate molecules are reduced to manganese(I1) by 1 dye molecule (a 9 e reaction). In the analytical oxidations, permanganate is present in excess, and reduction will not go to a lower oxidation state than manganese dioxide. The analytical reaction may be very complex, and all states from VI1 to I1 may be present, but a t the end of the reaction it is probable that only the stable VI1 and I V forms will be present. The I V (manganese dioxide) can also react with Fast Green. If permanganate is heated for a few minutes in 10 M sulfuric acid, it is decomposed to a red-yellow colloidal solution of manganese dioxide. A dilution of this in 2 M sulfuric acid, at the same manganese concentration as the 0.002 M permanganate reagent, is stable for 20 to 30 minutes. If this is used to react with Fast Green under the conditions described for permanganate, it is reduced to manganese(II), but decolorizes only half as much dye. Therefore 3.6 manganese dioxide molecules react with 1dye molecule (a 7.2 e reaction). If, in the primary oxidation reaction, the compound being oxidized transfers ne/molecule to the manganese( VII) to manganese(I1) system, each molecule will convert n/3 M n + + + + + + + t o n / 3 Rln++++. Then/3 i l l n + + + + + + + would have decolorized n/3 (1.8)dye molecules, while the n/3 Mn + + + + will still decolorize n/3(3.6) dye molecules. The number of dye molecules protected from oxidation by one molecule of oxidizable compound is therefore n/3(3.6) or n/10.8. 2. Analytical Procedure for Citric Acid and Other a-Hydroxycarboxylic Acids. Oxidation of citric acid by 4 potassium permanganate t o 3 oxalic acid (a 12 e reaction) gives a theoretical P value of 3.06 for hydrated trisodium citrate (molecular weight 294). The primary reaction with compounds of the type RiRzC(0H)-COOH seems to be a cleavage to HOOC-COOH R1-OH R2-OH. The last two products may be further oxidized.
+
+
A solution of 100 micrograms of sodium citrate dihydrate per ml. in water was prepared. Aliquots of 0, 0.05, 0.10, and 0.15 ml. were pipetted into about 0.5 ml. of water in a series of Klett tubes. To each tube, 0.15 ml. of permanganate reagent was added, and gently ehaken to mix. After 15 to 20 minutes at room
temperature, 0.15 ml. of dye reagent was added to each tube. After 2 minutes, 1.5 ml. of 1 M sodium sulfate were added, and water to 10 ml. Duplicate readings agreed within 2%, and duplicates mere averaged and residual dye was estimated from the standard curve. Observed P values for 5, 10, and 15 micrograms of trisodium citrate were 3.1, 3.0, and 2.96, all within 4% of the theoretical. In a replication, P values were 3.05, 2.95, and 2.85. It is important not to allow permanganate oxidations to go on longer than 20 minutes at room temperature. The colloidal manganese dioxide formed turns the reaction mixture yellouish, then reddish and cloudy, and manganese dioxide eventually precipitates. 3. Analytical Procedure for Crotonic Acid and Other Compounds with Reactive Double Bonds. Oxidation of a double bond may form a glycol, or may proceed to a cleavage into two carboxyl groups. Oxidation of crotonic acid to acetic and oxalic acid will require 2.67 potassium permanganate molecules (an 8 e reaction). The theoretical P is 6.95. Using an ~nalyticalprocedure like that described for citric acid, observed P values for 4.5 and 9 micrograms of crotonic acid were 5.8 and 5.8, about 85% of the theoretical. The discrepancy is not easy t o interpret, is partial formation of dihydroxybutyric acid, an a-hydroxy acid, is improbable. The crotonic acid used had been recrystallized once from water solution, but the purity WRS not checked. Oxidation of allyl alcohol to formic and oxalic acid would have a theoretical P of 15.5, but partial formation of glycerol, which is resistant to further oxidation, can occur. Using an analytical procedure like that described for citric acid, observed P values for 5, 7.5, and 10 micrograms of allyl alcohol were 6.0, 5.95, and 6.0, about 38% of the theoretical. This can be interpreted as a 26% complete oxidation to formic and oxalic acid, with 74% incomplete oxidation to glycerol. It is clear that reactions having several alternate pathways are not likely to be stoichiometric, but a constant empirical P value is nevertheless attainable. ANALYTICAL PROCEDURES USING CERIC SULFATE
