- Email: [email protected]
A comparison of pulsed amperometric detection and conductivity detection of underivatized amino-acids in liquid chromatography
Talanta.Vol. 37, No. 4, pp. 377-380, 1990 Printedin Great Britain. All rights reserved
0039-9140/90 53.00 + 0.00 Copyright 0 1990 Pergamon Press plc
A COMPARISON OF PULSED AMPEROMETRIC DETECTION AND CONDUCTIVITY DETECTION OF UNDERIVATIZED AMINO-ACIDS IN LIQUID CHROMATOGRAPHY LAWRENCE E. WELCH*
Department of Chemistry, Knox College, Galesburg, IL 61401-4999, U.S.A. WILLIAM R. LACOURSE, DAVID A. MEAD, JR.?
and
DENNIS C. JOHNSON
Department of Chemistry, Iowa State University, Ames, IA 50011, U.S.A. (Received 26 July 1989. Accepted I September
1989)
Summary-Pulsed amperometric detection (PAD) in tandem with conductivity detection (CD) has been applied to the direct detection of amino-acids by liquid chromatography. Although sensitive, PAD has a limited linear range of response. Sequential use of conductimetric detection and then PAD extends the dynamic range of amino-acid determination,
Recent progress in amino-acid determination can be attributed to technological advances in liquid chromatography (LC) and chromatographic detectors. Cation-exchange stationary phases’ have long been the standard for LC separations of amino-acids, and the use of anion-exchange separations* is more recent. Numerous pre-column derivatization schemes produce adducts that can be separated by reversed-phase stationary phases.3 Post-column derivatization is employed in most ion-exchange methods to overcome the lack of response of traditional LC detectors towards amino-acids. Alternatively, “indirect” methods can be used.4 Direct and sensitive amino-acid detection without a derivatization step is a desirable further alternative to traditional methods. Pulsed amperometric detections,6 uses a triplestep potential waveform to combine amperometric detection with alternating anodic and cathodic polarizations to clean and reactivate the electrode surface. This waveform exploits the surface-catalyzed oxidation of the amine group, activated by the transient formation of surface oxides on noble metals. Use of pulsed amperometric detection following liquid chromatography has gained prominence as a selective and sensitive technique for the determination of alcohols, polyalcohols, carbo*Author for correspondence. ?Commonwealth Edison, Maywood, IL 66046, U.S.A.
hydrates,‘-” amino-alkanols,” many inorganic and organic sulfur-containing compounds’* and amino-acids.13 Although sensitive, amino-acid determinations by use of PAD exhibit a limited linear response range. Conductimetric detection has been utilized successfully in ion-chromatography for the determination of inorganic and organic ions.‘“‘6 Suppressed CD of amino-acids has been shown to be feasible in work on alternative ionchromatographic eluents,” but no amino-acid separations were attempted. Recently, Johnson ef al. applied unsuppressed CD in tandem with PAD for carbohydrate determinations.” The dual detection provided linear calibration over a wide dynamic range. This paper compares the application of PAD and CD to the simple, direct determination of amino-acids by isocratic chromatography. No suppressor columns were used with the CD system. In addition, CD was applied before PAD, to provide a non-destructive method of extending the linear range of amino-acid determination. EXPERIMENTAL
Reagents All solutions were prepared from reagent grade chemicals. Amino-acid standards were obtained from Aldrich Co. (Milwaukee, WI), Fisher Scientific Co. (Springfield, NJ), and 377
LAWRENCE E. WELCHet al.
378
Pierce (Rockford, IL). L-Lysine and Amino Acid Complex dietary supplements were obtained from American Dietary Laboratories (Pasadena, CA). Water was purified either in a MILLI-Q system (Millipore Corp., Milford, MA) or a Barnstead NANOpure II system (Newton, MA), followed by filtration (0.2 pm). Apparatus
Chromatographic separations were performed with an AS-6 anion-exchange column preceded by an AG-6 guard column in an isocratic liquid chromatography system (Dionex Corp., Sunnyvale, CA). All mobile phases were filtered with 0.45 pm Nylon-66 filters (Rainin Corp., Woburn, MA) and a solvent filtration kit (Millipore) before use. The injection volume was 50 /ll. PAD was performed with the Model PAD-2 electrochemical detector (Dionex) with homemade gold and platinum flow-through cells.19 A saturated calomel reference electrode and a platinum wire counter-electrode were used. Conductimetric detection was done with the CD module from a Model-10 Ion Chromatograph (Dionex). For tandem CD and PAD experiments, the detector cells were placed in series, with the conductivity detector cell first. Procedure
Calibration data were plotted as log[(A, - a)/ bC] t’s. log concentration,20 where A, is the peak area, C the analyte concentration and a and b are the intercept and slope obtained from modified regression analysis2’ of the linear region of a plot of A,, us. C. Accordingly, for data which fall exactly on the regression line, log[(A, - a)/bC] = 0.0. This plotting scheme has several advantages over the traditional plotting of A, us. C, as follows: relative deviations from the regression line are quantitatively depicted throughout the concentration range, the upper (and lower) limits of linearity are readily apparent, and calibration data for two or more detection techniques can be compared easily on the same relative scale. RESULTS AND DISCUSSION
Pulsed amperometric detection
Pulsed amperometric detection for aminoacids at gold electrodes in basic solutions was successful when a three-step potential waveform was used. The anodic current was sampled for 16.7 msec following a 540 msec delay at 0.5 V.
