Anal. Chem. 2002, 74, 3809-3818
Retention of Ionizable Compounds on HPLC. 12. The Properties of Liquid Chromatography Buffers in Acetonitrile-Water Mobile Phases That Influence HPLC Retention Sonia Espinosa, Elisabeth Bosch, and Martı´ Rose´s*
Departament de Quı´mica Analı´tica, Universitat de Barcelona, Diagonal 647, E-08028 Barcelona, Spain
The addition of acetonitrile to aqueous buffers to prepare RP HPLC mobile phases changes the buffer properties (pH and buffer capacity). This variation is studied for acetate, phosphate, phthalate, citrate, and ammonia buffers in acetonitrile-water mixtures up to 60% in acetonitrile (v/v). Equations are proposed to relate pH and buffer capacity change of these buffers to the initial aqueous pH value and to the volume fraction of acetonitrile added. It is demonstrated that the pH change of the buffer depends not only on the initial aqueous pH of the buffer and on the percentage of acetonitrile added but also on the particular buffer used. The proposed equations allow an accurate prediction of this ionization for the studied buffers. Since the retention of acid/base compounds shows a strong dependence of their degree of ionization, the equations are used to predict the change in this ionization with addition of acetonitrile when the RP HPLC mobile phase is prepared. This prediction allows estimation of the retention of an acid/base compound in a particular acetonitrile-water buffered mobile phase. Buffers are widely used for pH control of chemical processes.1 In reversed-phase high-performance liquid chromatography (RP HPLC) separations of compounds with acid/base properties, appropriate buffered solutions are needed for calibration of the electrode system used to measure pH and for control of the mobile-phase pH, since the latter influences analyte ionization and thus retention. pH standardization of electrode systems in water and in the most used RP HPLC mobile phases has been substantially achieved. The IUPAC has endorsed rules for pH standardization in water and in water-organic solvent mixtures of moderate to high permittivities and proposed some buffered solutions for pH standardization in these solvents.2-4 Some additional buffers in * Corresponding author. Fax: 34 93 402 12 33. E-mail: marti@apolo. qui.ub.es. (1) Bates, R. G. Determination of pH: Theory and Practice, 2nd ed.; Wiley: New York, 1964. (2) IUPAC Compendium of Analytical Nomenclature. Definitive Rules 1997, 3rd ed.; Blackwell: Oxford, U.K., 1998. (3) Mussini, T.; Covington, A. K.; Longhi, P.; Rondinini, S. Pure Appl. Chem. 1985, 57, 865-876. (4) Mussini, P. R.; Mussini, T.; Rondinini, S. Pure Appl. Chem. 1997, 69, 10071014. 10.1021/ac020012y CCC: $22.00 Published on Web 07/03/2002
© 2002 American Chemical Society
acetonitrile-water have been studied by Barbosa et al.5-7 On the basis of IUPAC rules and recommendations, we have studied the different pH scales that are employed in pH measurement of RP HPLC mobile phases. Three different pH scales have been used. The most common is the aqueous pH scale (wwpH), which is obtained when the electrode system is calibrated with aqueous buffers and the pH measured in the RP HPLC aqueous buffer before mixing it with the organic modifier. If the electrode system is calibrated with aqueous buffers, but the pH is measured in the mobile phase obtained after mixing the aqueous buffers with the organic modifier, the pH scale in the mobile-phase solvent (s) relative to water (w) as standard-state solvent is obtained (swpH). Finally, if the electrode system is calibrated with buffers prepared in the same mixed solvent used as mobile phase (s) and the pH is measured in the mobile phase (s), the pH scale in the mobile phase referred to the same mobile phase as standard-state solvent is obtained (sspH). Whenever possible, we recommend sspH and s wpH scales because they give much better general relationships between pH and retention.8-10 The swpH scale is specially recommended because of its simplicity of measurement, since it does not require pH standards for each different mobile-phase composition. However, the main acid/base properties of the buffers used to control the pH of RP HPLC mobile phases has not been systematically studied. In a pioneer work, Bates discussed the properties of acid/base aqueous buffers and its applicability to chemical processes.1 Bates remarked that in choosing a suitable buffer system, one should not only consider the pH required but should also take into account the nature of the reaction to be regulated. A high buffer capacity is essential for the control of an acid/base reaction, and a low dilution value is desirable if dilution of the medium is likely to occur. The dilution value is defined as the increase of pH suffered by a solution when it is diluted with an equal volume of pure solvent. Salt effects and temperature changes may be important in some instances. The chemical nature (5) Barbosa, J.; Sanz-Nebot, V. Anal. Chim. Acta 1993, 283, 320-325. (6) Barbosa, J.; Butı´, S.; Sanz-Nebot, V. Talanta 1994, 41, 825-831. (7) Barbosa, J.; Sanz-Nebot, V. Mikrochim. Acta 1994, 116, 131-141. (8) Canals, I.; Portal, J. A.; Bosch, E.; Rose´s, M. Anal. Chem. 2000, 72, 18021809. (9) Espinosa, S.; Bosch, E.; Rose´s, M. Anal. Chem. 2000, 72, 5193-5200. (10) Canals, I.; Oumada, F. Z.; Rose´s, M.; Bosch, E. J. Chromatogr., A 2001, 911, 191-202.
