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Optimized reverse-phase high-performance liquid chromatographic separation of cinnamic acids and related compounds
ANALYTICAL BIOCHEMISTRY 93, 233-237 (1979)
Optimized Reverse-Phase High-Performance Liquid Chromatographic Separation of Cinnamic Acids and Related Compounds W I L L I A M P . PRICE, JR., RANDY E D E N S , D O N A L D L . H E N D R I X , 1 AND STANLEY N . D E M I N G
Department of Chemistry, University of Houston, Houston, Texas 77004 Received July 10, 1978 The effect of mobile-phase pH on reverse-phase high-performance liquid chromatographic separation is studied for a nine-component sample containing cinnamic, ferulic, hydrocinnamic, p-coumaric, caffeic, phenylacetic, vanillic, and fl-phenylpyruvic acids and phenylethylamine. A systematic optimization strategy is utilized: Retention times of each component are measured for mobile phases buffered with citric acid at pH's of 3.0, 4.0, 5.0, and 6.0; a mathematical model is fit to the chromatographic data; the model parameters are used to construct a window diagram which provides an estimate of the mobilephase pH required for optimum separation.
Reverse-phase high-performance liquid chromatography (hplc) 2 is becoming a popular analytical tool for the separation of complex mixtures of metabolic intermediates (1-4). The more complex the sample mixture, the more difficult is the task of obtaining adequate chromatographic resolution among all components in a reasonable time. Under these circumstances, the development of chromatographic methods by empirical experimental strategies is usually inefficient; a systematic optimization strategy is often preferred. Deming and Turoff (5) have shown that, with modification, a gas chromatographic optimization method of Laub and Purnell (6-9) can be successfully used for the optimization of hplc separations by controlling mobile-phase pH. This paper describes the application of this optimization method to the separation of a mixture containing cinnamic, ferulic, hydrocinnamic, p-coumaric, caffeic, phenylacetic, vanillic, 1 Department of Biology, University of Houston, Tex. 77004. z Abbreviation used: hplc, high-performance liquid chromatography.
233
and fl-phenylpyruvic acids and phenylethylamine. THEORY
Figure 1 illustrates the partitioning of a dilute phenolic acid solute between the polar, aqueous mobile phase (buffered by a more concentrated weak acid) and the nonpolar, organic stationary phase of a reverse-phase hplc column. A relatively basic mobile phase shifts the solute acidbase equilibrium toward the unprotonated form, thereby causing a shift in the phasedistribution equilibrium so that (in this illustration) more of the total solute appears in the mobile phase. A relatively acidic mobile phase has an opposite effect on the solute partitioning. As the fraction of solute dissolved in the mobile phase increases, the solute retention time (ta) decreases. For a solute such as that shown in Fig. 1, tn may be described by the simple relationship ta = tHAfHA + tAfA,
[1]
where tHA is the retention time of the solute when it is completely in the protonated 0003-2697/79/040233-05502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any form reserved.
234
PRICE ET AL.
+
i~
H+
Mobile,Aqueousphase Stationary,Organicphase
HO~-CH=CH-C-OH FIG. l. Partitioning of a dilute phenolic acid solute between the polar, aqueous mobile phase, and the nonpolar organic phase in a reverse-phase hplc column.
