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[47] Microanalytical method for acetic and other volatile acids
[47]
ACETIC AND OTHER VOLATILE ACIDS
263
Mter cooling to 3 to 5 ° and standing for several hours, the fine crystalline precipitate is filtered out, washed with cold water, and dried as above.
[47] Microanalytical Method for Acetic and Other Volatile Acids By SIMON BLACK Principle. Volatile acids such as acetic diffuse from an acidified sample in the main chamber of a microdiffusion vessel to an alkaline solution in the center cup. After liberation of COs from the cup by an acid buffer, a microtitration is made. The latter measures volatile acid plus the excess of acid buffer over initial alkali. A blank titration must therefore be subtracted from each determination to obtain the volatile acid value. Although the method is nonspecific, some distinction between the several volatile fatty acids can be made on the basis of their distribution behavior between ether and water. Extraction of the acid into ether prior to its determination also serves to eliminate many interfering substances. Special Equipment Glass-stoppered centrifuge tubes, 30-ml. capacity. These may be obtained from the Wilkins-Anderson Co., 111 N. Canal St., Chicago, Ill., as Maizel-Gerson reaction vessels. A 10-ml. pipet with its tip bent 90 ° with respect to the pipet axis. The curved tip facilitates withdrawal of supernatant ether, leaving an aqueous layer undisturbed. Glass-stoppered Erlenmeyers, 50-ml. capacity. Center cups. These are made by cutting the lower 15-mm. portion from shell vials (15 mm. o.d.) which are obtainable from most scientific supply companies. They should be discarded after they have been used once or twice. For greater precision steel cups may be used.1 A microburet which delivers with an accuracy of +0.1 #l. A magnetic stirrer and a stirring bar made by sealing a 4-ram. length of iron wire in glass.
Reagents Alkaline trapping solution. Dissolve 0.124 g. of K2CO3-1.5 H20 plus 0.25 g. of KNO3 (to increase hygroscopicity) in distilled water, and dilute to 50 ml. 1S. Black, Arch. B/avhvm. 25~ 347 (1949).
264
CAR~OHYVRATES
[47]
80% H2S04. Add 400 ml. of concentrated H2S04 (c.p. or reagent) to 100 ml. of distilled water. 0.1 M monosodium maleate. Dilute 5 ml. of 1.0 N NaOH and 1.5 ml. of isoamyl alcohol (to reduce surface tension) plus 0.58 g. of maleic acid with distilled water to 50 mh 0.1 N standard NaOH. Indicator solution. Dilute 1 mh of phenolphthaiein indicator, 0.1% in 95 % alcohol, to 25 mh with CO~-free (boiled) distilled water. This solution should be made up on the day of use. 5 N H2S04. Alkaline Na2S04. Dissolve 20 g. of anhydrous Na~S04 (c.p. or reagent) plus 10 mh of 1 N NaOH and dilute to 100 ml. Sodium hydroxide solutions without Na~S04 do not remove fatty acids quantitatively from ether. Ethyl ether. This reagent should be of good quality, substantially free of peroxides. 10% sodium tungstate. Procedure. One milliliter of a copper-lime filtrate, ~ containing not more than 2 microequivalents of volatile acid, is pipetted into a glass-stoppered Erlenmeyer. One milliliter of 80% H2SO~ plus about 10 rag. of Ag2S04 (to retain chloride) are added, and the flask is shaken gently to ensure mixing. A center cup, containing 100.0 ~l. of the alkaline trapping solution, is placed centrally on the floor of the flask with the aid of forceps. The cup stands directly in the acidified sample. The glass stopper is put loosely in place (do not twist or "lock "), and the flask set in a 100 to 105 ° oven. After 14 to 16 hours the center cup is removed and 40.0 ~1. of monosodium maleate solution is added. The cup is then tilted gently, if necessary, to ensure distribution of this reagent over its bottom. After 10 to 20 minutes, during which CO2 escapes, about 0.3 ml. of indicator solution and a magnetic stirrer are added. A titration is then made in the cup, with stirring, with 0.1 N NaOH. The amount of volatile acid is calculated by substrating a blank titration value. When the amount of interfering materials make necessary a preliminary separation from the volatile acid, the following ether extraction procedure may be used: Three milliliters of a protein-free filtrate is placed in a 30-ml. glass-stoppered centrifuge tube. Two-tenths milliliter of 5 N H2S04 is added, 15 ml. of ether run in, and the tube shaken vigorously for 2 minutes. Ten milliliters of the supernatant ether is transferred with the special pipet to a second stoppered tube containing 1.2 ml. of the alkaline Na2SO~ solution. This tube is shaken for 1 minute. One milliliter of the A. L. Lehninger and S. W. Smith, J. Biol. Chem. 173, 773 (1948).