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002 3809
of the buffer materials is a key factor, because the buffer cannot form insoluble compounds or undesired side reactions. All these factors, cited by Bates, should be considered in the selection of RP HPLC buffers. In addition, the compatibility of the buffer with the detection method used must be also considered; e.g., the buffer must not absorb in the UV region in analyte UV detection or form volatile compounds for mass spectrometry detection. In this work, we shall study the acid/base properties of some of the buffers most used to control the pH of acetonitrile-water RP HPLC mobile phases. RP HPLC buffers are commonly prepared in water and later mixed with the organic modifier (acetonitrile in this instance). When an aqueous buffer is diluted with acetonitrile, its acid/base properties (namely, pH and buffer capacity) are altered because of both the dilution and the change of solvent. The properties change depends on the particular type and composition of the buffer prepared. An understanding of the changes produced by addition of acetonitrile to the most typical RP HPLC aqueous buffers seems essential in order to foresee the suitability of the buffer for a practical RP HPLC separation. EXPERIMENTAL SECTION Apparatus. Potentiometric measurements were taken with a Ross combination electrode Orion 8102 (glass electrode and a reference electrode with a 3.0 mol‚L-1 KCl solution in water as salt bridge) in a Crison micropH 2002 potentiometer with a precision of (0.1 mV. All the solutions were thermostated externally at 25 ( 0.1 °C. The retention data were measured on a 15 cm × 4.6 mm i.d. Xterra MS C18 5-µm (Waters) column with a flow rate of 1 mL/min in an Isco (Lincoln, NE) model 2350 dualpump system with a 20-µL loop valve. A Shimadzu (Kyoto, Japan) model SPD-10Avp UV-visible detector was used set at 254 nm. All data were taken by triplicate at 25 °C with the potentiometric cell and the column thermostated with water jackets. Chemicals. Acetonitrile was RP HPLC gradient grade from Merck and water purified by the Milli-Q plus system from Millipore. The studied buffers were prepared from orthophosphoric acid (Merck, 85%, for analysis), citric acid (Fluka Chemica, for analysis), ammonia (Merck, 25%, for analysis), or sodium dihydrogenphosphate monohydrate (Merck, for analysis) using hydrochloric acid (Merck, 25%, for analysis) and sodium hydroxide (Merck, for analysis) to adjust the pH to the desired value. The chromatographied samples were 2-nitrophenol (Fluka, >99%), 3-bromophenol (Schuchardt, 90%), 2,4,6-trimethylpyridine (Merck, 96%) and N,N-dimethylbenzylamine (Merck-Schuchardt, for synthesis). Care should be taken in the handling and disposal of these samples. They are harmful by inhalation, in contact with skin, and if swallowed. Wear suitable protective clothing. Neutralize 2,4,6trimethylpyridine and N,N-dimethylbenzylamine with acid before its disposal. N,N-dimethylbenzylamine is harmful to aquatic organisms and may cause long-term adverse effects. Avoid its release to the environment. Procedure. Aqueous buffers were prepared by addition until the desired pH of concentrated solutions of sodium hydroxide or hydrochloric acid to 0.01 mol‚L-1 aqueous solutions of phosphoric acid, citric acid, sodium dihydrogenphosphate, or ammonia. Acetonitrile-water buffers were prepared by addition of acetonitrile to the aqueous buffers. In all instances, the electrode system was calibrated using the usual aqueous standard reference buffers of potassium hydrogenphthalate (wwpH 4.00) and potassium dihy3810
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
Table 1. Properties of Relevant Interest for pH Measurement in Acetonitrile-Water Mixtures at 25 °Ca % MeCN
A
a0B
s spKap
δ
0 10 20 30 40 50 60
0.528 0.566 0.604 0.655 0.712 0.791 0.877
1.52 1.55 1.59 1.63 1.68 1.74 1.80
14.00 14.24 14.47 14.74 15.08 15.48 15.90
0.00 -0.01 -0.03 -0.04 -0.14 -0.22 -0.46
a A and a B, Debye-Hu ¨ ckel equation parameters; sspKap, autopro0 tolysis constant of the solvent mixture (pK values); δ, interconversion parameter between sspH and swpH scales (eq 1).