Method C1. Reaction of Fast Green with Ceric Sulfate. The two 0.007 M ceric sulfate reagents, one in 4 M and the other in 17.5 M sulfuric acid, react similarly. If 0.15 ml. of the reagent in 4 M acid is added to 0.5 ml. of water in a Klett tube, and then 0.15 ml. of dye reagent is added, a transient red color appears, changing within 1 minute to a stable yellow-green. On addition of 1.5 ml. of the alkaline sulfate solution and water to 10 ml., residual dye may be estimated from the colorimeter reading and the corrected standard curve. The reaction is nearly quantitative in the range of 0 to 1 micromole of ceric sulfate; an excess of 30 or more micrograms of dye must be present. One dye molecule reacts with 6 ceric ions. 2. Analytical Procedure for Oxalate. The ceric reagent in 4 M sulfuric acid reacts with readily oxidizable compounds, but i s less active than permanganate. I t does, however, oxidize sodium oxalate; the theoretical P for this reaction is 2.01. A solution of 160 micrograms of sodium oxalate per ml. in water was prepared. Aliquots of 0, 0.075, and 0.15 ml. of the solution were pipetted into 0.5 ml. of water in a series of Klett tubes. To each tube, 0.15 ml. of the ceric reagent in 4 i%fsulfuric acid was added, gently shaken to mix, and allowed to stand 15 minutes at room temperature. To each tube, 0.15 ml. of dye reagent was added, and, after 2 minutes, 1.5 ml. of alkaline sulfate solution and water to 10 ml. Duplicate colorimeter readings agreed within 2% and were averaged, and residual d e was estimated from the corrected standard curve. ObservecfP values for 12 and 24 micrograms of sodium oxalate were 2.25 and 2.12, 10 and 5% above the theoretical. I n a replication, P values were 2.15 and 2.10. 3. High-Temperature Oxidations Using Ceric Reagent in 17.5 M Sulfuric Acid. Ceric sulfate is stable in strong acid in the temperature range from 100" to 200°, and oxidizes a wide
1857
V O L U M E 23, NO. 12, D E C E M B E R 1 9 5 1 range of organic compounds. Oxidations a t high temperature, however, present many special problems ( 4 ) : There is a critical minimum reaction temperature, differing for different compounds; the concentration of sulfuric acid is also critical; the reaction may be complex and follow several alternate pathways; relatively volatile compounds may partly or wholly distill out of the mixture before the critical oxidation temperature is attained; and volatile oxidation products, such as formaldehyde or carbon monojdde, may escape from the reaction mixture. For these reasons, the over-all reaction may not be stoichiometric, and the errors may be large. The high-temperature analytical reactions are carried out in b,orosilicate glass culture tubes (No. 9820), 10 X 75 mm., without rim. The tubes must be thoroughly cleaned in hot chromic-sulfuric solution, rinsed, and dried, to ensure a zero blank reading. Heating is carried out on an electric hot plate, on which are placed three asbestos-center wire gauzes to serve as heat insulators and buffers. A cylindrical metal can, 65 mm. high and 100 mm. in diameter, with open bottom and the top perforated with circular holes 11 mm. in diameter, is placed on the topmost wire gauze, and serves as a holder for the tubes. The system is essentially a hot-air bath. A 250" thermometer is inserted through one of the holes in the top of the can and rests in a vertical position on the wire gauze. The temperature readin can be adjusted by varying the current throu h the heater, a n d at equilibrium will be constant within a few fegrees. In the ivork reported below, this final temperature was 165", but is lowered when cold tubes are inserted and while water is being evaporated from the reaction mixtures. By the end of the reaction, in 15 to 30 minutes, the temperature rises to 165O, high enough to drive out nearly all the water from a dilute sulfuric acid solution without causing evaporation of the acid. All reactions in these microprocedures were carried out in open tubes, not sealed tubes. This is more convenient, and the tubes can be cleaned and re-used. However, losses due to volatilization make the reactions only semiquantitative. The use of evacuated or nitrogen-filled sealed tubes would make possible complete oxidation and more precise analysis. 4. Analytical Procedure for Glycerol and Other Relatively Nonvolatile Water-Soluble Compounds. Oxidation of glycerol by 4 ceric ions to 2CHz0 and lHCOOH has a theoretical P value of 5.95, and oxidation by 8 ceric ions to 3HCOOH has a P value of 12.