injector (Waters) connected to a 10-cm × 2mm-i.d. Bondapak C18/Corasil precolumn (Waters) and a 30-cm × 4-mm-i.d. ~C18 Bondapak high efficiency column (Waters). Precolumn and column temperatures (10) were maintained at 25.0 __+0.1°C with a constant temperature circulating water bath (Model FK, Haake, Inc., Saddle Brook, log ([A]/[HA]) = (pH - pKa), [2] N. J.). A SP8200 uv detector (Spectra [A]/[HA] = 10(pH-pK~), [3] Physics, Santa Clara, Calif.) was operated at 254 nm; the absorbance out[HA] 1 put was recorded on a strip chart refHA -- [HA] + [A] 1 + 10(pH-p~") ' [4] corder (Model 281, Soltec, Encino, Calif.). Flow rate was 2.0 ml min-L Injection [A] 10('H-pK~) volumes were 5/zl. The time equivalent of fA -- [HA] + [A] 1 + 10('H-p~") ' [5] the void volume (to = 1.26 min) was measwhere pKa is the acid dissociation constant ured from the time of injection to the first for the weak acid HA. Substitution of Eqs. small deviation from signal baseline. Table 1 shows the composition of the [4] and [5] into Eq. [1] gives buffered mobile phases used to obtain tHA + tA 10(pH-pKa) tR = [6] experimental data for fitting Eq. [6]. Citric 1 + l 0 (pH-pKa) acid, NaOH, and NaCl were purchased from Fisher Scientific Company (Fair For each solute, the three parameters of this Lawn, N. J.). Spectrophotometric grade model (/HA, tA, and pKa) can be estimated methanol (Gold Label, Aldrich Chemical from experimental data obtained by measCo., Inc., Milwaukee, Wis.) was added, uring solute retention time at three or more 10% (v/v), to modify the aqueous mobile values of mobile-phase pH. The fitted phase for more rapid elution. The ionic models may then be used to predict solute strength of each buffered mobile phase was retention time as a function of the single 0.06 M. Each buffered mobile phase was independent variable pH. filtered through a 0.45-/zm cellulose acetate filter (Millipore Corp., Bedford, Mass.) and METHODS degassed. Reported pH values are those The chromatographic system consisted of calculated for an aqueous solution and are a Model 6000A pump (Waters Associates, based on pKa values of 3.128, 4.761, and Milford, Mass.) equipped with a U6K 6.396 for citric acid (11). Reported pH
form; t A is its retention time when it is completely in the unprotonated form; and fHg and fA are the fractions of solute in the protonated and unprotonated forms, respectively, for any intermediate condition (5). For a dilute monoprotic weak acid in aqueous solution,
235
O P T I M I Z E D HPLC S E P A R A T I O N TABLE l COMPOSITION OF BUFFERED MOBILE PHASES
Solvent p H ~
Citric acid, 2.0 Mb (ml)
N a O H , 2.0 Mb (ml)
NaCP (g)
Methanol ° (ml)
3.0 4.0 5.0 6.0
10.00 10.00 10.00 10.00
4.10 10.23 16.56 22.39
6.502 5.661 4.276 2.624
200.0 200.0 200.0 200.0
See text. b In 2.00-liter solution.
values probably do not accurately describe the true pH of the buffered mobile phases; the calculated pH scale does, however, serve as a practical operational variable for this study. The solutes cinnamic acid, p-coumaric acid (4-hydroxycinnamic acid), caffeic acid (3,4-dihydroxycinnamic acid), ferulic acid (4-hydroxy-3-methoxycinnamic acid), vanillic acid (4-hydroxy-3-methoxybenzoic acid), phenylacetic acid, fl-phenylpyruvic acid, hydrocinnamic acid (fl-phenylpropionic acid), and phenylethylamine (2-aminoethylbenzene) were purchased from Fisher or from Sigma Chemical Company (St. Louis, Mo.). Isomeric solutes were purchased as the t r a n s isomer. Solutes were first prepared at a concentration of approximately 5 mg m1-1 (in 10%, v/v, methanol-water) and diluted to an appropriate concentration.
Two mixtures of the nine solutes were also prepared. The parameters of Eq. [6] were fit to the experimental data for each of the solutes using a simplex, nonlinear, least-squares computer program similar to that described by O'Neill (12). RESULTS AND DISCUSSION
Observed retention times for each solute at each of the four different mobile-phase pH's are presented in Table 2. Table 3 contains fitted values of the parameters of Eq. [6] for each of the nine solutes. In Table 3, the value oftr~Ais greater than the value of tA for all solutes except phenylethylamine. This is not surprising: The protonated form of phenylethylamine is a charged species; it would be expected to have a greater affinity
TABLE2 OBSERVED RETENTION TIMES Retention time Solute
Letter code
p H 3.0 a
pH 4.0 a
pH 5.0 a
pH 6.0 a
Phe nylet hylamine fl-Phenylpyruvic acid Vanillic acid Phenylacetic acid Caffeic acid p-Coumaric acid Hydrocinnamic acid Ferulic acid Cinnamic acid
E P V A C M H F N
4.96 6.95 16.28 19.27 21.95 38.33 47.85 55.98 --
4.91 5.62 13.28 15.35 18.47 31.93 42.31 51.17 71.55
4.93 5.31 6.40 7.74 9.87 16.66 24.11 25.46 32.10
5.67 5.46 3.12 4.50 4.19 6.37 -9.39 13.16
a See text.