This detection step was followed by an anodic potential pulse to 1.05 V for 180 msec and then a cathodic pulse to -0.55 V for 240 msec. The anodic detection mechanism for lysine is believed to be electrocatalytic, requiring simultaneous formation of a surface oxide.13 All other essential amino-acids display electrochemical behavior nearly identical to that of lysine. Chromatography
For glycine, pK, of the carboxyl group is 2.35 and pK, of the amine group is 9.778.*’ Other amino-acid pK, values are similar to these.23 The traditional zwitterion form of glycine observed under neutral conditions is not present in the basic chromatographic eluents used in this work. At high pH the amine group is deprotonated, resulting in an anionic form suitable for separation by anion-exchange chromatography. Sodium hydroxide solution was used as an eluent since it provided the necessary high pH as well as serving as an excellent electrolyte for the electrochemical detector. Two dietary supplement pills were analyzed to illustrate the application of PAD to aminoacids. The contents of an L-lysine capsule were dissolved, and the solution was diluted and an aliquot injected without further pretreatment, along with a series of standards. The lysine content was found to be within 6% of the nominal value (625 mg). The contents of a capsule of Amino Acid Complex were analyzed in a similar manner: the chromatogram (with PAD) is shown in Fig. 1. No sample pretreatment was applied other than dissolution. The dissolution of atmospheric carbon dioxide in the sodium hydroxide eluent proved problematic when CD was used. A similar problem was encountered during CD of carbohydrates,‘* which was solved by using barium hydroxide instead of sodium hydroxide. Barium hydroxide proved to be the eluent of choice for amino-acids as well, and concentrations of I-5mM gave the best results. Conductimetric detection
The applicability of CD for amino-acids is due to the difference between the limiting equivalent ionic conductances (LEIC) of the eluting anion and the analyte anion. Hydroxide ions have a very high LEIC (198 phmo .cm2),24 whereas the values for amino-acid anions are much lower.” The elution of the anion of an amino-acid, with simultaneous sorption of OH-, results in a decrease in CD response to
Liquid chromatography
a
TimeFig. 1. Chromatogram for Amino Acid Complex. Conditions: contents of a single capsule diluted to 5 I., 50-p] injection, eluent 50mM NaOH at 1.0 ml/min, pulsed amperometric detection. Peaks: (a) arginine, (b) lysine, (c) leucine, (d) phenylalanine.
give a “negative” peak. The separation of a mixture of lysine, methionine, asparagine and glycine is shown in Fig. 2.
