Spectrophotometric Determination of Glycine in GIyci ne Potassium Trioxa Ia toc hromium(III) HORACE C. PERRIN and GEORGE H. SPAULDING Department o f Chemistry, Morgan State College, Baltimore, Md.
b The glycine in the complex reacts stoichiometrically and directly with copper phosphate, forming copper glycinate. The copper of this complex reacts with neocuproine to form a straw-colored solution with maximum absorption a t 457 mp.
S
methods of determining alpha-amino acids are based on the chelation (1) of cupric ion by the amino acids. I n all of them the amino acid which is brought into reaction with the cupric ion is in the free condition-Le., i t is not bound to another ion or molecule. I n a previous study (s), it was shown that the glycine of glycine potassium trioxalatochromiuni(II1) may be converted quantitatively to copper glycinate by reacting the glycine complex with a buffered copper phosphate suspension. It seemed possible that the copper in the copper glycinate might be determined spectrophotometrically with 2,9-dimethyl-l,lO-phenanthroline (neocuproine) (4). h borate-buffered solution of the copper glycine chelate is reacted directly with neocuproine under reducing conditions to form the strawcolored cuproin-copper complex. This complex obeys Beer’s law for low concentrations of copper. EVERAL
APPARATUS AND REAGENTS
All absorption measurements were made with a Bausch B: Lomb Spectronic20. Analytical reagent grade chemicals were used. Borate Buffer, prepared by the method of Blaedel and Todd (9). Boric acid (0.2M), 1Y sodium sulfate, is adjusted to p H 8.75 with 50% sodium hydroxide. For use, it is diluted 1 to 9. Copper Phosphate Stock Suspension ( 4 ) . Add 1 volume of trisodium phosphate solution (36.3 grams of Na3P04 1 2 H 2 0 per liter) t o 1 volume of cupric chloride solution (27.3 grams of CuClz.2 H 2 0 per liter). Then a d d 4 volumes of disodium phosphate solution (25.6 grams of NazHP04.7Hz0 per liter). Reflux the resulting slurry for 2 hours, permit it to age and settle for about 24 hours, then decant off the mother liquor. Store the remaining concentrated stock in a glass-stoppered bottle. Copper Phosphate Reagent (4). Centrifuge portions of t h e copper 196
ANALYTICAL CHEMISTRY
phosphate stock suspension and reject t h e mother liquor. Wash the residue with three consecutive portions of borate buffer diluted 1 t o 9 with Ivater. Use a stirring rod t o break u p t h e precipitate for each wash. After removal of the third wash portion, take u p the residue in 20 times its centrifuged volume of dilute borate buffer. Prepare this reagent fresh daily. Neocuproine Reagent. Prcpare 100 ml. by dissolving 7 5 mg. of neocuproine in diluted borate buffer solution. Hydroxylamine Hydrochloride Solution. Dissolve 10 grams of KH20H HC1 in 100 ml. of n a t e r . ilmmonium Hydroxide, 6111. Ethyl Alcohol, 95%. Potassium Trioxalatochromium (111) (KTCr) Solution. Dissolve 0.2 gram of the salt in 100 ml. of n a t e r . Standard Glycine Solution. Standardize an approximately 0.lM glycine solution iodometrically ( 3 ) . Blank Solution. Place 2 nil. of the potassium triovalatochromium(II1) solution in a 100-ml. volumetric flask, add 2 drops of phenolphthalein, and adjust t h e pH to approximately 8 (discharge of pink color n i t h very dilute acid). Add 10 ml. of t h e borate buffer and 10 ml. of the copper phosphate reagent, then dilute t o the mark. Allow the mixture to stand 30 minutes, shaking a t about 10-minute intervals. Centrifuge. Use the clear centrifugate as a blank in all measurements.
Solutions for Color Standards. GLYCINESTOCKSOLUTIOK.Measure 1.00 ml. of the standard solution of glycine into a 100-ml. volumetric flask, a d d 10 ml. of t h e K T C r solution, then dilute t o the mark. DEVELOPMENT OF COLOR. Measure into 100-ml. flasks 0.2-, 0.4-, 0.6-, 0.8-, and 1.0-ml. portions of the glycine stock solution. To each, add 10 ml. of water and 2 drops of phenolphthalein and adjust the p H until the first faint pink color is observed. Dilute with water to about 70 ml., then add 10 ml. of borate buffer and 10 ml. of the copper phosphate reagent, and dilute to the mark. Allow the mixture to stand 30 minutes, shaking a t about 10-minute intervals. Centrifuge. Transfer 3 ml. of the centrifuge to a 25-ml. volumetric flask and add 5 ml. of hydroxylamine hydrochloride solution. Adjust the p H to 4 to 6 with 6111 ammonium hydroxide, using p H paper. Add 3 ml. of the neocuproine solution, shake vigorously, then dilute to the mark with ethyl alcohol. Read the absorbance a t 457 mp against the prepared blank. Determination of Glycine in Glycine Potassium Trioxalatochromium(II1). Dissolve a sample of approximately 0.5 gram in about 20 ml. of water. Transfer it t o a 100-ml. volumetric flask and dilute to the mark. Measure 10 ml. of this solution into a 100-ml. flask and adjust the p H until the first faint pink color appears, using phenolphthalein as indicator. Continue as under Development of Color, starting with “Dilute with water to about 70 ml.”