236
PRICE
ET AL.
TABLE 3 FITTED VALUES OF MODEL PARAMETERS Solute
t HAa
t Aa
p Ka
Phenylethylamine fl-Phenylpyruvic acid Vanillic acid Phenylacetic acid Caffeic acid p-Coumaric acid Hydrocinnamic acid Ferulic acid Cinnamic acid
4.88 8.71 16.59 19.73 22.03 38.59 48.60 57.38 86.91
6.91 5.36 2.75 4.22 3.44 5.17 12.50 6.22 10.21
6.32 2.95 4.53 4.43 4.69 4.69 4.70 4.80 4.60
~: tl:
~-m, ,.2 0=
Uncorrected retention times in minutes. for the polar mobile phase and thus elute more rapidly than the unprotonated (uncharged) form (13). Observed and calculated tR values are plotted vs mobile-phase pH in Fig. 2. The relative retention (a) of two solutes, X and Y, is defined as a --
(tpo: -
to)
or
( t R y -- to)
( t R y -- to)
[7]
(tpo: -- to)
such that o~is always greater than or equal to unity. An ot = 1.0 indicates a pair of completely eclipsed solute peaks; as a mt.~'
i
7-
N
M
PH FIG. 2. Observed and calculated retention times vs pH for each of the nine solutes. (See Table 2 for letter codes.)
--
3.8
3.E
q.l~
4. zz
5Z.B
E.E
E,B
PH
FIo. 3. Window diagram of relative retention (c~ values) vs pH for important pairs of solutes. (See Table 2 for letter codes.)
increases, the resolution of the two peaks improves. Plotting relative retention (o0 vs pH for each of the 36 different pairs of nine solutes produces the "window diagram" (6) shown in Fig. 3. At any pH, the resolution of the least resolved pair of solutes is represented by the lowest curve. The greatest possible resolution of the least resolved pair(s) may be obtained at the pH corresponding to the top of the tallest shaded window (6-9). In practice, the optimum separation for a mixture is often a compromise between maximum resolution and minumum analysis time. Information from Figs. 2 and 3 can be combined to select the mobile-phase pH required for the "optimum" separation of the nine-component mixture in this study. As seen in Fig. 3, a pH of 3.65 should produce the best possible resolution for the worst separated pairs (or = 1.18), but Fig. 2 shows that it would require an analysis time longer than an hour. A mobile-phase pH of 4.7 is a reasonable compromise, predicting a minimum resolution of o~ = 1.12 and a maximum analysis time of approximately 45 min. (A means of decreasing the analysis
237
OPTIMIZED HPLC SEPARATION
E {= V
'
f
2B
3~B TIMEr MINUTES
,
q~t~
FIG. 4. Chromatogram of nine-component mixture at pH = 4.7. (See Table 2 for lettering codes.)
time and still maintaining optimal relative separation might be to carry out the separation with increased methanol concentration in a mobile phase buffered at the optimal pH of 3.65. This approach assumes that the values would not be affected by the change in concentration of methanol.) The shape of the particular window used is also helpful in assessing the ruggedness of the method with respect to mobile phase pH. At pH = 4.7, the top of the window is relatively fiat and small changes in mobilephase pH will not seriously degrade the performance of the method. A pH = 4.7 buffered mobile phase was prepared by adding 10.00 ml 2.0 M citric acid, 14.53 ml 2.0 M NaOH, 4.758 g NaC1, 200.0 ml methanol, and sufficient distilled water to make 2.00 liters of solution. Figure 4 illustrates the chromatogram obtained with this mobile phase for a mixture of the nine solutes. The initial solute mixture was spiked with fl-phenylpyruvic, phenylacetic, hydrocinnamic, and cinnamic acids and the peak assignments shown in Fig. 4 were confirmed. ACKNOWLEDGMENTS L. R. Parker, Jr., assisted with the computer programming. Primary financial support for this work
was provided by Grant E-644 from the Robert A. Welch Foundation. Additional support was provided by Grant NSG7300 from the National Aeronautics and Space Administration.