drogenphosphate/disodium hydrogenphosphate (wwpH 7.02). The pH readings provide directly the swpH value of the solution. RESULTS AND DISCUSSION Relationships between the pH of a Buffer Measured in the Different pH Scales. As pointed out in the introduction, the three procedures used in RP HPLC to measure the pH of buffers lead to three different pH quantities. If one calibrates the electrode system with aqueous buffers and measures the pH of the RP HPLC aqueous buffer before mixing it with the organic modifier, w wpH is obtained. However, the pH of the solution changes after dilution of the aqueous buffer with the organic modifier. If the electrode system is calibrated with aqueous buffers, but the pH is measured after mixing the RP HPLC aqueous buffers with the organic modifier, swpH is directly obtained. Finally, if the electrode system is calibrated with buffers prepared in the same solvent composition used as mobile phase, and the pH is measured in this same mobile-phase composition (i.e., after mixing aqueous buffer and organic modifier), the quantity obtained is sspH. s s wpH can be easily converted to spH by means of eq 1 which
δ)E h j - log(swγH0) ) swpH - sspH
(1)
includes the difference of the liquid-junction potentials (or Ej, expressed in pH units and assumed to be constant), together with the primary medium effect - log(swγ0H). The addition of the two terms leads to the useful quantity δ.1,8-10 The δ term is a constant value for each mobile-phase composition. Table 1 reports these values for some acetonitrile-water mixtures, as well as other parameters of interest for pH estimation in acetonitrile-water mixtures. The literature reports equations to estimate these parameters for other acetonitrile-water compositions not given in the table.9,11,12 However, the difference between wwpH and sspH (or swpH) depends not only on the mobile-phase composition but also on the particular buffering solution employed. Buffer solutions may be prepared from strong or weak acids or bases.1 If an aqueous buffering solution is prepared from a strong monoprotic acid of ca concentration, the pH is (11) Rose´s, M.; Ra`fols, C.; Bosch, E. Anal. Chem. 1993, 65, 2294-2299. (12) Bosch, E.; Fonrodona, G.; Ra`fols, C.; Rose´s, M. Anal. Chim. Acta 1997, 349, 367-376.