of amino-acids
379
pmho.sec, slope 2.71 x lo5 ~mho.sec.l.mole-‘, S, = 0.0002 x 105) throughout the range O.l-1OmM studied, with a higher limit of linearity (LOL) but a poorer limit of detection (LOD), compared to PAD. The two detectors used in tandem offer linear detection over more than three decades of concentration. The estimated limit of detection (S/N = 3) for lysine by PAD at a gold electrode was 200 ng/ml (10 ng, 70 pmole). Response for other primary amino-acids was between 0.3 and 3 times that for lysine. The secondary amino-acids, hydroxyproline and proline, also gave reasonable PAD detection limits (3 and 4@t4 respectively). The limit of detection for lysine by CD was 2 pg/ml (100 ng, 700 pmole). The coupling of PAD at a platinum electrode and CD has been shown to work satisfactorily as well, but this system was not studied extensively. The long-term stability of the PAD response was confirmed by injecting a lysine solution every hour for 6 hr. The responses showed only a 1.3% relative standard deviation over this time span. CONCLUSION
Analytical response
The calibration data for lysine obtained by PAD and CD are shown in Fig. 3. The PAD response for lysine was linear (intercept 1.77 PC, slope =’ 6.79 x IO4 pC.l.mole-I, S,, = 1.6 x 10e3) over nearly one decade, with significant deviation from linearity for concentrations greater than 0.1 1OmM. The CD response was linear (intercept = -0.872
A
The determination of underivatized aminoacids by anion-exchange separation with pulsed amperometric detection is direct, sensitive and simple, with detection limits superior to those obtained by spectrophotometric detection of ninhydrin adducts. PAD is much more sensitive than CD but has a response which deviates from linearity for concentrations above ca. O.lmM. Use of the two detectors in tandem can give a
t; T
Time -
.L
.E
a
t
T
e
Time -
I
Fig. 2. Sequential chromatograms obtained by CD and PAD. Conditions: eluent 2.0mM Ba(OH), at 1.0 ml/min. Detection: (A) conductivity, (B) pulsed amperometry. Peaks: (a) sample matrix, (b) 2.2 pg of lysine, (c) 2.8 ng of methionine, (d) 2.5 pg of asparagine, (e) 2.9 pg of glycine.
LAWRENCEE. WELCH et al.
380
i? -0.5 -
(PAD) LOD
LOD (CD)
I -0.7 L ' -6
1 -5
I -3
I -4
0
I -2
log (concentration,Ml Fig. 3. Calibration plots for lysine. Conditions: eluent 2.OmM Ba(OH), at 1.0 ml/mitt. + Conductivity detection, l pulsed amperomettic detection. The solid horizontal line represents log[(A, - a)/bC] = 0. The parallel dashed lines delineate relative errors (A) of 10, 30 and 50%.
combined linear dynamic decades for lysine.
range of cu. three
Acknowledgement-This research was supported by a grant from Dionex Corp., Sunnyvale, CA.
REFERENCES 1. G. Ogden and P. Foldi, LC-GC, 1987, 5, 28. 2. J. A. Polta and D. C. Johnson, J. Liq. Chromatog., 1983, 6, 1727. 3. D. W. Hill, F. H. Walters, T. D. Wilson and J. D. Stuart, Anal. Chem., 1979, 51, 1338. 4. W. G. Kuhr and E. S. Yeung, ibid., 1988, 60, 1832. 5. S. Hughes, P. L. Meschi and D. C. Johnson, Anal. Chim. Acta, 1981, 132, 1. 6. J. A. Polta, Ph.D. Dissertation, Iowa State University, 1985. I. S. Hughes and D. C. Johnson, Anal. Chim. Acta, 1981, 132, 11. 8. P. Edwards and K. K. Haak, Am. Lab., 1983, 15, No. 4, 78.
9. R. D. Rocklin and C. A. Pohl, J. Liq. Chromatog., 1983, 6, 1577.
10. D. C. Johnson, Nature, 1986, 321, 451.
11. W. R. LaCourse, W. A. Jackson and D. C. Johnson,
Anal. Chem., submitted for publication. 12. D. C. Johnson and T. Z. Polta, Chromatog. Forum, 1986, 1, 37. 13. L. E. Welch, W. R. LaCourse, D. A. Mead, Jr., D. C. Johnson and T. Hu, Anal. Chem., 1989, 61, 555. 14. H. Small, T. Stevens and W. Bauman, ibid., 1975, 47, 1801.
15. D. Gjerde, J. S. Fritz and G. J. Schmuckler, J. Chromatog., 1979, 186, 509. 16. J. S. Fritz and K. Tanaka, ibid., 1987, 409, 271. 17. 0. A. Shpigun, L. N. Voloshik and Yu. A. Zolotov, Anal. Sci., 1985, 1, 335. 18. L. E. Welch, D. A. Mead and D. C. Johnson, Anal. Chim. Acta, 1988, 204, 323.
19. T. Z. Polta, Ph.D. Dissertation, Iowa State University, 1986. 20. D. C. Johnson, Anal. Chim. Acta, 1988, 204, 1. 21. M. Naturella, Experimental Statistics, pp. 6-19. NBS, Washington, DC, 1963. 22. L. G. Hargis, Analytical Chemistry, Prentice-Hall, Englewood Cliffs, NJ, 1988. 23. A. Martell and R. Smith, Critical Stability Constants, Vol. 3, Plenum Press, New York, 1977. 24. R. C. Weast (ed.), Handbook of Chemistry and Physics, 65th Ed., CRC Press, Boca Raton, FL, 1984-5.
