- Email: [email protected]
Cleansing ninhydrin reaction coils in automatic amino acid analyzers
ANALYTICAL
Cleansing
BIOCHEMISTRY
88,332-333
Ninhydrin
(1978)
Reaction Coils in Automatic Acid Analyzers’
Amino
Ninhydrin solutions, which are used in automatic amino acid analysis (I), are light sensitive and develop a brownish tinge when exposed to air. It is, therefore, necessary to add antioxidants and store the ninhydrin reservoir in a dark container under nitrogen at low temperatures in order to preserve this reagent. Stannous chloride has been routinely used as a reducing agent. However, as a consequence of the addition of stannous chloride to ninhydrin reagent in the reservoir of a Beckman 121 automatic amino acid analyzer, a fine white precipitate progressively accumulates in the analyzer fluid lines and cuvettes and seriously interferes with quantification of amino acids. The precipitate is sparingly soluble or almost insoluble in water, which is generally used to flush the coil in most amino acid analyzers. Flushing the coil with 0.2 to 1 N NaOH does not reduce or eliminate the precipitate formed in the lines. Washing the fluid line system with methyl Cello-solve or an aqueous solution of Methyl Cellosolve (IO-50%) or ethyl alcohol (70-95%) partially frees the lines of the precipitate. However, washing with aqueous ethanol creates tiny air bubbles inside the lines, which eventually interfere with quantitative measurements. It was reported that titanous chloride (2,3), when used in ninhydrin solution, was beneficial in reducing the interfering precipitate in fluid lines and in stabilizing the baseline of the recorder. We have tried to replace stannous chloride with titanous chloride (Pierce Chemical Co.) as a reducing agent in ninhydrin solutions with little success. During the first 4 to 6 days, the color yield of ninhydrin solution is almost the same as that of stannous chloride, and there is only a minimal amount of color quenching. However, after 5 to 6 additional days, the reagent turns to dark brown, which seriously quenches the color development in the reaction coil. Sometimes the color yield drops as low as 50% that of the fresh ninhydrin solution. Occasional precipitation and subsequent blocking of fluid lines have also been observed, even when hydrindantin and methyl Cellosolve are used in the ninhydrin reagent (4,5). This problem can be reduced if dimethyl sulfoxide is used instead of methyl Cellosolve, but some equipment modifications are required to accommodate this solvent system (6). We have found that flushing the coil with 0.1 to 2 N HCl for 1 to 2 hr after the end of the run and/or before shutting down the instrument helps to ’ Supported
in part
by NIH
Grants
0003-2697/78/0881-0332$02.00/O Copyright 0 1978 by Academic Ress, Inc. All rights of reproduction in any form reserved.
5S07RR07097-13 332
and GM-19,972.
SHORT COMMUNICATIONS
333
remove all of the interfering particles in the instrument coil system. However, even though the precipitate is totally eliminated, this procedure creates tiny air bubbles, which accumulate in the pump valve and calorimeter systems and interfere with subsequent analyses. Other appropriate acids such as formic, dilute sulfuric, and dilute acetic acids have been tried. Dilute acetic acid (1 part glacial acetic and 3-4 parts water) gives excellent results in totally eliminating the precipitate from the fluid lines and does not result in air bubbles. We now routinely use dilute acetic acid as a solvent to flush the coil at the end of analysis or before shutting down of the instrument. We have made more than 1000 analyses in the last 12 months without encountering the problem of an interfering precipitate in the ninhydrin reagent in our Beckman 121 automatic amino acid analyzer. Our procedure totally eliminates the unwanted formation of precipitate in the ninhydrin reagent and thus permits use of stannous chloride as a reducing agent. REFERENCES 1. 2. 3. 4. 5.