w wpH
) -log aH ) -log ca - log wwγH
(2)
where γH stands for the activity coefficient of the hydrogen ion, which is usually estimated by means of the Debye-Hu¨ckel equation,
log γH ) -AI1/2/(1 + a0BI1/2)
(3)
where I is the ionic strength of the solution. Table 1 reports A and a0B values for acetonitrile-water mixtures. Later, the solution is diluted with the organic modifier and the concentration of the acid changes to φH2Oca (neglecting volume contraction), and the activity coefficient is ssγH, where φH2O is the volume fraction of water in the mobile phase. The pH in the scale relative to the acetonitrile-water mixtures is then s spH
) -log aH ) -log ca - log φH2O - logssγH
(4)
the medium on a ∆pKap term (see Table 1). The overall pH change is
∆pH ) ∆pKap + log φH2O
(7)
where ∆pKap is the difference between the negative logarithms of the autoprotolysis constants of the solvent used as mobile phase and water. Table 1 reports pKap values for acetonitrile-water mixtures, which show that the term is important for these solutions because the pH of the buffer will change more than 1 unit for high acetonitrile percentages. A common case in RP HPLC is buffers prepared from a weak acid, e.g., acetic acid. The hydrogen activity is approximately13-15 aH ) (Kaca)1/2, where Ka is the acidity constant of the acid. It can be derived that
∆pH ) (∆pKa - log φH2O)/2
(8)
where ∆pKa is the difference between the pKa values of the acid in the mobile-phase solvent and in water:
and the difference ∆pH is
∆pH ) sspH - wwpH ) -log φH2O - ∆ logγH
(5)
∆log γH ) log ssγH - log wwγH
(6)
∆pKa ) sspKa - wwpKa
(9)
where
The variation on the activity coefficients is very small because the increase in the Debye-Hu¨ckel A parameter with acetonitrile content (Table 1) is balanced by the decrease in the acid concentration (ionic strength). For example, an initial acid concentration of 0.1 mol‚L-1 in water has log wwγH equal to -0.11, whereas when it is diluted to 50% acetonitrile, the term log ssγH is -0.12. Thus, ∆log γH is meaningless and it will not further be considered in this paper. Notice that we use the same IUPAC nomenclature for ionic activity coefficients as for the related pH and pK terms. A left-hand superscript indicates the medium where the quantity is measured or determined (w for water and s for an acetonitrile-water mixture). A left-hand subscript indicates the standard-state medium (the solvent where activity coefficients are taken as equal to unity at infinite dilution). If the aqueous buffer is prepared from a strong monoprotic base (e.g., KOH), the pH will depend also on the autoprotolysis constant of the medium (Kap), because the hydrogen ion activity is directly calculated from this constant and from the activity of the lyate ion of the solvent (aOH in water, aS in general), i.e., aH ) Kap/aS.13-15 The activity of the lyate ion is equivalent to the concentration of the strong base ([OH-]) corrected by the activity coefficient. Addition of the organic modifier to the aqueous buffer changes the concentration and activity of the lyate ions in the term log φH2O, but it also changes the autoprotolysis constant of (13) Su ¨cha, L.; Kotrly, S. Solution Equilibria in Analytical Chemistry; Van Nostrand Reinhold: London, 1972. (14) Skoog, D. A.; West, D. M.; Holler, F. J. Fundamentals of Analytical Chemistry, 7th ed.; Saunders: Forth Worth, TX, 1996. (15) Laitinen, H. A.; Harris, W. E. Chemical Analysis. An Advanced Text and Reference, 2nd ed.; McGraw-Hill: New York, 1975.
If the initial aqueous solution is a weak base (e.g., NH3), the ∆pH also includes the variation in the autoprotolysis constant:
∆pH ) ∆pKap - (∆pKb - log φH2O)/2 ) (∆pKap +∆pKa + log φH2O)/2 (10)
The most common RP HPLC buffers are prepared from an acid at concentration ca and its conjugated base at concentration cb (e.g., acetic/acetate, NH4+/NH3, dihydrogenphosphate/hydrogenphosphate, etc.). The Henderson-Hasselbach equation can usually be applied:13-15
pH ) pKa + log(cb/ca)
(11)
and the pH variation is
∆pH ) ∆pKa
(12)