0039-9140/90 53.00 + 0.00 Copyright 0 1990 Pergamon Press plc
A COMPARISON OF PULSED AMPEROMETRIC DETECTION AND CONDUCTIVITY DETECTION OF UNDERIVATIZED AMINO-ACIDS IN LIQUID CHROMATOGRAPHY LAWRENCE E. WELCH*
Department of Chemistry, Knox College, Galesburg, IL 61401-4999, U.S.A. WILLIAM R. LACOURSE, DAVID A. MEAD, JR.?
and
DENNIS C. JOHNSON
Department of Chemistry, Iowa State University, Ames, IA 50011, U.S.A. (Received 26 July 1989. Accepted I September
1989)
Summary-Pulsed amperometric detection (PAD) in tandem with conductivity detection (CD) has been applied to the direct detection of amino-acids by liquid chromatography. Although sensitive, PAD has a limited linear range of response. Sequential use of conductimetric detection and then PAD extends the dynamic range of amino-acid determination,
Recent progress in amino-acid determination can be attributed to technological advances in liquid chromatography (LC) and chromatographic detectors. Cation-exchange stationary phases’ have long been the standard for LC separations of amino-acids, and the use of anion-exchange separations* is more recent. Numerous pre-column derivatization schemes produce adducts that can be separated by reversed-phase stationary phases.3 Post-column derivatization is employed in most ion-exchange methods to overcome the lack of response of traditional LC detectors towards amino-acids. Alternatively, “indirect” methods can be used.4 Direct and sensitive amino-acid detection without a derivatization step is a desirable further alternative to traditional methods. Pulsed amperometric detections,6 uses a triplestep potential waveform to combine amperometric detection with alternating anodic and cathodic polarizations to clean and reactivate the electrode surface. This waveform exploits the surface-catalyzed oxidation of the amine group, activated by the transient formation of surface oxides on noble metals. Use of pulsed amperometric detection following liquid chromatography has gained prominence as a selective and sensitive technique for the determination of alcohols, polyalcohols, carbo*Author for correspondence. ?Commonwealth Edison, Maywood, IL 66046, U.S.A.
hydrates,‘-” amino-alkanols,” many inorganic and organic sulfur-containing compounds’* and amino-acids.13 Although sensitive, amino-acid determinations by use of PAD exhibit a limited linear response range. Conductimetric detection has been utilized successfully in ion-chromatography for the determination of inorganic and organic ions.‘“‘6 Suppressed CD of amino-acids has been shown to be feasible in work on alternative ionchromatographic eluents,” but no amino-acid separations were attempted. Recently, Johnson ef al. applied unsuppressed CD in tandem with PAD for carbohydrate determinations.” The dual detection provided linear calibration over a wide dynamic range. This paper compares the application of PAD and CD to the simple, direct determination of amino-acids by isocratic chromatography. No suppressor columns were used with the CD system. In addition, CD was applied before PAD, to provide a non-destructive method of extending the linear range of amino-acid determination. EXPERIMENTAL
Reagents All solutions were prepared from reagent grade chemicals. Amino-acid standards were obtained from Aldrich Co. (Milwaukee, WI), Fisher Scientific Co. (Springfield, NJ), and 377
LAWRENCE E. WELCHet al.
378
Pierce (Rockford, IL). L-Lysine and Amino Acid Complex dietary supplements were obtained from American Dietary Laboratories (Pasadena, CA). Water was purified either in a MILLI-Q system (Millipore Corp., Milford, MA) or a Barnstead NANOpure II system (Newton, MA), followed by filtration (0.2 pm). Apparatus
Chromatographic separations were performed with an AS-6 anion-exchange column preceded by an AG-6 guard column in an isocratic liquid chromatography system (Dionex Corp., Sunnyvale, CA). All mobile phases were filtered with 0.45 pm Nylon-66 filters (Rainin Corp., Woburn, MA) and a solvent filtration kit (Millipore) before use. The injection volume was 50 /ll. PAD was performed with the Model PAD-2 electrochemical detector (Dionex) with homemade gold and platinum flow-through cells.19 A saturated calomel reference electrode and a platinum wire counter-electrode were used. Conductimetric detection was done with the CD module from a Model-10 Ion Chromatograph (Dionex). For tandem CD and PAD experiments, the detector cells were placed in series, with the conductivity detector cell first. Procedure
Calibration data were plotted as log[(A, - a)/ bC] t’s. log concentration,20 where A, is the peak area, C the analyte concentration and a and b are the intercept and slope obtained from modified regression analysis2’ of the linear region of a plot of A,, us. C. Accordingly, for data which fall exactly on the regression line, log[(A, - a)/bC] = 0.0. This plotting scheme has several advantages over the traditional plotting of A, us. C, as follows: relative deviations from the regression line are quantitatively depicted throughout the concentration range, the upper (and lower) limits of linearity are readily apparent, and calibration data for two or more detection techniques can be compared easily on the same relative scale. RESULTS AND DISCUSSION
Pulsed amperometric detection
Pulsed amperometric detection for aminoacids at gold electrodes in basic solutions was successful when a three-step potential waveform was used. The anodic current was sampled for 16.7 msec following a 540 msec delay at 0.5 V.
