- Email: [email protected]
Determination of tyrosine and phenylalanine by derivatization with nitric acid and differential pulse polarography
MICROCHEMICAL
JOURNAL
36, 113- 117 (1987)
Determination of Tyrosine and Phenylalanine by Derivatization with Nitric Acid and Differential Pulse Polarography G. RAMIS RAMOS,’ A. R. MAURI AUCEJO, M. C. GARCIA ALVAREZ-C• AND C. MONGAY FERNANDEZ Department
of Analytical
Chemistry,
Faculty of Chemistry, Valencia, Spain
University
of Valencia,
QUE,
Burjasot,
Received November 24, 1986; accepted February 7, 1987 The determination of tyrosine and phenylalanine by differential pulse polarography, after separation by thin-layer chromatography and derivatization with nitric acid to form the nitro compounds, is proposed. Experimental conditions for the derivatization treatment are established and the polarographic determination is optimized. o 1987 Academic press, IEX
Amino acid analysis usually involves chromatographic separation followed by a suitable derivatization reaction that permits the application of a given detection method. Good derivatization reactions must be simple, rapid, and selective. They must use inexpensive reagents and give easily detectable compounds with the usual techniques. These requirements are not entirely met by any reaction described and each has its own application fields (I). Aromatic compounds can be determined polarographically after derivatization with nitric acid (2). Methods have been proposed for the determination of mixtures of tyrosine, phenylalanine, and tryptophan using classic polarography (3-ZO).
In this work the use of differential pulse polarography (DPP) in the determination of tyrosine and phenylalanine after nitro derivatization is studied. Experimental conditions for the formation of the derivatives are established. EXPERIMENTAL Reagents and Apparatus
Analytical grade reagents and deionized and distilled water were used in all experiments. DL-Phenylalanine and L-tyrosine solutions were prepared shortly before use. DPP polarograms were obtained using a Metrohm E-506 apparatus provided with a time-controlled drop mercury electrode, a 3 M KC1 silver/silver chloride reference electrode (Qr = 0.196 V), and a Pt auxiliary electrode. Solutions (4 M) of acetic acid/sodium acetate, citric acid/sodium citrate, or ammonium chloride/ ammonia were used for pH control and as background electrolytes. A Crison 501 pH-meter provided with an Ingold combined glass electrode was used for pH 1 To whom correspondence
should be addressed. 113 0026-265X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
114
RAMOS ET AL.
measurements. Thin-layer chromatographic lulose sheets from Merck (Ref. 5552).
separations
were carried out on cel-
Procedures Samples for TLC were prepared by dissolving several amino acids in 5% HCl. Pure compounds and mixtures were spotted on the layers by means of a I-PI Hamilton syringe and were developed twofold using the n-butanol:acetic acid:water mixture (8:2:2 v/v/v) as the eluant. The substances were located using a UV lamp and the tyrosine and phenylalanine zones were stripped off into a funnel provided with a glass wool plug and washed with 20 ml of nitric acid to remove the amino acids from the adsorbent, which interferes with the polarographic determination. The solution was collected in a loo-ml spherical glass vessel and heat was applied. During the derivatization treatment, reflux was provided by using the same funnel atop the vessel. After this treatment the excess acid was neutralized with an equivalent amount of NaOH, and the solution was transferred to a loo-ml volumetric flask and diluted with water to the mark. Aliquots were taken and introduced into the polarographic cell together with the buffer solution. The polarograms were usually obtained at pH 9.5, using the NH&l/NH, buffer. Air was removed with a nitrogen stream and the following set of conditions was usually adopted: potential sweep in the cathodic direction at a speed of 10 mV/ set, negative potential pulses of 60 mV, and a drop lifetime oft,., = 0.4 sec. RESULTS
AND DISCUSSION
Polarograms In a first series of experiments, the amino acids were treated with 0.3 and 10 it4 boiling nitric acid for 15 and 90 min. The results are summarized in Table 1. When a high electrical sensitivity is used, a stepped and irregular baseline is obtained. However, if an appropriate baseline criterion is adopted, the sensitivity of the measurements can be kept constant at very low concentrations, even near TABLE 1 DPP Peaks of the Nitro Derivatives Treatment conditions 0.3 M nitric acid and 15-min boiling time 10 M nitric acid and 90-min boiling time