,4solution of 50 micrograms of glycerol per ml. in water was prepared. A series of borosilicate glass tubes was set up in triplicate, with the following additions: 0.1 ml. of water, 0.05 ml. of water plus 0.05 ml. of glycerol solution, and 0.1 ml. of glycerol solution. To each tube, 0.15 ml. of the ceric reagent in 17.5 M sulfuric acid was added, and the tubes were shaken to mix. The tubes were placed on the hot plate for 15 minutes, then cooled in running water, and 1 ml. of water and 0.15 ml. of dye reagent were added to each. The tubes were shaken and let stand 2 minutes, then water was added nearly to the brim and the contents were poured into a series of Klett tubes; the borosilicate glass tubes were rinsed and the rinsings poured into the corresponding Klett tubes. Then 1ml. of 4.1 M sodium hydroxide was added to each tube, and water to 10 ml. Observed P values for 2 . 5 and 5 micrograms of lycerol were 5.6 and 9.6. I n a replication P values were 6.3 anf9.5. The glycerol analysis was repeated as described above, but about 80% of the water was evaporated from the tubes, by heating on the hot plate, before the ceric reagent was added. Observed P values were 7.2 and 10.0. This phenomenon of a low P value for small quantities of oxidizable compound is common in open-tube reactions, especially when a high proportion of water is present. As there are millions of water molecules to each molecule of oxidizable compound, loss by entrainment in the vapor stream, either of the substance itself or of volatile fragments of the primary oxidation stages, is probable. Compounds having a relatively low boiling point, such aa ethanol or even cyclohexanol, completely escape being oxidized
under the conditions used for glycerol, for the rate of loss far exceeds the oxidation rate. Readings of triplicates in the glycerol oxidation agreed within 3%. Because the P value is both empirical and variable, however, unknowns could probably not be determined to an accuracy better than 10%. 5. Analytical Procedure for Palmitic Acid and Other Relatively Nonvolatile Water-Insoluble Compounds. Oxidation of palmitic acid to the CHzO stage would give a P value of nearly 17, and to the HCOOH stage a P value of 33.
A solution of 10 micrograms of palmitic acid per ml. in purified carbon tetrachloride was prepared. A series of borosilicate glass tubes was set up in triplicate, with the following additions: none, 0.15 ml. of carbon tetrachloride, 0.05 ml. of palmitic acid solution, and 0.1 ml. of palmitic acid solution. Each of the tubes containing carbon tetrachloride was placed in a water bath at No, and a pipet connected to a water pump was inserted into the tube to allow a stream of air to flow over the surface of the liquid and cause evaporation. The tube was withdrawn as soon as the last trace of liquid disappeared. To each of the dry tubes, 0.15 ml. of the ceric reagent in 17.5 M sulfuric acid was added, and the tubes were kept on the hot plate for 30 minutes, then cooled in running water. To each, 1 ml. of water and 0.15 ml. of dye reagent were added. Quantitative transfer to Klett tubes was carried out as in the procedure for glycerol; likewise addition of 1ml. of 4.1 M sodium hydroxide and dilution to 10 ml. Triplicate readings agreed within 5%; occasionally, however, in reactions of this kind, contamination will cause one of the readings to differ greatly from the other two of a triplicate set, and such a reading is not included in the average. Observed P values for 0.5 and 1 microgram of palmitic acid were 22.5 and 29.5. In a replication, P values were 23.8 and 29.0. The discrepancy between these values is not as great as for the glycerol procedure, probably because much less water was present in the palmitic oxidation mixture. If 1.5 micrograms of palmitic acid are added to a borosilicate glass tube by the above procedure, and 0.1 ml. of water is added also before the ceric reagent, the oxidizing conditions are the same as in the glycerol procedure. The observed P value, however, is only 5.0, or about 20% of the value to be expected from the procedure in which water is omitted. Although readings of triplicates usually agree within 5%, experimental P values fluctuate in replicate tests from 5 to 10% or more, and accuracy to better than 10% is not to be expected. The main sources of variation are probably in the pipetting of the carbon tetrachloride and the 17.5 M acid solutions, and in the air-bath heating conditions. ANALYTICAL PROCEDURE USING BROMATE