This detection step was followed by an anodic potential pulse to 1.05 V for 180 msec and then a cathodic pulse to -0.55 V for 240 msec. The anodic detection mechanism for lysine is believed to be electrocatalytic, requiring simultaneous formation of a surface oxide.13 All other essential amino-acids display electrochemical behavior nearly identical to that of lysine. Chromatography
For glycine, pK, of the carboxyl group is 2.35 and pK, of the amine group is 9.778.*’ Other amino-acid pK, values are similar to these.23 The traditional zwitterion form of glycine observed under neutral conditions is not present in the basic chromatographic eluents used in this work. At high pH the amine group is deprotonated, resulting in an anionic form suitable for separation by anion-exchange chromatography. Sodium hydroxide solution was used as an eluent since it provided the necessary high pH as well as serving as an excellent electrolyte for the electrochemical detector. Two dietary supplement pills were analyzed to illustrate the application of PAD to aminoacids. The contents of an L-lysine capsule were dissolved, and the solution was diluted and an aliquot injected without further pretreatment, along with a series of standards. The lysine content was found to be within 6% of the nominal value (625 mg). The contents of a capsule of Amino Acid Complex were analyzed in a similar manner: the chromatogram (with PAD) is shown in Fig. 1. No sample pretreatment was applied other than dissolution. The dissolution of atmospheric carbon dioxide in the sodium hydroxide eluent proved problematic when CD was used. A similar problem was encountered during CD of carbohydrates,‘* which was solved by using barium hydroxide instead of sodium hydroxide. Barium hydroxide proved to be the eluent of choice for amino-acids as well, and concentrations of I-5mM gave the best results. Conductimetric detection
The applicability of CD for amino-acids is due to the difference between the limiting equivalent ionic conductances (LEIC) of the eluting anion and the analyte anion. Hydroxide ions have a very high LEIC (198 phmo .cm2),24 whereas the values for amino-acid anions are much lower.” The elution of the anion of an amino-acid, with simultaneous sorption of OH-, results in a decrease in CD response to
Liquid chromatography
a
TimeFig. 1. Chromatogram for Amino Acid Complex. Conditions: contents of a single capsule diluted to 5 I., 50-p] injection, eluent 50mM NaOH at 1.0 ml/min, pulsed amperometric detection. Peaks: (a) arginine, (b) lysine, (c) leucine, (d) phenylalanine.
give a “negative” peak. The separation of a mixture of lysine, methionine, asparagine and glycine is shown in Fig. 2.
of amino-acids
379
pmho.sec, slope 2.71 x lo5 ~mho.sec.l.mole-‘, S, = 0.0002 x 105) throughout the range O.l-1OmM studied, with a higher limit of linearity (LOL) but a poorer limit of detection (LOD), compared to PAD. The two detectors used in tandem offer linear detection over more than three decades of concentration. The estimated limit of detection (S/N = 3) for lysine by PAD at a gold electrode was 200 ng/ml (10 ng, 70 pmole). Response for other primary amino-acids was between 0.3 and 3 times that for lysine. The secondary amino-acids, hydroxyproline and proline, also gave reasonable PAD detection limits (3 and 4@t4 respectively). The limit of detection for lysine by CD was 2 pg/ml (100 ng, 700 pmole). The coupling of PAD at a platinum electrode and CD has been shown to work satisfactorily as well, but this system was not studied extensively. The long-term stability of the PAD response was confirmed by injecting a lysine solution every hour for 6 hr. The responses showed only a 1.3% relative standard deviation over this time span. CONCLUSION
Analytical response
The calibration data for lysine obtained by PAD and CD are shown in Fig. 3. The PAD response for lysine was linear (intercept 1.77 PC, slope =’ 6.79 x IO4 pC.l.mole-I, S,, = 1.6 x 10e3) over nearly one decade, with significant deviation from linearity for concentrations greater than 0.1 1OmM. The CD response was linear (intercept = -0.872
A
The determination of underivatized aminoacids by anion-exchange separation with pulsed amperometric detection is direct, sensitive and simple, with detection limits superior to those obtained by spectrophotometric detection of ninhydrin adducts. PAD is much more sensitive than CD but has a response which deviates from linearity for concentrations above ca. O.lmM. Use of the two detectors in tandem can give a
t; T
Time -
.L
.E
a
t
T
e
Time -
I
Fig. 2. Sequential chromatograms obtained by CD and PAD. Conditions: eluent 2.0mM Ba(OH), at 1.0 ml/min. Detection: (A) conductivity, (B) pulsed amperometry. Peaks: (a) sample matrix, (b) 2.2 pg of lysine, (c) 2.8 ng of methionine, (d) 2.5 pg of asparagine, (e) 2.9 pg of glycine.