Peak potentials (V) and sensitivities (pA/ppm) Tyrosine -0.52 - 0.40 - 0.72 -0.48 -0.35
(0.12) (0.080) (0.050) (0.12) (0.029)
Phenylalanine No peaks -0.52 (0.080)
DETERMINATION
OF TYROSINE
AND PHENYLALANINE
115
the quantitation limit. The adopted criterion is shown in Fig. 1, where a straight line has been drawn between the minimum at the right of the peaks and the point at -0.68 V. Nitration
Conditions
Tyrosine can be nitrated at room temperature if concentrated nitric acid is used (at least a 10 A4 concentration). However, it takes more than 1 hr to reach a constant sensitivity. Using 0.3 M boiling nitric acid, 5 min of treatment is enough to obtain a constant sensitivity for the -0.52 V peak of tyrosine, whereas the -0.40 V peak appears more slowly. In this medium, both peaks reach the sensitivities indicated in Table 1; no further changes are produced with a longer boiling time, at least for 90 min. Similar results are obtained by using 0.15 and 1 A4 nitric acid, although with 0.15 M sensitivity increases very slowly and with 1 M 5 min is enough to obtain the maximum sensitivity for the two peaks. The reproducibility is worse in a 10 M nitric acid medium and when direct fire is used to heat the reaction vessel, a brown solid residue on the walls sometimes remains, which is never observed in diluted acid medium. On the other hand, 20 min of treatment with 10 M boiling nitric acid is needed to reach a constant sensitivity of 0.08 uA/ppm with phenylalanine.
b I - 0.6
- 0.6
!
- 0.4
-a2 E (VI
k -0.6
-0.6
I
-0.4
-0.2 E (‘4)
FIG. 1. Baseline criteria: (a) Tyrosine treated with 0.3 M nitric acid; (b) phenylalanine treated with 10 M nitric acid. Concentrations in the polarographic cell were 0.3 ppm in both cases.
116
RAMOS
ET AL.
At low amino acid concentrations the nitration reactions become slower and, thus, for given treatment conditions (a fixed acid concentration and boiling time) there is a minimum amino acid concentration below which the sensitivity falls quickly. This minimum for tyrosine using 0.3 M nitric acid and a 15-min treatment time is about 1.5 ppm (- 0.52 V peak). For phenylalanine using 10 M nitric acid and a 20-min treatment time the limit is about 4 ppm. These two sets of treatment conditions are adopted for the optimization of the polarographic determinations. The nitro derivatives obtained are very stable, and no losses of peak intensities are observed for at least 3 weeks. Reciprocal Influence of the Amino Acids during Treatment Experiments performed with a mixture of 50 pg of both substances using 0.3 and 1 M nitric acid indicated that the formation of tyrosine derivatives is inhibited by the presence of phenylalanine. No peaks were observed even after 15 min of treatment in 0.3 M acid medium, and the sensitivity fell to 50% in 1 M acid medium. When a great excess of phenylalanine, e.g., 100 to 600 times the amount of tyrosine, is used sensitivity losses are smaller, about 20%. Similarly, when a 10 M acid medium is used, a strong reciprocal influence that depends both on the phenylalanine/tyrosine ratio and on their concentrations is observed. Tryptophan and glycine produce similar effects on tyrosine. Therefore, the derivatives must be formed after a separation step. Optimum Conditions in the Determination by DPP Peak potentials shift linearly toward more negative values when pH increases. Tyrosine derivative peaks show 45 mV/pH-unity shifts, whereas the shift amounts to more than 60 mV/pH-unity for the phenylalanine peak. Sensitivity does not change with pH for any case. Linear losses of sensitivity amounting to 25 and 20% are observed for tyrosine and phenylalanine, respectively, when the potential sweep rate increases between 5 and 40 mV/sec. At the same time, peak potentials shift slightly in the direction of the sweep, that is, toward more negative values. Sensitivity increases linearly with tds, approximately 70% between 0.54 and 1.59 set@ (0.4 and 2 set drop lifetime) for both substances. Sensitivity and pulse amplitude are also linearly related below amplitudes of about 50 mV. The sensitivity/pulse amplitude