The bromate reagent is exceptional in that it will not decolorize Fast Green, even in strongly acid solution a t 100" C, unless 2 or 3 drops of 0.2 M sodium bromide are added to the reaction mixture (total volume about 1 ml.). The bromate reacts quantitatively with hydrobromic acid in 1 M sulfuric acid a t looo, the reaction taking 2 to 3 minutes, and the 6 bromine atoms formed by each bromate ion react immediately with the dye. No analytical use has yet been developed for bromate, but its stability in acid a t 100O may make it useful for some reactions. It is included here only because i t is one of the few oxidants that can be detected by Fast Green at very low concentration. DISCUSSION
Specificity of Oxidimetric Reactions. The most serious limitation of oxidimetry is lack of specificity, and it cannot be used with complex mixtures unless separation by physical methods such as differential solubility, adsorption, or microdistillation, is possible. In the procedures described in the preceding sections, hoxever, both the oxidant and the compound being oxidized are present
ANALYTICAL CHEMISTRY
1858 a t very low concentrations, and the reaction rate is 1/1000th t o 1/1,000,000th of the rate a t the concentrations commonly used. This magnifying of the time scale confers some selectivity t o oxidations and makes it possible to oxidize certain compounds specifically in the presence of other, more stable compounds; useful differences in reaction rate, however, will exist only between compounds in different structural classes, not between compounds in the same class. Selectivity can also be attained by using different oxidants and by varying the pH and temperature. The bromine and hypochlorite reagents are relatively specific, attacking only certain reactive groups such as double bonds, SH, and amino groups. Acid permanganate is less selective, but it is possible that alkaline permanganate (not tried so far) would prove to be more selective; an extensive series of macrovolumetric organic oxidations using alkaline permanganate was developed by Stamm (IO). A summary of macrovolumetric oxidation procedures is available (9). The problem of acid permanganate oxidation is discussed by Conway ( 2 ) . The specificity of oxidimetric procedures may also be increased by devising “conversion” reactions that will render a compound oxidizable-e g., the splitting of the lactal ring of glucose to the free aldehyde by heating in alkaline solution; the aldehyde is oxidizable by hypochlorite. Glycerol, if converted to its phosphate ester, becomes Oxidizable by hypochlorite. “Masking” of an oxidizable group bv a conversion reaction can make specific differential analysis possible-e.g., the benzoylation of glycine to hippuric acid, or its methylation to betaine, renders it unoxidizable by hypochlorite. Table I lists a number of organic compounds, and their reaction in the several oxidimetric procedures described above.
Table I.
Oxidimetric Reactions
-_
PO++++ Compound
Br2, Acid
+
FaOC1, hlk., A
;t
++ +
t
t
KMnOa. Acid,
B
+
Ce++++ 4 .If Acid, C2
17.5 M ’
HzSO4 C4, 5 , 165‘ C.
Cysteines Tyrosine b GlycineC Formaldehyde 0 0 Glucosed Glycerol6 0 0 0 0 Glycerophosphate 0 Citric acidf Crotonic acidg 0 0 Cholesterolh 0 0 $ 0 Acetic acid 0 Palmitic a c i d i 0 0 0 Other compounds in the same structural class have been found t o react in microgram quantities (in most cases in rough tests only).
+
++
t
++ ++ -+
+
a Methinnine . ..~.. .. b Histidine, tryptophan,
casein. c ArginiRe, asi)aragine. aspartic acid. l e x i n e . lysine, isoleucine. phenylalanine, serine, threonine, valine. d Fructose, xylose. lactose. e Mannitol, inositol, glucose, sucrose. / Lactic acid, tartaric acid, malic acid. I Sorbic acid. h Ergosterol, oleic acid. i Stearic acid, beeswax, cetyl alcohol. Quantitative reaction. 0. No reaction. Reaction aftw preliminary conversion reaction.
+. *.