Spackman, D. H., Stein. W. H., and Moore, S. (1958) Anal. Chem. 30, 1190-1206. James, L. B. (1971)J. Chromatogr. 59, 178-180. Pierce General Catalog (197611977) p. 49, Rockford, III. Moore, S. and Stein, W. H. (1954)J. Bid/. Chem. 211, 907-913. Schmidt, D. 1. (1966) Techniques in Amino Acid Analysis, p. 127-131, Technion Instruments Company, Chertsey, England. 6. Moore, S. (1968) J. Biol. Chem. 243, 6281-6283. S. GURUSIDDAIAH B. A. MCFADDEN
Bioanalyticul Center Troy Hall 304, Washington Stute University Pullman. Washington 99164 Received July 25, 1977; accepted
February
IO. 1978
Cleansing
BIOCHEMISTRY
88,332-333
Ninhydrin
(1978)
Reaction Coils in Automatic Acid Analyzers’
Amino
Ninhydrin solutions, which are used in automatic amino acid analysis (I), are light sensitive and develop a brownish tinge when exposed to air. It is, therefore, necessary to add antioxidants and store the ninhydrin reservoir in a dark container under nitrogen at low temperatures in order to preserve this reagent. Stannous chloride has been routinely used as a reducing agent. However, as a consequence of the addition of stannous chloride to ninhydrin reagent in the reservoir of a Beckman 121 automatic amino acid analyzer, a fine white precipitate progressively accumulates in the analyzer fluid lines and cuvettes and seriously interferes with quantification of amino acids. The precipitate is sparingly soluble or almost insoluble in water, which is generally used to flush the coil in most amino acid analyzers. Flushing the coil with 0.2 to 1 N NaOH does not reduce or eliminate the precipitate formed in the lines. Washing the fluid line system with methyl Cello-solve or an aqueous solution of Methyl Cellosolve (IO-50%) or ethyl alcohol (70-95%) partially frees the lines of the precipitate. However, washing with aqueous ethanol creates tiny air bubbles inside the lines, which eventually interfere with quantitative measurements. It was reported that titanous chloride (2,3), when used in ninhydrin solution, was beneficial in reducing the interfering precipitate in fluid lines and in stabilizing the baseline of the recorder. We have tried to replace stannous chloride with titanous chloride (Pierce Chemical Co.) as a reducing agent in ninhydrin solutions with little success. During the first 4 to 6 days, the color yield of ninhydrin solution is almost the same as that of stannous chloride, and there is only a minimal amount of color quenching. However, after 5 to 6 additional days, the reagent turns to dark brown, which seriously quenches the color development in the reaction coil. Sometimes the color yield drops as low as 50% that of the fresh ninhydrin solution. Occasional precipitation and subsequent blocking of fluid lines have also been observed, even when hydrindantin and methyl Cellosolve are used in the ninhydrin reagent (4,5). This problem can be reduced if dimethyl sulfoxide is used instead of methyl Cellosolve, but some equipment modifications are required to accommodate this solvent system (6). We have found that flushing the coil with 0.1 to 2 N HCl for 1 to 2 hr after the end of the run and/or before shutting down the instrument helps to ’ Supported
in part
by NIH
Grants
0003-2697/78/0881-0332$02.00/O Copyright 0 1978 by Academic Ress, Inc. All rights of reproduction in any form reserved.
5S07RR07097-13 332
and GM-19,972.
SHORT COMMUNICATIONS
333
remove all of the interfering particles in the instrument coil system. However, even though the precipitate is totally eliminated, this procedure creates tiny air bubbles, which accumulate in the pump valve and calorimeter systems and interfere with subsequent analyses. Other appropriate acids such as formic, dilute sulfuric, and dilute acetic acids have been tried. Dilute acetic acid (1 part glacial acetic and 3-4 parts water) gives excellent results in totally eliminating the precipitate from the fluid lines and does not result in air bubbles. We now routinely use dilute acetic acid as a solvent to flush the coil at the end of analysis or before shutting down of the instrument. We have made more than 1000 analyses in the last 12 months without encountering the problem of an interfering precipitate in the ninhydrin reagent in our Beckman 121 automatic amino acid analyzer. Our procedure totally eliminates the unwanted formation of precipitate in the ninhydrin reagent and thus permits use of stannous chloride as a reducing agent. REFERENCES 1. 2. 3. 4. 5.
Spackman, D. H., Stein. W. H., and Moore, S. (1958) Anal. Chem. 30, 1190-1206. James, L. B. (1971)J. Chromatogr. 59, 178-180. Pierce General Catalog (197611977) p. 49, Rockford, III. Moore, S. and Stein, W. H. (1954)J. Bid/. Chem. 211, 907-913. Schmidt, D. 1. (1966) Techniques in Amino Acid Analysis, p. 127-131, Technion Instruments Company, Chertsey, England. 6. Moore, S. (1968) J. Biol. Chem. 243, 6281-6283. S. GURUSIDDAIAH B. A. MCFADDEN
Bioanalyticul Center Troy Hall 304, Washington Stute University Pullman. Washington 99164 Received July 25, 1977; accepted
February
IO. 1978