ratio increases to a lesser extent in the pulse range 50100 mV. Analytical Figures of Merit Using the experimental conditions given for the procedure, the -0.52 V peak of tyrosine leads to a nearly linear calibration plot between 0.1 and 300 ppm. Average sensitivities are 0.126 and 0.101 pA/ppm in the ranges 0.1-3 and 3-300 ppm, respectively. On the other hand, phenylalanine has an excellent straight line with a slope of 0.078 pMppm in the same ranges. Reproducibility was established from series of six replicates, two series with 0.3 and 2.7 ppm of tyrosine and another two series with 0.6 and 2.9 ppm of phenylalanine. The four series lead to a standard deviation of about 0.002 PA. Using
DETERMINATION
OF TYROSINE
AND PHENYLALANINE
117
the 3-s criterion, the corresponding detection limits are 0.05 ppm for tyrosine and 0.08 ppm for phenylalanine. The whole procedure involving TLC, derivatization, and DPP was applied to a mixture of amino acids containing about 20 mg/ml of glycine, phenylalanine, and tyrosine. Ten experiments with OS-2 ~1 gave a coeffkient of variation of 2.5% for the determination of tyrosine. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Barret, G. C. (Ed.) Chemistry and Biochemistry of the Amino Acids. Chapman, London, 1985. Kolthoff, I. M.; Lingane, J. J. Polarography, Vol. 2, 2nd ed. Interscience, New York, 1952. Monnier, D.; Besso, 2. Helv. Chim. Acta, 1952, 35, 777-180. Monnier, D.; Besso, Z. Helv. Chim. Acta, 1951, 34, 1842-1848. Monnier, D.; Gueme, R. Anal. Chim. Acta, 1958, 19, 90- 100. Monnier, D.; Rusconi, Y. Helv. Chim. Acta, 1951, 34, 1297-1307. Monnier, D.; Rusconi, Y. Anal. Chim. Acta, 1952, I, 567. Monnier, D.; Vogel, J.; Wenger, P. E. Anal. Chim. Acta, 1960, 22, 369-383. Snell, F. D.; Ettre, B. S. (Eds.) Encyclopedia of Industrial Chemical Analysis, Vol. 8. Interscience, New York, 1969. 10. Wenger, P. E.; Monnier, D.; Vogel, J. Mikrochim. Acta, 1957, 405-416.
JOURNAL
36, 113- 117 (1987)
Determination of Tyrosine and Phenylalanine by Derivatization with Nitric Acid and Differential Pulse Polarography G. RAMIS RAMOS,’ A. R. MAURI AUCEJO, M. C. GARCIA ALVAREZ-C• AND C. MONGAY FERNANDEZ Department
of Analytical
Chemistry,
Faculty of Chemistry, Valencia, Spain
University
of Valencia,
QUE,
Burjasot,
Received November 24, 1986; accepted February 7, 1987 The determination of tyrosine and phenylalanine by differential pulse polarography, after separation by thin-layer chromatography and derivatization with nitric acid to form the nitro compounds, is proposed. Experimental conditions for the derivatization treatment are established and the polarographic determination is optimized. o 1987 Academic press, IEX
Amino acid analysis usually involves chromatographic separation followed by a suitable derivatization reaction that permits the application of a given detection method. Good derivatization reactions must be simple, rapid, and selective. They must use inexpensive reagents and give easily detectable compounds with the usual techniques. These requirements are not entirely met by any reaction described and each has its own application fields (I). Aromatic compounds can be determined polarographically after derivatization with nitric acid (2). Methods have been proposed for the determination of mixtures of tyrosine, phenylalanine, and tryptophan using classic polarography (3-ZO).
In this work the use of differential pulse polarography (DPP) in the determination of tyrosine and phenylalanine after nitro derivatization is studied. Experimental conditions for the formation of the derivatives are established. EXPERIMENTAL Reagents and Apparatus
Analytical grade reagents and deionized and distilled water were used in all experiments. DL-Phenylalanine and L-tyrosine solutions were prepared shortly before use. DPP polarograms were obtained using a Metrohm E-506 apparatus provided with a time-controlled drop mercury electrode, a 3 M KC1 silver/silver chloride reference electrode (Qr = 0.196 V), and a Pt auxiliary electrode. Solutions (4 M) of acetic acid/sodium acetate, citric acid/sodium citrate, or ammonium chloride/ ammonia were used for pH control and as background electrolytes. A Crison 501 pH-meter provided with an Ingold combined glass electrode was used for pH 1 To whom correspondence
should be addressed. 113 0026-265X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
114
RAMOS ET AL.