The pH difference does not include the dilution term log φH2O because it affects to ca and cb to the same degree.8 Often, a buffer is prepared with a solution of an acidic salt. For instance, the pH of 0.05 mol‚kg-1 potassium hydrogenphthalate solution has been adopted as the primary reference value standard for pH calibration,2-4 sodium hydrogencarbonate buffer is used in many biological studies,1 and sodium dihydrogencitrate and sodium hydrogencitrate solutions are used in RP HPLC. The pH of these solutions is practically equal to the average of the two pKa values involved, and thus the pH variation is
∆pH ) (∆pKa1 + ∆pKa2)/2 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
(13) 3811
Table 2. swpKa Values of Acids in Acetonitrile-Water Mixtures s wpKa
in % of acetonitrile by volume
acid/base pair
0
10
20
30
40
50
60
mpK
acetic/acetate phosphoric/dihydrogenphosphate dihydrogenphosphate/hydrogenphosphate phthalic/hydrogenphthalate hydrogenphthalate/phthalate citric/dihydrogencitrate dihydrogencitrate/hydrogencitrate hydrogencitrate/citrate ammonium/ammonia
4.74 2.21 7.23 2.92 5.39 3.16 4.79 6.42 9.29
4.93 2.38 7.39 3.07 5.73 3.30 4.94 6.61 9.26
5.14 2.59 7.57 3.23 6.08 3.46 5.11 6.82 9.18
5.40 2.76 7.78 3.41 6.49 3.64 5.31 7.07 9.13
5.62 2.97 7.94 3.54 6.85 3.76 5.46 7.26 9.05
5.93 3.20 8.16 3.71 7.27 3.94 5.69 7.52 8.99
6.16 3.29 8.27 3.77 7.60 3.99 5.82 7.67 8.88
2.30 1.87 1.78 1.51 3.68 1.48 1.73 2.12 -0.60
The above examples show that the pH difference between the true pH of the mobile phase and the pH of the aqueous buffer is very dependent on the buffer solution and especially on the pKa variation. Even if buffers of the same type are used (e.g., acid/ conjugated base), different acids and bases with different ∆pKa values will be needed to cover an extensive pH range. All ∆pH terms in the equations above are given in the sspH scale. ∆pH variations in the swpH scale can be easily calculated from them by adding the δ value for the corresponding acetonitrile-water mixture (eq 1). Variation with Solvent Composition of the pKa of Acids and Bases Used To Prepare Buffers. Solutions prepared from strong acids and bases have larger buffer capacity than solutions prepared from weak acids or bases at the same concentration.1 However, the pH ranges buffered by solutions of strong acids and bases lay in the extremes of the pH scale (pH 11 for strong bases in the aqueous wwpH scale). These pH ranges are seldom used in practical RP HPLC, and thus, common RP HPLC buffers are prepared from weak acids and bases that may have an acceptable buffer capacity at pH values close to the pKa of the acid/base pair employed. We studied the variation of the pKa values of some acids and bases commonly used to prepare RP HPLC buffers (acetic, phosphoric, and citric acids and ammonia) with the addition of acetonitrile up to 60% in volume. We also included phthalic acid because 0.05 m potassium hydrogenphthalate is the reference value standard adopted by the IUPAC for pH standardization of potentiometric systems.2-4 The pKa values were taken from the literature,16,17 except for the first pKa of phosphoric acid, which was not available, and it has been determined here for the acetonitrile-water mixtures studied. The procedure used to determine the pKa values of phosphoric acid in acetonitrile-water mixtures is reported elsewhere.18 The third pKa value of phosphoric acid in acetonitrile-water has not been studied because it lays in the most basic region of the pH scale, which is seldom used in RP HPLC separations. This complicates the potentiometric determination of pK, and probably because of this reason, it has not been reported in the literature. The pKa values of the studied acids in the swpH scale are presented in Table 2, and the variation of the pKa values with the volume fraction of acetonitrile added is presented in Figure 1. The (16) Barbosa, J.; Beltra´n, J. L.; Sanz-Nebot, V. Anal. Chim. Acta 1994, 288, 271278. (17) Rondinini, S.; Nese, A. Electrochim. Acta 1987, 32, 1499-1505. (18) Espinosa, S.; Bosch, E.; Rose´s, M. Anal. Chim. Acta 2002, 454, 157-166.