Possible Application. AIicro-oxidimetric procedures may be useful as classification tests for certain reactive groups, applicable to a few micrograms of an unknown. They may also serve to detect certain compounds present in very low concentration in eluates from adsorption columns, distillates, etc., wherever microgram sensitivity is needed. If a pure compound is available only in minute quantities, micro-oxidimetrymay be useful in estimationof minimal molecular weight, especially if the compound contains groups that react with bromine or hypochlorite. The molecular weight, M , is calculated from the observed protection ratio ( P = micrograms
of Fast Green protected from oxidation by 1 microgram of compound) by the formula: M = n(809)/a(P),where n is the number of electrons transferred to the oxidant from 1 molecule of the compound, and a is 2 for bromine and hypochlorite reactions, 10.8 for acid permanganate oxidations, and 6 for ceric sulfate oxidations. If n is unknown, it is set equal to 2 (the minimum number of electrons transferable in most organic oxidations) and M is the minimal molecular weight. As most of the analytical oxidation reactions are slow, requiring 5 to 20 minutes for completion, differences in reaction rate can be determined by measuring the per cent completion at 1, 2, etc., minutes following addition of the oxidant. Such information can help in determination of structure. Reaction rates differ significantly for many compounds only slightly different in structure (S, 6). In preliminary conversion reactions, like those described above for glucose and lactose, the temperature, pH, and time required for maximal conversion differ for various carbohydrates, and may aid in qualitative identification of an unknown. The method can be applied to oxidizable inorganic compounds also. Iodide, sulfite, sulfide, and sulfur have been found to react quantitatively in the bromine procedure. Serial Oxidation. A sequence of oxidations can be used to elucidate structure and identify unknowns-for example, serine, H O CH2-CH(NH2)-COOHJ is oxidized by sodium hypochlorite to glycolic acid, HO-CH2-COOH ( P = 15.4)) which is oxidized by acid permanganate to oxalic acid (P = 3.0), which is oxidized by ceric sulfate to 2 carbon dioxide ( P = 3.0). Aliquots of a serine solutioncan be degraded to equivalent glycolic and oxalic solutions by Methods A2 and B2, excess oxidant being destroyed by heating to 100” for a few minutes (instead of adding dye). If an aliquot of the unchanged serine solution protects A micrograms of dye in Method A 2 the equivalent glycolic solution will protect (3/15.4)A micrograms in Method B 2, and the equivalent oxalic solution will protect (3/15.4)A micrograms in Method C2. These two dye ratios are characteristic for serine, which can thereby be identified in pure solutions of unknown concentration (such as chromatographic eluates); once identified, the concentration is calculable. Even when identification is not possible, the molecular weight of the unknown can be calculated, if oxalic acid is formed in the permanganate degradation step (oxalic acid is the only compound so far known that is formed in permanganate oxidation, and can be subeequently oxidized by ceric sulfate). If n oxalic acid molecules are formed from 1 molecule of unknown, the molecular weight, MI of the unknown is calculated from the formula M = 270 ( n ) ( U ) / ( F ) where , U is the micrograms of unknown in the aliquot used for the degradation t o oxalic acid, and F is the micrograms of Fast Green protected by the oxalic in Method C2. If n is unknown, it is set equal to 1, and &I is the minimal molecular weight. LITERATURE CITED
(1) Benedetti-Pichler, A. 8.,“Microtechnique of Inorganic Analysis,” New York, John Wiley & Sons, 1942. (2) Conway, E. J., “Micro-Diffusion Analysis and Volumetric Error,” 2nd ed., Chap. XXVIII, London, Crosby Lockwood 8: Sons, 1947. (3) Hockett, R. C., Dienes, M. T., Fletcher, H. G., and Ramsden, H. E., J . Am. Chem. Soc., 66,467-8 (1944). (4) H u r k a , W., Mikrochemie per. Mikrochim. Acta, 30,228 (1942). (5) Ibid., p. 259. (6) Kirk, P. L., “Quantitative ~ltramicroanalysis,”Kew York, John Wiley & Sons, 1950. (7) Kul’berg, L., and Rlatveev, L., J . Gen. Chem. (U.S.S.R.), 17, 457-9 (1947). ( 8 ) Smith, G. F., and Bliss, H. H., J . Am. Chem. Soc., 53, 2091 (1931). (9) Smith, G. F., and Duke, F. R., IND.ENG.CHEX.,ASAL. ED., 15,120 (1943). (10) Stamm, H., 2.anal. Chem., 48,710 (1935). RECEIVED April 12, 1951.