measurements. Thin-layer chromatographic lulose sheets from Merck (Ref. 5552).
separations
were carried out on cel-
Procedures Samples for TLC were prepared by dissolving several amino acids in 5% HCl. Pure compounds and mixtures were spotted on the layers by means of a I-PI Hamilton syringe and were developed twofold using the n-butanol:acetic acid:water mixture (8:2:2 v/v/v) as the eluant. The substances were located using a UV lamp and the tyrosine and phenylalanine zones were stripped off into a funnel provided with a glass wool plug and washed with 20 ml of nitric acid to remove the amino acids from the adsorbent, which interferes with the polarographic determination. The solution was collected in a loo-ml spherical glass vessel and heat was applied. During the derivatization treatment, reflux was provided by using the same funnel atop the vessel. After this treatment the excess acid was neutralized with an equivalent amount of NaOH, and the solution was transferred to a loo-ml volumetric flask and diluted with water to the mark. Aliquots were taken and introduced into the polarographic cell together with the buffer solution. The polarograms were usually obtained at pH 9.5, using the NH&l/NH, buffer. Air was removed with a nitrogen stream and the following set of conditions was usually adopted: potential sweep in the cathodic direction at a speed of 10 mV/ set, negative potential pulses of 60 mV, and a drop lifetime oft,., = 0.4 sec. RESULTS
AND DISCUSSION
Polarograms In a first series of experiments, the amino acids were treated with 0.3 and 10 it4 boiling nitric acid for 15 and 90 min. The results are summarized in Table 1. When a high electrical sensitivity is used, a stepped and irregular baseline is obtained. However, if an appropriate baseline criterion is adopted, the sensitivity of the measurements can be kept constant at very low concentrations, even near TABLE 1 DPP Peaks of the Nitro Derivatives Treatment conditions 0.3 M nitric acid and 15-min boiling time 10 M nitric acid and 90-min boiling time
Peak potentials (V) and sensitivities (pA/ppm) Tyrosine -0.52 - 0.40 - 0.72 -0.48 -0.35
(0.12) (0.080) (0.050) (0.12) (0.029)
Phenylalanine No peaks -0.52 (0.080)
DETERMINATION
OF TYROSINE
AND PHENYLALANINE
115
the quantitation limit. The adopted criterion is shown in Fig. 1, where a straight line has been drawn between the minimum at the right of the peaks and the point at -0.68 V. Nitration
Conditions
Tyrosine can be nitrated at room temperature if concentrated nitric acid is used (at least a 10 A4 concentration). However, it takes more than 1 hr to reach a constant sensitivity. Using 0.3 M boiling nitric acid, 5 min of treatment is enough to obtain a constant sensitivity for the -0.52 V peak of tyrosine, whereas the -0.40 V peak appears more slowly. In this medium, both peaks reach the sensitivities indicated in Table 1; no further changes are produced with a longer boiling time, at least for 90 min. Similar results are obtained by using 0.15 and 1 A4 nitric acid, although with 0.15 M sensitivity increases very slowly and with 1 M 5 min is enough to obtain the maximum sensitivity for the two peaks. The reproducibility is worse in a 10 M nitric acid medium and when direct fire is used to heat the reaction vessel, a brown solid residue on the walls sometimes remains, which is never observed in diluted acid medium. On the other hand, 20 min of treatment with 10 M boiling nitric acid is needed to reach a constant sensitivity of 0.08 uA/ppm with phenylalanine.
b I - 0.6
- 0.6
!
- 0.4
-a2 E (VI
k -0.6
-0.6
I
-0.4
-0.2 E (‘4)
FIG. 1. Baseline criteria: (a) Tyrosine treated with 0.3 M nitric acid; (b) phenylalanine treated with 10 M nitric acid. Concentrations in the polarographic cell were 0.3 ppm in both cases.
116
RAMOS
ET AL.