REFERENCES 1. Wulf, L. W., and Nagel, C. W. (1976) J. Chromatogr. 116, 271-279. 2. Callmer, K., Edholm, L. E., and Smith, B. E. F. (1977) J. Chromatogr. 136, 45-62. 3. Becker, H., and Gudrun, W. (1977)J. Chromatogr. 136, 174-175. 4. Rouseff, R. L. (1978) in Trends in Fluorescence, Vol. 1, pp. 10-13, Perkin-Elmer Corporation, Norwalk, Conn. 5. Deming, S. N., and Turoff, M. L. H. (1978)Anal. Chem. 50, 546-548. 6. Laub, R. J., and Purnell, J. H. (1975)J. Chromatogr. 112, 71-79. 7. Laub, R. J., and PurneU, J. H. (1976)Anal. Chem. 48, 799-803. 8. Lanb, R. J., and Purnell, J. H. (1976)Anal. Chem. 48, 1720-1724. 9. Laub, R. J., Purnell, J. H., and Williams, P. S. (1977) J. Chromatogr. 134, 249-261. 10. Kirkland, J. J. (1971) Modern Practice of Liquid Chromatography, p. 183, Wiley, New York. 11. Kortum, G., Vogel, W., and Andrussow, K. (1961) Dissociation Constants of Organic Acids in Aqueous Solution, p. 314, International Union of Pure and Applied Chemistry, Section of Analytical Chemistry, Commission on Electrochemical Data, Butterworths, London. 12. O'Neill, R. (1971)Appl. Statist. 20, 338-345. 13. Horvath, C., Melander, W., and Molnar, I. (1977) Anal. Chem. 49, 142-154.
Optimized Reverse-Phase High-Performance Liquid Chromatographic Separation of Cinnamic Acids and Related Compounds W I L L I A M P . PRICE, JR., RANDY E D E N S , D O N A L D L . H E N D R I X , 1 AND STANLEY N . D E M I N G
Department of Chemistry, University of Houston, Houston, Texas 77004 Received July 10, 1978 The effect of mobile-phase pH on reverse-phase high-performance liquid chromatographic separation is studied for a nine-component sample containing cinnamic, ferulic, hydrocinnamic, p-coumaric, caffeic, phenylacetic, vanillic, and fl-phenylpyruvic acids and phenylethylamine. A systematic optimization strategy is utilized: Retention times of each component are measured for mobile phases buffered with citric acid at pH's of 3.0, 4.0, 5.0, and 6.0; a mathematical model is fit to the chromatographic data; the model parameters are used to construct a window diagram which provides an estimate of the mobilephase pH required for optimum separation.
Reverse-phase high-performance liquid chromatography (hplc) 2 is becoming a popular analytical tool for the separation of complex mixtures of metabolic intermediates (1-4). The more complex the sample mixture, the more difficult is the task of obtaining adequate chromatographic resolution among all components in a reasonable time. Under these circumstances, the development of chromatographic methods by empirical experimental strategies is usually inefficient; a systematic optimization strategy is often preferred. Deming and Turoff (5) have shown that, with modification, a gas chromatographic optimization method of Laub and Purnell (6-9) can be successfully used for the optimization of hplc separations by controlling mobile-phase pH. This paper describes the application of this optimization method to the separation of a mixture containing cinnamic, ferulic, hydrocinnamic, p-coumaric, caffeic, phenylacetic, vanillic, 1 Department of Biology, University of Houston, Tex. 77004. z Abbreviation used: hplc, high-performance liquid chromatography.