3812
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
Figure 1. Variation of the pKa values of buffer components with the addition of acetonitrile in the absolute pH scale (swpKa): (]) acetic acid, (0) phosphoric acid, (9) dihydrogenphosphate, (2) phthalic acid, (4) hydrogenphthalate, (×) citric acid, ([) dihydrogencitrate, (/) hydrogencitrate, and (O) ammonium.
s wpKa
variations with volume fraction of acetonitrile are close to linear, as we have been already observed in previous work.18-20 In this instance, the swpKa variation (∆swpKa) can be approximately described by the equation
∆swpKa ) mpKφMeCN
(14)
where φMeCN is the volume fraction of acetonitrile in the mixture and mpK the slope of the plots of Figure 1, which has been included in Table 2. mpK values are positive for neutral (acetic, phosphoric, phthalic, citric) and anionic (dihydrogenphosphate, dihydrogencitrate, hydrogencitrate, hydrogenphthalate) acids and negative for cationic acids (ammonium). This has been already explained in terms of the electrostatic interactions that contribute to the pKa value.15,18,21 The swpH values of buffers prepared from these acid/ base pairs are expected to change in a similar way with the addition of acetonitrile. Variation with Solvent Composition of the swpH of Buffers. To study the pH variation when acetonitrile is added to an aqueous buffer of a fixed pH value in water, we calculated the pH (19) Espinosa, S.; Bosch, E.; Rose´s, M. J. Chromatogr., A 2002, 945, 83-96. (20) Espinosa, S.; Bosch, E.; Rose´s, M. J. Chromatogr., A 2002, 947, 47-58.
an equation similar to the one obtained for the variation of swpKa values (eq 14), i.e.
∆swpH ) mpHφMeCN
Figure 2. Variation of swpH of aqueous buffers prepared from phthalic acid with the addition of acetonitrile. wwpH: (+) 2.54, (]) 3.00, (2) 3.50, (×) 4.00, (0) 4.49, (O) 4.99, (b) 5.47, (4) 5.97, (f) 6.46, (9) 7.17, and ([) 8.55. Dashed lines are for swpH variation of solutions of strong acids and bases. Overall aqueous buffer concentrations are 0.01 mol‚L-1.
of several of the acid/conjugate base buffer solutions of Table 2. The overall ca + cb concentration has been fixed to 0.01 mol‚L-1. The swpH values for different ca/cb ratios were calculated through a procedure similar to that described by De Levie22 for acid/base titrations of arbitrary mixtures, except that it incorporates the effect of the ionic activity coefficients.9,23 Figure 2 presents the results obtained for phthalic/hydrogenphthalate. The plots are close to linear. For initial aqueous pH (wwpH) values between about 4.5 and 7, the slopes of the plots are maximum and almost independent of the initial aqueous pH (the lines are parallel). In this pH range, the hydrogenphthalate/ phthalate acid/base pair buffers the solution, and according to the approximate eq 12, the ∆swpH variation should be equal to the ∆swpKa variation of hydrogenphthalate in Figure 1 and Table 2. A similar trend is observed for initial pH values between 3 and 3.5. These solutions are buffered by the phthalic/hydrogenphthalate pair, which has a lower mpK value than that of hydrogenphthalate/phthalate (Table 2), and thus the swpH variation is smaller. Between these two pH ranges, the slope of the plot moves between the two mpK values of phthalic/hydrogenphthalate and hydrogenphthalate/phthalate. In fact, the pH variation of a solution of pure hydrogenphthalate should follow eq 13, and thus, the slope of this variation should be the average of the mpK values of phthalic and hydrogenphthalate in Table 2 (pH 4 in Figure 2). Therefore, the ∆swpH values obtained for buffers prepared from phthalic acid/hydrogenphthalate/phthalate can be fitted to (21) Rived, F.; Canals, I.; Bosch, E.; Rose´s, M. Anal. Chim. Acta 2001, 439, 315-333. (22) De Levie, R. Anal. Chem. 1996, 68, 585-590. (23) Bosch, E.; Espinosa, S.; Rose´s, M. J. Chromatogr., A 1998, 824, 137-146.