At low amino acid concentrations the nitration reactions become slower and, thus, for given treatment conditions (a fixed acid concentration and boiling time) there is a minimum amino acid concentration below which the sensitivity falls quickly. This minimum for tyrosine using 0.3 M nitric acid and a 15-min treatment time is about 1.5 ppm (- 0.52 V peak). For phenylalanine using 10 M nitric acid and a 20-min treatment time the limit is about 4 ppm. These two sets of treatment conditions are adopted for the optimization of the polarographic determinations. The nitro derivatives obtained are very stable, and no losses of peak intensities are observed for at least 3 weeks. Reciprocal Influence of the Amino Acids during Treatment Experiments performed with a mixture of 50 pg of both substances using 0.3 and 1 M nitric acid indicated that the formation of tyrosine derivatives is inhibited by the presence of phenylalanine. No peaks were observed even after 15 min of treatment in 0.3 M acid medium, and the sensitivity fell to 50% in 1 M acid medium. When a great excess of phenylalanine, e.g., 100 to 600 times the amount of tyrosine, is used sensitivity losses are smaller, about 20%. Similarly, when a 10 M acid medium is used, a strong reciprocal influence that depends both on the phenylalanine/tyrosine ratio and on their concentrations is observed. Tryptophan and glycine produce similar effects on tyrosine. Therefore, the derivatives must be formed after a separation step. Optimum Conditions in the Determination by DPP Peak potentials shift linearly toward more negative values when pH increases. Tyrosine derivative peaks show 45 mV/pH-unity shifts, whereas the shift amounts to more than 60 mV/pH-unity for the phenylalanine peak. Sensitivity does not change with pH for any case. Linear losses of sensitivity amounting to 25 and 20% are observed for tyrosine and phenylalanine, respectively, when the potential sweep rate increases between 5 and 40 mV/sec. At the same time, peak potentials shift slightly in the direction of the sweep, that is, toward more negative values. Sensitivity increases linearly with tds, approximately 70% between 0.54 and 1.59 set@ (0.4 and 2 set drop lifetime) for both substances. Sensitivity and pulse amplitude are also linearly related below amplitudes of about 50 mV. The sensitivity/pulse amplitude ratio increases to a lesser extent in the pulse range 50100 mV. Analytical Figures of Merit Using the experimental conditions given for the procedure, the -0.52 V peak of tyrosine leads to a nearly linear calibration plot between 0.1 and 300 ppm. Average sensitivities are 0.126 and 0.101 pA/ppm in the ranges 0.1-3 and 3-300 ppm, respectively. On the other hand, phenylalanine has an excellent straight line with a slope of 0.078 pMppm in the same ranges. Reproducibility was established from series of six replicates, two series with 0.3 and 2.7 ppm of tyrosine and another two series with 0.6 and 2.9 ppm of phenylalanine. The four series lead to a standard deviation of about 0.002 PA. Using
DETERMINATION
OF TYROSINE
AND PHENYLALANINE
117
the 3-s criterion, the corresponding detection limits are 0.05 ppm for tyrosine and 0.08 ppm for phenylalanine. The whole procedure involving TLC, derivatization, and DPP was applied to a mixture of amino acids containing about 20 mg/ml of glycine, phenylalanine, and tyrosine. Ten experiments with OS-2 ~1 gave a coeffkient of variation of 2.5% for the determination of tyrosine. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Barret, G. C. (Ed.) Chemistry and Biochemistry of the Amino Acids. Chapman, London, 1985. Kolthoff, I. M.; Lingane, J. J. Polarography, Vol. 2, 2nd ed. Interscience, New York, 1952. Monnier, D.; Besso, 2. Helv. Chim. Acta, 1952, 35, 777-180. Monnier, D.; Besso, Z. Helv. Chim. Acta, 1951, 34, 1842-1848. Monnier, D.; Gueme, R. Anal. Chim. Acta, 1958, 19, 90- 100. Monnier, D.; Rusconi, Y. Helv. Chim. Acta, 1951, 34, 1297-1307. Monnier, D.; Rusconi, Y. Anal. Chim. Acta, 1952, I, 567. Monnier, D.; Vogel, J.; Wenger, P. E. Anal. Chim. Acta, 1960, 22, 369-383. Snell, F. D.; Ettre, B. S. (Eds.) Encyclopedia of Industrial Chemical Analysis, Vol. 8. Interscience, New York, 1969. 10. Wenger, P. E.; Monnier, D.; Vogel, J. Mikrochim. Acta, 1957, 405-416.