233
and fl-phenylpyruvic acids and phenylethylamine. THEORY
Figure 1 illustrates the partitioning of a dilute phenolic acid solute between the polar, aqueous mobile phase (buffered by a more concentrated weak acid) and the nonpolar, organic stationary phase of a reverse-phase hplc column. A relatively basic mobile phase shifts the solute acidbase equilibrium toward the unprotonated form, thereby causing a shift in the phasedistribution equilibrium so that (in this illustration) more of the total solute appears in the mobile phase. A relatively acidic mobile phase has an opposite effect on the solute partitioning. As the fraction of solute dissolved in the mobile phase increases, the solute retention time (ta) decreases. For a solute such as that shown in Fig. 1, tn may be described by the simple relationship ta = tHAfHA + tAfA,
[1]
where tHA is the retention time of the solute when it is completely in the protonated 0003-2697/79/040233-05502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any form reserved.
234
PRICE ET AL.
+
i~
H+
Mobile,Aqueousphase Stationary,Organicphase
HO~-CH=CH-C-OH FIG. l. Partitioning of a dilute phenolic acid solute between the polar, aqueous mobile phase, and the nonpolar organic phase in a reverse-phase hplc column.
injector (Waters) connected to a 10-cm × 2mm-i.d. Bondapak C18/Corasil precolumn (Waters) and a 30-cm × 4-mm-i.d. ~C18 Bondapak high efficiency column (Waters). Precolumn and column temperatures (10) were maintained at 25.0 __+0.1°C with a constant temperature circulating water bath (Model FK, Haake, Inc., Saddle Brook, log ([A]/[HA]) = (pH - pKa), [2] N. J.). A SP8200 uv detector (Spectra [A]/[HA] = 10(pH-pK~), [3] Physics, Santa Clara, Calif.) was operated at 254 nm; the absorbance out[HA] 1 put was recorded on a strip chart refHA -- [HA] + [A] 1 + 10(pH-p~") ' [4] corder (Model 281, Soltec, Encino, Calif.). Flow rate was 2.0 ml min-L Injection [A] 10('H-pK~) volumes were 5/zl. The time equivalent of fA -- [HA] + [A] 1 + 10('H-p~") ' [5] the void volume (to = 1.26 min) was measwhere pKa is the acid dissociation constant ured from the time of injection to the first for the weak acid HA. Substitution of Eqs. small deviation from signal baseline. Table 1 shows the composition of the [4] and [5] into Eq. [1] gives buffered mobile phases used to obtain tHA + tA 10(pH-pKa) tR = [6] experimental data for fitting Eq. [6]. Citric 1 + l 0 (pH-pKa) acid, NaOH, and NaCl were purchased from Fisher Scientific Company (Fair For each solute, the three parameters of this Lawn, N. J.). Spectrophotometric grade model (/HA, tA, and pKa) can be estimated methanol (Gold Label, Aldrich Chemical from experimental data obtained by measCo., Inc., Milwaukee, Wis.) was added, uring solute retention time at three or more 10% (v/v), to modify the aqueous mobile values of mobile-phase pH. The fitted phase for more rapid elution. The ionic models may then be used to predict solute strength of each buffered mobile phase was retention time as a function of the single 0.06 M. Each buffered mobile phase was independent variable pH. filtered through a 0.45-/zm cellulose acetate filter (Millipore Corp., Bedford, Mass.) and METHODS degassed. Reported pH values are those The chromatographic system consisted of calculated for an aqueous solution and are a Model 6000A pump (Waters Associates, based on pKa values of 3.128, 4.761, and Milford, Mass.) equipped with a U6K 6.396 for citric acid (11). Reported pH
form; t A is its retention time when it is completely in the unprotonated form; and fHg and fA are the fractions of solute in the protonated and unprotonated forms, respectively, for any intermediate condition (5). For a dilute monoprotic weak acid in aqueous solution,
235
O P T I M I Z E D HPLC S E P A R A T I O N TABLE l COMPOSITION OF BUFFERED MOBILE PHASES
Solvent p H ~
Citric acid, 2.0 Mb (ml)
N a O H , 2.0 Mb (ml)
NaCP (g)
Methanol ° (ml)
3.0 4.0 5.0 6.0
10.00 10.00 10.00 10.00
4.10 10.23 16.56 22.39
6.502 5.661 4.276 2.624
200.0 200.0 200.0 200.0
See text. b In 2.00-liter solution.