[47]
ACETIC AND OTHER VOLATILE ACIDS
265
aqueous layer is then transferred with a fine-tipped pipet to a 50-ml. Erlenmeyer flask, and the analysis carried out as described above b y the microdiffusion procedure. T o calculate the a m o u n t of volatile acid present in the original 3 ml. of filtrate, the q u a n t i t y found is divided b y the factor 0.55 multiplied b y whatever fraction of the acid in question the table indicates should be extracted b y this procedure. EXTRACTION OF VOLATILE ACIDS FROM WATER WITH ETHYL ETHER
I. 15 ml. of ether used to extract 3.2 ml. of aqueous solution2 II. 10 ml. of ether used to extract 10 ml. of aqueous solution. Per cent of acid found in ether layer Acid extracted Acetic Propionic Butyric Valeric Caproic Caprylic
I
II
71 87 95 98 100 100
32 60 83 93 98 100
The small amount of calcium in copper-lime filtrates causes additional retention in the aqueous layer of about 0.1 micromole of volatile acid; this amount is independent of the total volatile acid in the sample. Addition of 0.1 ml. of 10% Na~WO4 prior to extraction causes precipitation of the calcium (which need not be removed) and elimination of this source of error. Specificity. The method is nonspecific for volatile acids. However, the different distribution characteristics between water and ether (see the table) can be used as an aid in identification. I t is also possible to replace the titration procedure with a colorimetric test specific for acetic acid. ~ Numerous sources of interference with this colorimetric method are eliminated b y prior use of the microdiffusion procedure. A method for acetic acid which employs paper chromatography is also available. 4 Some interference is also given in the titrimetric procedure by lactic, f~-hydroxybutyric, and pyruvic acids. Diffusion of the latter can be eliminated by HgSO4 (about 10 mg.). When HgSO4 is used in conjunction with the extraction procedure, ether should first be eliminated b y drying the flask in an oven; 1 ml. of water plus solid HgSO4 are then added before the 80% H~SO4. Precision. K n o w n quantities of acetic acid can be recovered from tissue samples, b y means of the ether extraction and microdiffusion procedures, with an average deviation of 0.03 ~M.
O. Hutchens and B. M. Kass, J. Biol. Chem. 177, 571 (1949). 4 E. P. Kennedy and H. A. Barker, Anal. Chem. 23, 1033 (1951).
3 j.
266
CARBOHYDRATSS
[48]
Advantages and Disadvantages. The advantages of this method, compared to the Duclaux procedure, ~ are in the possibility of analyzing many samples simultaneously with simple apparatus, and in its suitability for small quantities of sample. Its low specificity, however, limits its usefulness in experiments involving crude extracts, or in other cases where the samples contain potential sources of interference. T. E. Friedeman, J. Biol. Chem. 123, 161 (1938). See Vol. III [61].
[48] An Enzymatic Micromethod for the Determination of Acetate B y MORRm SOODAK
Acetate + ATP -{- CoA + Sulfanilamide-~ Acetyl-Sulfanilamide ~- AMP ~- CoA + Pyrophosphate Assay Method Principle. In the enzymatic acetylation of sulfanilamide by a partially purified pigeon liver extract preparation, ~,~ acetate may be made the limiting factor2 This system has been adapted for the quantitative determination of small amounts of acetate. If proper care is taken to ensure acetate-free reactants, the system permits the estimation of 0.025 to 0.20 micromole of acetate in 0.24 ml. In this range the acetylation of sulfanilamide measured as a disappearance of sulfanilamide (Bratton and Marshal procedure 4) bears a linear or near-linear relationship to the acetate concentration. Reagents
0.02 M sulfanilamide. 0.1 N crystalline Na ATP. 0.1 M 1VIgC12. 1.0 M K citrate. CoA solution, 500 units/ml. Assay mixture. The above solutions are mixed in the following proportions, dispensed in approximately 3-ml. portions, and stored in the deep-freeze: 2 ml. of sulfanilamide, 3 ml. of ATP, N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174, 37 (1948). 2 N. O. Kaplan and F. Lipmann, Federation Proc. 6, 266 (1947). s M. Soodak and F. Lipmann, Federation Proc. 7, 190 (1948). 4A. C. Bratton and E. K. Marshall~ Jr., J. Biol. Chem. 128, 537 (1939).