(15)
with an mpH value that depends on the initial wwpH of the buffer. The most acidic buffer solution is the solution of pure phthalic acid. The pH variation for this buffer is given by eq 8 and the slope should be close to half the mpK value for phthalic/ hydrogenphthalate in Table 2, corrected by the change in the log φH2O value. Figure 2 shows that the variation for this solution (pH 2.5) is the smallest observed. If a more acidic buffer is prepared by addition of a strong acid, the pH would be determined by this strong acid and the pH variation by eq 5. This depends only on the log φH2O variation and for the swpH scale on the δ variation. These terms do not show a linear dependence on φMeCN, although their combination produces a very small pH variation, between 0.11 and -0.06 pH units in the studied range of 0-60% acetonitrile. This would indicate that the swpH value of buffers prepared from strong acids practically does not change with the addition of acetonitrile (bottom dashed line in Figure 2). The most basic buffer prepared from phthalic is the 0.01 mol‚L-1 solution of phthalate (pH 8.5 in Figure 2). The pH variation for this solution is given by eq 10, which points out that the slope obtained is intermediate between those obtained for hydrogenphthalate/phthalate buffers and buffers prepared with a strong base. According to eqs 7 and 1, the pH variation for a strong base should be
∆swpH ) ∆swpKap + log φH2O
(16)
There is a good linear correlation between this ∆swpKap + log φH2O term and the volume fraction of acetonitrile, φMeCN, in the solvent mixture, which is
∆swpH ) (1.81 ( 0.03)φMeCN
r ) 0.997 SD ) 0.03
F ) 1046 (17)
Therefore, the mpH slope for a strong base is 1.81 (top dashed line in Figure 2). The mpH values obtained for the swpH variation of the studied buffers are presented in Figure 3. The interpretation of the mpH variation for the different studied buffers follows the same trends explained for phthalate buffers. Constant mpH values, close to mpK values of Table 2, are obtained for initial wwpH values close to each of the corresponding wwpKa values of the acid/base pair. For buffers prepared from neutral or anionic acid/base pairs, the mpH values are positive, and for initial wwpH values lower than the first w wpKa of the acid, the mpH values are between the mpK that corresponds to this first wwpKa and zero. For buffers prepared by a mixture of a cationic acid and the corresponding neutral base (ammonium/ammonia), mpH values are negative and close to the mpK values for the acid/base pair in the initial pH range close to the pKa value. For higher pH values, the mpH value increases toward the one that would be obtained for a buffer solution prepared from a strong base (mpH ) 1.81). Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
3813
Figure 3. Variation of the slope of the swpH vs volume fraction of acetonitrile plot (Figure 2) with the initial aqueous pH of the buffer (wwpH): (O) phthalic acid, (]) acetic acid, (0) citric acid, (4) phosphoric acid, and (×) ammonium. Continuous lines fitted through eq 18.
The type of variation with pH (initial wwpH) obtained for the mpH values indicates that it should fit well the following model:
in Table 3. a0 and an+1 were fixed to the theoretical values of 0 and 1.81 and all the other parameters were adjusted by nonlinear regression. Table 3 shows that ai values are close to the corresponding mpK values of Table 2 and agree with the mpH values of the plateaus of the plots of Figure 3. The bi values agree with the pH value of the inflection points of the plots and they are related to the wwpKa values of the acid/base pair. For instance, for citric acid, b1 ) 2.62 ≈ 2.58 ) (pKa1 - log c)/2; b2 - b1 ) 3.92 ≈ 3.98 ) (pKa1 + pKa2)/2; b3 - b2 ) 5.29 ≈ 5.60 ) (pKa2 + pKa3)/2; b4 - b3 ) 8.87 ≈ (pKa3 + pKap + log c)/2, taking into account that c ) 0.01 mol‚L-1 and pKap ) 14. Very good fits have been obtained for all studied buffers. Equation 18 offers an easy way to predict how the pH of an aqueous buffer will change with the addition of acetonitrile. It helps to choose the best aqueous buffer that will give a desired s wpH value in a particular acetonitrile-water mobile phase. The exact swpH required could be later finely adjusted by addition of a small amount of a strong acid or base. The other buffer property that must be considered is the buffer capacity of the aqueous buffer and how it changes with the addition of acetonitrile. Variation of Buffer Capacity with Solvent Composition. The buffer capacity of the studied buffer systems at the different pH values has been calculated for several acetonitrile-water compositions obtained by addition of acetonitrile to an initial aqueous buffer at overall 0.01 mol‚L-1 concentration. Buffer capacity is defined by the differential eq 19;1,13 i.e., it is the reverse