values probably do not accurately describe the true pH of the buffered mobile phases; the calculated pH scale does, however, serve as a practical operational variable for this study. The solutes cinnamic acid, p-coumaric acid (4-hydroxycinnamic acid), caffeic acid (3,4-dihydroxycinnamic acid), ferulic acid (4-hydroxy-3-methoxycinnamic acid), vanillic acid (4-hydroxy-3-methoxybenzoic acid), phenylacetic acid, fl-phenylpyruvic acid, hydrocinnamic acid (fl-phenylpropionic acid), and phenylethylamine (2-aminoethylbenzene) were purchased from Fisher or from Sigma Chemical Company (St. Louis, Mo.). Isomeric solutes were purchased as the t r a n s isomer. Solutes were first prepared at a concentration of approximately 5 mg m1-1 (in 10%, v/v, methanol-water) and diluted to an appropriate concentration.
Two mixtures of the nine solutes were also prepared. The parameters of Eq. [6] were fit to the experimental data for each of the solutes using a simplex, nonlinear, least-squares computer program similar to that described by O'Neill (12). RESULTS AND DISCUSSION
Observed retention times for each solute at each of the four different mobile-phase pH's are presented in Table 2. Table 3 contains fitted values of the parameters of Eq. [6] for each of the nine solutes. In Table 3, the value oftr~Ais greater than the value of tA for all solutes except phenylethylamine. This is not surprising: The protonated form of phenylethylamine is a charged species; it would be expected to have a greater affinity
TABLE2 OBSERVED RETENTION TIMES Retention time Solute
Letter code
p H 3.0 a
pH 4.0 a
pH 5.0 a
pH 6.0 a
Phe nylet hylamine fl-Phenylpyruvic acid Vanillic acid Phenylacetic acid Caffeic acid p-Coumaric acid Hydrocinnamic acid Ferulic acid Cinnamic acid
E P V A C M H F N
4.96 6.95 16.28 19.27 21.95 38.33 47.85 55.98 --
4.91 5.62 13.28 15.35 18.47 31.93 42.31 51.17 71.55
4.93 5.31 6.40 7.74 9.87 16.66 24.11 25.46 32.10
5.67 5.46 3.12 4.50 4.19 6.37 -9.39 13.16
a See text.
236
PRICE
ET AL.
TABLE 3 FITTED VALUES OF MODEL PARAMETERS Solute
t HAa
t Aa
p Ka
Phenylethylamine fl-Phenylpyruvic acid Vanillic acid Phenylacetic acid Caffeic acid p-Coumaric acid Hydrocinnamic acid Ferulic acid Cinnamic acid
4.88 8.71 16.59 19.73 22.03 38.59 48.60 57.38 86.91
6.91 5.36 2.75 4.22 3.44 5.17 12.50 6.22 10.21
6.32 2.95 4.53 4.43 4.69 4.69 4.70 4.80 4.60
~: tl:
~-m, ,.2 0=
Uncorrected retention times in minutes. for the polar mobile phase and thus elute more rapidly than the unprotonated (uncharged) form (13). Observed and calculated tR values are plotted vs mobile-phase pH in Fig. 2. The relative retention (a) of two solutes, X and Y, is defined as a --
(tpo: -
to)
or
( t R y -- to)
( t R y -- to)
[7]
(tpo: -- to)
such that o~is always greater than or equal to unity. An ot = 1.0 indicates a pair of completely eclipsed solute peaks; as a mt.~'
i
7-
N
M
PH FIG. 2. Observed and calculated retention times vs pH for each of the nine solutes. (See Table 2 for letter codes.)