ACETIC AND OTHER VOLATILE ACIDS
263
Mter cooling to 3 to 5 ° and standing for several hours, the fine crystalline precipitate is filtered out, washed with cold water, and dried as above.
[47] Microanalytical Method for Acetic and Other Volatile Acids By SIMON BLACK Principle. Volatile acids such as acetic diffuse from an acidified sample in the main chamber of a microdiffusion vessel to an alkaline solution in the center cup. After liberation of COs from the cup by an acid buffer, a microtitration is made. The latter measures volatile acid plus the excess of acid buffer over initial alkali. A blank titration must therefore be subtracted from each determination to obtain the volatile acid value. Although the method is nonspecific, some distinction between the several volatile fatty acids can be made on the basis of their distribution behavior between ether and water. Extraction of the acid into ether prior to its determination also serves to eliminate many interfering substances. Special Equipment Glass-stoppered centrifuge tubes, 30-ml. capacity. These may be obtained from the Wilkins-Anderson Co., 111 N. Canal St., Chicago, Ill., as Maizel-Gerson reaction vessels. A 10-ml. pipet with its tip bent 90 ° with respect to the pipet axis. The curved tip facilitates withdrawal of supernatant ether, leaving an aqueous layer undisturbed. Glass-stoppered Erlenmeyers, 50-ml. capacity. Center cups. These are made by cutting the lower 15-mm. portion from shell vials (15 mm. o.d.) which are obtainable from most scientific supply companies. They should be discarded after they have been used once or twice. For greater precision steel cups may be used.1 A microburet which delivers with an accuracy of +0.1 #l. A magnetic stirrer and a stirring bar made by sealing a 4-ram. length of iron wire in glass.
Reagents Alkaline trapping solution. Dissolve 0.124 g. of K2CO3-1.5 H20 plus 0.25 g. of KNO3 (to increase hygroscopicity) in distilled water, and dilute to 50 ml. 1S. Black, Arch. B/avhvm. 25~ 347 (1949).
264
CAR~OHYVRATES
[47]
80% H2S04. Add 400 ml. of concentrated H2S04 (c.p. or reagent) to 100 ml. of distilled water. 0.1 M monosodium maleate. Dilute 5 ml. of 1.0 N NaOH and 1.5 ml. of isoamyl alcohol (to reduce surface tension) plus 0.58 g. of maleic acid with distilled water to 50 mh 0.1 N standard NaOH. Indicator solution. Dilute 1 mh of phenolphthaiein indicator, 0.1% in 95 % alcohol, to 25 mh with CO~-free (boiled) distilled water. This solution should be made up on the day of use. 5 N H2S04. Alkaline Na2S04. Dissolve 20 g. of anhydrous Na~S04 (c.p. or reagent) plus 10 mh of 1 N NaOH and dilute to 100 ml. Sodium hydroxide solutions without Na~S04 do not remove fatty acids quantitatively from ether. Ethyl ether. This reagent should be of good quality, substantially free of peroxides. 10% sodium tungstate. Procedure. One milliliter of a copper-lime filtrate, ~ containing not more than 2 microequivalents of volatile acid, is pipetted into a glass-stoppered Erlenmeyer. One milliliter of 80% H2SO~ plus about 10 rag. of Ag2S04 (to retain chloride) are added, and the flask is shaken gently to ensure mixing. A center cup, containing 100.0 ~l. of the alkaline trapping solution, is placed centrally on the floor of the flask with the aid of forceps. The cup stands directly in the acidified sample. The glass stopper is put loosely in place (do not twist or "lock "), and the flask set in a 100 to 105 ° oven. After 14 to 16 hours the center cup is removed and 40.0 ~1. of monosodium maleate solution is added. The cup is then tilted gently, if necessary, to ensure distribution of this reagent over its bottom. After 10 to 20 minutes, during which CO2 escapes, about 0.3 ml. of indicator solution and a magnetic stirrer are added. A titration is then made in the cup, with stirring, with 0.1 N NaOH. The amount of volatile acid is calculated by substrating a blank titration value. When the amount of interfering materials make necessary a preliminary separation from the volatile acid, the following ether extraction procedure may be used: Three milliliters of a protein-free filtrate is placed in a 30-ml. glass-stoppered centrifuge tube. Two-tenths milliliter of 5 N H2S04 is added, 15 ml. of ether run in, and the tube shaken vigorously for 2 minutes. Ten milliliters of the supernatant ether is transferred with the special pipet to a second stoppered tube containing 1.2 ml. of the alkaline Na2SO~ solution. This tube is shaken for 1 minute. One milliliter of the A. L. Lehninger and S. W. Smith, J. Biol. Chem. 173, 773 (1948).