Table 1. Determination of Glycine in Glycine Potassium Trioxalatochromium
RESULTS
Batch No.
KTCr.G,
1
45.45 55.85 65.80
h4g.
2
43.52 56.12 67.41
3
40.00 55.15 62.00
(111) Glycine Found, 70 SpectroBy photoKjeldahl metrically 14.18 14.26 14.20 14.28 Av. 14.22 12.08 12.12 12.01 12.00 .4v. 12.03 13.55 13.70 13.76 13.70 Av. 13.67
The results in Table I indicate that the glycine content of the glycine complex of potassium trioxalatochromium (111) may be determined spectrophotometrically through the use of neocuproine. DISCUSSION
I n preparing the neocuproine reagent in borate buffer solution, the p H should be adjusted to about 7 . 5 ; otherwise complete solution may not be obtained. I n some instances, during the development of the color, the solution was cloudy after the addition of the neocuproine followed by dilution with ethyl alcohol. A few drops of water
before reaching the mark Fill remedy this. In earl it^ work ( 4 ,the color of the trioxalatochromium(II1) ion interfered with the end point in the iodometric determination of copper. The incorporation of KTCr in the blank solution in approximately the same concentration as was present in the solutions
( 2 ) Blaedel, W. J., Todd, J. W., ANAL.
being measured corrected for this interference. This procedure is recommended for the determinat'ion of glycine in glycinecopper complex because of its simplicity and speed.
CHEM.32,1018(1960). (3) Schroeder, W.A., Kay, L. & Mills, 'I., R. S., Ibid., 22, 760 (1950). (4) Smith, G. F., McCurdy, W. H., Ibid., 24,371 (1952). (5) Spaulding, G. H., Ibid., 31, 1109 (1909).
LITERATURE CITED
(1) Albert, A., Biochem. J . 47, (1950) ; 50, 690 (1952).
531
RECEIVED for review September 27, 1961. Accepted November 30, 1961.
Ultraviolet Spectrophotometric Determination of Benzoic Acid in Refined Phthalic Anhydride RAITA MURNIEKS and C. E. GONTER Research and Development Department, Pittsburgh Chemical Co., Neville Island, Pittsburgh 25, Pa.
b Benzoic acid in chloroform can be determined spectrophotometrically at 274 mp. The determination requires complete removal of phthalic anhydride, phthalic acid, and 1,4-naphthoquinone, which also absorb strongly at this wavelength. Standard methods for separation of benzoic acid from phthalic anhydride were found inapplicable at concentrations of less than 0.570 benzoic acid. Good separation was achieved at pH 4.00 b y conversion of phthalic anhydride to the monosodium salt of phthalic acid followed by chloroform extraction. 1,4-Naphthoquinone can be removed b y acid potassium permanganate. The described method determines benzoic acid in refined phthalic anhydride in the concentration range below 0.5y0 with a relative error of =k2Oa/,.
0.1 -
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-
-
253
control of small amounts of impurities in refined phthalic anhydride (PAX) becomes increasingly important when the anhydride is used for the manufacture of plasticizers. Because the odor of the esters of benzoic acid is particularly objectionable, it is necessary to keep this acid impurity a t a minimum in the P=iA-i.e., less than 0.275, Ideally, a spectrophotometric procedure would appear to be the most practical for determining low concentrations of benzoic acid, since the acid shows some absorption throughout most of the ultraviolet region with a me11 defined peak of maximum absorption a t 274 mp. However, PAA, phthalic acid, and 1,4-naphthoquinone also show maximum absorption a t this wavelength (Figure 1) and must therefore be removed. Quantitative separation of amounts greater than 2% of benzoic acid from HE
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270
L%NE LEhGTY
Figure 1.
T
A
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-
U
3lG
233
330
MiLLIVCROhS
Absorption spectra in chloroform
- Benzoic acid - -.- - - - -. Phthalic acid -_1,4-Naphthoquinone phthalic acid can be effected by chloroform extraction and subsequent titration with standard alkali ( 2 ) . The separation proposed by Kappelmeier (3) and refined by Swann, Adams, and Weil (6) employing anhydrous potassium hydroxide is applicable below 2% but is not sufficiently sensitive for complete separation of less than 0.5% benzoic acid from PAA. Aforeover, no means could be found in this procedure for eliminating interference from the basic hydrolysis product of 1,4-naphthoquinone. The present investigation describes an ultraviolet spectrophotometric procedure for the determination of benzoic acid in P A S containing less than 0.5% benzoic acid and up to 50 p.p.m. of (5). Greater 1,4-naphthoquinone
amounts of the latter are removed by pretreatment with acidic potassium permanganate (4). APPARATUS AND REAGENTS
SDectroDhotometer. Beckman hfodel
DK'-2.
A
AbsorDtion cells. silica. 1.0-cm. 6N hydrochloric acid. ' Phthalic acid anhydride, recrystallized from chloroform. Benzoic acid, National Bureau of Standards. Benzoic acid standard solution, 1.0000 gram of NBS benzoic acid in 1 liter of distilled water. CALIBRATION PROCEDURE
Pipet 0.0 (reference), 2.0, 4.0, 6.0, and 8.0 ml. of benzoic acid standard VOL. 34, NO. 2, FEBRUARY 1962
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