--
3.8
3.E
q.l~
4. zz
5Z.B
E.E
E,B
PH
FIo. 3. Window diagram of relative retention (c~ values) vs pH for important pairs of solutes. (See Table 2 for letter codes.)
increases, the resolution of the two peaks improves. Plotting relative retention (o0 vs pH for each of the 36 different pairs of nine solutes produces the "window diagram" (6) shown in Fig. 3. At any pH, the resolution of the least resolved pair of solutes is represented by the lowest curve. The greatest possible resolution of the least resolved pair(s) may be obtained at the pH corresponding to the top of the tallest shaded window (6-9). In practice, the optimum separation for a mixture is often a compromise between maximum resolution and minumum analysis time. Information from Figs. 2 and 3 can be combined to select the mobile-phase pH required for the "optimum" separation of the nine-component mixture in this study. As seen in Fig. 3, a pH of 3.65 should produce the best possible resolution for the worst separated pairs (or = 1.18), but Fig. 2 shows that it would require an analysis time longer than an hour. A mobile-phase pH of 4.7 is a reasonable compromise, predicting a minimum resolution of o~ = 1.12 and a maximum analysis time of approximately 45 min. (A means of decreasing the analysis
237
OPTIMIZED HPLC SEPARATION
E {= V
'
f
2B
3~B TIMEr MINUTES
,
q~t~
FIG. 4. Chromatogram of nine-component mixture at pH = 4.7. (See Table 2 for lettering codes.)
time and still maintaining optimal relative separation might be to carry out the separation with increased methanol concentration in a mobile phase buffered at the optimal pH of 3.65. This approach assumes that the values would not be affected by the change in concentration of methanol.) The shape of the particular window used is also helpful in assessing the ruggedness of the method with respect to mobile phase pH. At pH = 4.7, the top of the window is relatively fiat and small changes in mobilephase pH will not seriously degrade the performance of the method. A pH = 4.7 buffered mobile phase was prepared by adding 10.00 ml 2.0 M citric acid, 14.53 ml 2.0 M NaOH, 4.758 g NaC1, 200.0 ml methanol, and sufficient distilled water to make 2.00 liters of solution. Figure 4 illustrates the chromatogram obtained with this mobile phase for a mixture of the nine solutes. The initial solute mixture was spiked with fl-phenylpyruvic, phenylacetic, hydrocinnamic, and cinnamic acids and the peak assignments shown in Fig. 4 were confirmed. ACKNOWLEDGMENTS L. R. Parker, Jr., assisted with the computer programming. Primary financial support for this work
was provided by Grant E-644 from the Robert A. Welch Foundation. Additional support was provided by Grant NSG7300 from the National Aeronautics and Space Administration.
REFERENCES 1. Wulf, L. W., and Nagel, C. W. (1976) J. Chromatogr. 116, 271-279. 2. Callmer, K., Edholm, L. E., and Smith, B. E. F. (1977) J. Chromatogr. 136, 45-62. 3. Becker, H., and Gudrun, W. (1977)J. Chromatogr. 136, 174-175. 4. Rouseff, R. L. (1978) in Trends in Fluorescence, Vol. 1, pp. 10-13, Perkin-Elmer Corporation, Norwalk, Conn. 5. Deming, S. N., and Turoff, M. L. H. (1978)Anal. Chem. 50, 546-548. 6. Laub, R. J., and Purnell, J. H. (1975)J. Chromatogr. 112, 71-79. 7. Laub, R. J., and PurneU, J. H. (1976)Anal. Chem. 48, 799-803. 8. Lanb, R. J., and Purnell, J. H. (1976)Anal. Chem. 48, 1720-1724. 9. Laub, R. J., Purnell, J. H., and Williams, P. S. (1977) J. Chromatogr. 134, 249-261. 10. Kirkland, J. J. (1971) Modern Practice of Liquid Chromatography, p. 183, Wiley, New York. 11. Kortum, G., Vogel, W., and Andrussow, K. (1961) Dissociation Constants of Organic Acids in Aqueous Solution, p. 314, International Union of Pure and Applied Chemistry, Section of Analytical Chemistry, Commission on Electrochemical Data, Butterworths, London. 12. O'Neill, R. (1971)Appl. Statist. 20, 338-345. 13. Horvath, C., Melander, W., and Molnar, I. (1977) Anal. Chem. 49, 142-154.