[47]
ACETIC AND OTHER VOLATILE ACIDS
265
aqueous layer is then transferred with a fine-tipped pipet to a 50-ml. Erlenmeyer flask, and the analysis carried out as described above b y the microdiffusion procedure. T o calculate the a m o u n t of volatile acid present in the original 3 ml. of filtrate, the q u a n t i t y found is divided b y the factor 0.55 multiplied b y whatever fraction of the acid in question the table indicates should be extracted b y this procedure. EXTRACTION OF VOLATILE ACIDS FROM WATER WITH ETHYL ETHER
I. 15 ml. of ether used to extract 3.2 ml. of aqueous solution2 II. 10 ml. of ether used to extract 10 ml. of aqueous solution. Per cent of acid found in ether layer Acid extracted Acetic Propionic Butyric Valeric Caproic Caprylic
I
II
71 87 95 98 100 100
32 60 83 93 98 100
The small amount of calcium in copper-lime filtrates causes additional retention in the aqueous layer of about 0.1 micromole of volatile acid; this amount is independent of the total volatile acid in the sample. Addition of 0.1 ml. of 10% Na~WO4 prior to extraction causes precipitation of the calcium (which need not be removed) and elimination of this source of error. Specificity. The method is nonspecific for volatile acids. However, the different distribution characteristics between water and ether (see the table) can be used as an aid in identification. I t is also possible to replace the titration procedure with a colorimetric test specific for acetic acid. ~ Numerous sources of interference with this colorimetric method are eliminated b y prior use of the microdiffusion procedure. A method for acetic acid which employs paper chromatography is also available. 4 Some interference is also given in the titrimetric procedure by lactic, f~-hydroxybutyric, and pyruvic acids. Diffusion of the latter can be eliminated by HgSO4 (about 10 mg.). When HgSO4 is used in conjunction with the extraction procedure, ether should first be eliminated b y drying the flask in an oven; 1 ml. of water plus solid HgSO4 are then added before the 80% H~SO4. Precision. K n o w n quantities of acetic acid can be recovered from tissue samples, b y means of the ether extraction and microdiffusion procedures, with an average deviation of 0.03 ~M.
O. Hutchens and B. M. Kass, J. Biol. Chem. 177, 571 (1949). 4 E. P. Kennedy and H. A. Barker, Anal. Chem. 23, 1033 (1951).
3 j.
266
CARBOHYDRATSS
[48]
Advantages and Disadvantages. The advantages of this method, compared to the Duclaux procedure, ~ are in the possibility of analyzing many samples simultaneously with simple apparatus, and in its suitability for small quantities of sample. Its low specificity, however, limits its usefulness in experiments involving crude extracts, or in other cases where the samples contain potential sources of interference. T. E. Friedeman, J. Biol. Chem. 123, 161 (1938). See Vol. III [61].
[48] An Enzymatic Micromethod for the Determination of Acetate B y MORRm SOODAK
Acetate + ATP -{- CoA + Sulfanilamide-~ Acetyl-Sulfanilamide ~- AMP ~- CoA + Pyrophosphate Assay Method Principle. In the enzymatic acetylation of sulfanilamide by a partially purified pigeon liver extract preparation, ~,~ acetate may be made the limiting factor2 This system has been adapted for the quantitative determination of small amounts of acetate. If proper care is taken to ensure acetate-free reactants, the system permits the estimation of 0.025 to 0.20 micromole of acetate in 0.24 ml. In this range the acetylation of sulfanilamide measured as a disappearance of sulfanilamide (Bratton and Marshal procedure 4) bears a linear or near-linear relationship to the acetate concentration. Reagents
0.02 M sulfanilamide. 0.1 N crystalline Na ATP. 0.1 M 1VIgC12. 1.0 M K citrate. CoA solution, 500 units/ml. Assay mixture. The above solutions are mixed in the following proportions, dispensed in approximately 3-ml. portions, and stored in the deep-freeze: 2 ml. of sulfanilamide, 3 ml. of ATP, N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174, 37 (1948). 2 N. O. Kaplan and F. Lipmann, Federation Proc. 6, 266 (1947). s M. Soodak and F. Lipmann, Federation Proc. 7, 190 (1948). 4A. C. Bratton and E. K. Marshall~ Jr., J. Biol. Chem. 128, 537 (1939).