A Spectrophotometric Method for Quantitative Determination of ...

In the Laboratory

A Spectrophotometric Method for Quantitative Determination of Bromine Using Tris(2-carboxyethyl)phosphine Joan Han, Troy Story, and Grace Han* Department of Chemistry, Morehouse College, Atlanta, GA 30314-3773

We have recently employed tris(2-carboxyethyl)phosphine (TCEP) for quantitation of hydrogen peroxide, iodine, iodate, and hypochlorite (1–3). We report here that TCEP is also useful for quantitation of bromine. This method is more sensitive and convenient than the standard method of bromine assay with iodide and thiosulfate (4, 5). In addition to its usefulness in research, this method is considered suitable for use in undergraduate student laboratories.

concentration of bromine initially present. Incubation beyond 20 s (up to 60 min) did not result in further oxidation of TCEP (data not shown), indicating that the stoichiometric oxidation of TCEP by bromine is essentially complete within 20 s. The reaction of TCEP with bromine was also investigated in 5 mM HCl and essentially the same results were obtained (data not shown). The data indicate that one molecule of TCEP reacts with one molecule of bromine: TCEP + Br2 + H2O → TCEPO + 2HBr

Materials and Methods 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) was obtained from Sigma Chemical Company (St. Louis, MO). Bromine, potassium iodide, and sodium thiosulfate volumetric standard (0.1 N solution in water) were purchased from Aldrich (Milwaukee, WI). Bromine dissolved in water was determined by reaction with excess potassium iodide followed by titration with standard sodium thiosulfate (4, 5). TCEP?HCl was obtained from Pierce Chemical Company (Rockford, IL). The concentration of TCEP was determined by measuring the amount of 2-nitro-5-thiobenzoate (NTB) formed after reaction with DTNB (6 ). TCEP reduces DTNB rapidly and stoichiometrically to generate two moles of NTB per mole of TCEP (Scheme I). A molar absorptivity (1-cm light path) of 14,150 at 412 nm for the NTB dianion (7) was used for all calculations.

Discussion The results of this study demonstrate that bromine can be conveniently determined by reaction with TCEP. In this method, the sample containing bromine is first incubated with an excess amount of TCEP, and the residual concentration of TCEP then determined by measuring the amount of NTB formed after reaction with DTNB (6 ). The concentration of bromine is equivalent to the decrease in the concentration of TCEP. This method does not require the use of a standard bromine solution because the assay is based on the well-established DTNB/NTB system. The method has a detection limit of about 0.5 µ M. The detection limit of this assay for bromine can be further decreased Table 1. Stoichiometric Reaction of Bromine with TCEP

COOCH2CH2COO-OOCCH CH P 2 2

NO2

S

+

CH2CH2COO-

Initial Concn in Mixture a

+

H 2O

Bromine

COODTNB (II)

3.00 mM

0.50 mM

2.48 ± 0.05 mM

1.04

3.00 mM

1.00 mM

2.06 ± 0.04 mM

0.94

3.00 mM

1.50 mM

1.45 ± 0.08 mM

1.03

Experiment II

CH2CH2COO-OOCCH CH P O 2 2 CH2CH2COOTCEPO (III)

COO-

+

2 -S

NO2

+

2H

+

Scheme I: Reduction of DTNB(II) by TCEP(I)

Results Changes in the concentration of TCEP after incubation with various concentrations of bromine were determined as described in the legend to Table 1. The results are shown in Table 1. In both experiments I and II, the concentration of TCEP was found to decrease by an amount equivalent to the 976

e

20.00 µM

1.00 µM

18.88 ± 0.65 µM

1.12

20.00 µM

3.00 µM

16.76 ± 0.54 µM

1.08

20.00 µM

6.00 µM

13.75 ± 0.49 µM

1.04

aReaction

NTB (IV)

Mol TCEP Oxidized/ Mol Br2c

Experiment I d

NO2

S

TCEP (I)

TCEP

Residual Concn of TCEP b

of TCEP with bromine was carried out at room temperature in 50 mM phosphate buffer (pH 7.5) containing 50 µM EDTA. bDetermined by reaction with DTNB (6) after incubation for 20 s. Mean ± SD of triplicate determinations. cCalculated by dividing the decrease in concentration of TCEP by the initial concentration of bromine. d10 µL of each reaction mixture was removed and incubated in a cuvette with 990 µL of 50 mM phosphate buffer (pH 7.5 containing 0.1 mM DTNB and 50 µM EDTA. The increase in absorbance at 412 nm was measured after 2 min. TCEP concentration was determined on the basis of amount of NTB formed as described in Materials and Methods. e900 µL of each reaction mixture was removed and incubated in a cuvette with 100 µL of 50 mM phosphate buffer (pH 7.5) containing 1.0 mM DTNB and 50 µM EDTA. The increase in absorbance at 412 nm was measured after 2 min. TCEP concentration was determined on the basis of amount of NTB formed as described in Materials and Methods.

Journal of Chemical Education • Vol. 76 No. 7 July 1999 • JChemEd.chem.wisc.edu

In the Laboratory

by about 40% if 4,4′-dithiodipyridine (DTDP) is used instead of DTNB because the reduced product of DTDP, 4-thiopyridone, has a molar absorptivity of 19,800 at 324 nm (8), compared with 14,150 for NTB at 412 nm (7). Acknowledgment This work was supported by NIH Grant S06GM45199. Literature Cited 1. Han, J. C.; Yen, S.; Han, G. Y.; Han, P. Anal. Biochem. 1996, 234, 107–109.

2. Han, J. C.; Payne, V.; Yen, S.; Han, G. Y.; Han, P. Anal. Biochem. 1996, 242, 150–152. 3. Han, J. C.; Chu, T. C.; Browne, J.; Brown, I.; Han, G. Y.; Han, P. Microchem. J. 1998, 58, 218–224. 4. Skoog, D. A.; West, D. M.; Holler, F. J. Fundamentals of Analytical Chemistry, 7th ed.; Saunders: Fort Worth, TX, 1995; pp 375–378. 5. Harris, D. C. Quantitative Chemical Analysis, 4th ed.; Freeman: New York, 1995; p 443. 6. Han, J. C.; Han, G. Y. Anal. Biochem. 1994, 220, 5–10. 7. Riddles, P. W.; Blakeley, R. L.; Zerner, B. Anal. Biochem. 1979, 94, 75–81. 8. Grassetti, D. R.; Murray, J. E. Arch. Biochem. Biophys. 1967, 119, 41–49.

† Incumbent of the Benno Gitter & Ilana Ben-Ami chair of Biotechology, Technion. Also in the Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037.

JChemEd.chem.wisc.edu • Vol. 76 No. 7 July 1999 • Journal of Chemical Education

977