LAWRENCEE. WELCH et al.
380
i? -0.5 -
(PAD) LOD
LOD (CD)
I -0.7 L ' -6
1 -5
I -3
I -4
0
I -2
log (concentration,Ml Fig. 3. Calibration plots for lysine. Conditions: eluent 2.OmM Ba(OH), at 1.0 ml/mitt. + Conductivity detection, l pulsed amperomettic detection. The solid horizontal line represents log[(A, - a)/bC] = 0. The parallel dashed lines delineate relative errors (A) of 10, 30 and 50%.
combined linear dynamic decades for lysine.
range of cu. three
Acknowledgement-This research was supported by a grant from Dionex Corp., Sunnyvale, CA.
REFERENCES 1. G. Ogden and P. Foldi, LC-GC, 1987, 5, 28. 2. J. A. Polta and D. C. Johnson, J. Liq. Chromatog., 1983, 6, 1727. 3. D. W. Hill, F. H. Walters, T. D. Wilson and J. D. Stuart, Anal. Chem., 1979, 51, 1338. 4. W. G. Kuhr and E. S. Yeung, ibid., 1988, 60, 1832. 5. S. Hughes, P. L. Meschi and D. C. Johnson, Anal. Chim. Acta, 1981, 132, 1. 6. J. A. Polta, Ph.D. Dissertation, Iowa State University, 1985. I. S. Hughes and D. C. Johnson, Anal. Chim. Acta, 1981, 132, 11. 8. P. Edwards and K. K. Haak, Am. Lab., 1983, 15, No. 4, 78.
9. R. D. Rocklin and C. A. Pohl, J. Liq. Chromatog., 1983, 6, 1577.
10. D. C. Johnson, Nature, 1986, 321, 451.
11. W. R. LaCourse, W. A. Jackson and D. C. Johnson,
Anal. Chem., submitted for publication. 12. D. C. Johnson and T. Z. Polta, Chromatog. Forum, 1986, 1, 37. 13. L. E. Welch, W. R. LaCourse, D. A. Mead, Jr., D. C. Johnson and T. Hu, Anal. Chem., 1989, 61, 555. 14. H. Small, T. Stevens and W. Bauman, ibid., 1975, 47, 1801.
15. D. Gjerde, J. S. Fritz and G. J. Schmuckler, J. Chromatog., 1979, 186, 509. 16. J. S. Fritz and K. Tanaka, ibid., 1987, 409, 271. 17. 0. A. Shpigun, L. N. Voloshik and Yu. A. Zolotov, Anal. Sci., 1985, 1, 335. 18. L. E. Welch, D. A. Mead and D. C. Johnson, Anal. Chim. Acta, 1988, 204, 323.
19. T. Z. Polta, Ph.D. Dissertation, Iowa State University, 1986. 20. D. C. Johnson, Anal. Chim. Acta, 1988, 204, 1. 21. M. Naturella, Experimental Statistics, pp. 6-19. NBS, Washington, DC, 1963. 22. L. G. Hargis, Analytical Chemistry, Prentice-Hall, Englewood Cliffs, NJ, 1988. 23. A. Martell and R. Smith, Critical Stability Constants, Vol. 3, Plenum Press, New York, 1977. 24. R. C. Weast (ed.), Handbook of Chemistry and Physics, 65th Ed., CRC Press, Boca Raton, FL, 1984-5.