n
a0 + mpH )
∑a 10
s(ipH-bi)
i
i)1
∑10
s(ipH-bi)
+ 10
s((n+1)pH-bn+1)
i)1
The first term of numerator and denominator (a0 and 1) predominates at low-pH values (pH , b1) and it is the limiting mpH value for buffer solutions prepared from strong acids; i.e., a0 must be practically equal to zero. The last term (the n + 1 term) predominates at very basic pH values (pH . bn), and it stands out for the buffer solutions prepared from strong bases, i.e., an+1 ) 1.81 (eq 17). The intermediate terms stand out for the different acid/conjugate base pairs that can form buffer solutions and there are as many as pKa values for the compound studied (one for acetic acid and ammonia, two for phthalic and phosphoric acids, and three for citric acids). Each one of these terms prevails in the pH zone close to the pKa that relates the concentrations of the acid/base pair. The ai values should be close to the mpK values reported in Table 2, and the bi values a combination of the pKa values of the corresponding acid/base system. s is a fitting parameter that accounts for the velocity of transition between the different pH zones buffered by the different acid/base pairs of the system. Equation 18 is analogous to the equation used to fit retention time and chromatographic hydrophobicity index (CHI) to aqueous pH during gradient elution.24,25 A similar equation with two terms and s ) 1 was used to fit the swpH change of ammonium acetate buffers in methanol-water mobile phases.24 The results obtained by application of the model to the mpH values for the different acid/base systems studied are reported 3814
(19)
(18)
n
1+
β ) dcb/dpH
+ an+110s((n+1)pH-bn+1)
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
of the pH variation observed for the buffer when a small concentration of a strong base is added. It can be easily calculated with the algorithm used to calculate the pH of the buffer by simple calculation of the pH change observed for the buffer when the concentration of conjugate base (cb) is increased by a small amount (e.g., 0.1%). The results obtained are plotted in Figure 4. The type of β versus pH plots observed for buffers in acetonitrile-water are very similar to the ones commonly observed in water, which is a solvent extensively studied,1,13 and much simpler than those observed in low dielectric constant or poor solvating solvents, such as pure acetonitrile.26-28 Taking activity coefficients equal to unity, the function that describes the buffer capacity for an acid/base buffer with up to n acid/base equilibria (n Ka constants) can be written as1,13
[
β ) 2.303 [H+] +
n
∑
Kaici[H+]
i)1 (K
+ 2 ai + [H ])
+
Kap
]
[H+]
(20)
where ci is the concentration of one of the acid/base pairs of the buffer. It is an additive function of all acid/base pairs that compose the buffer. (24) Canals, I.; Valko´, K.; Bosch, E.; Hill, A. P.; Rose´s, M. Anal. Chem. 2001, 73, 4937-4945. (25) Valko´, K.; Espinosa, S.; Du, C. M.; Bosch, E.; Rose´s, M.; Bevan, C.; Abraham, M. H. J. Chromatogr., A 2001, 933, 73-81. (26) Bosch, E.; Rose´s, M. Talanta 1989, 36, 615-621. (27) Barbosa, J.; Bosch, E.; Cortina, J. L.; Rose´s, M. Anal. Chim. Acta 1992, 256, 211-220. (28) Rose´s, M. Anal. Chim. Acta 1994, 285, 391-399.
Table 3. Parameters of Eq 18 for the Variation of the swpH Values of Buffers with the Addition of Acetonitrile acid
s
a0
b1
a1
b2
a2
ammonium acetic phosphoric phthalic citric
2.88 3.10 1.62 2.83 2.11
0.00 0.00 0.00 0.00 0.00
5.77 3.36 2.21 2.53 2.62
-0.61 2.29 1.42 1.35 1.41
16.41 11.69 6.85 6.62 6.54
1.81 1.81 1.75 3.64 1.69
b3
16.78 15.16 11.83
a3
1.81 1.81 2.02
b4
20.70
a4
N
SD
r
F
1.81
23 19 21 18 24
0.01 0.02 0.02 0.04 0.01
0.998 0.995 0.997 0.999 1.000
2824 1097 939 3151 6921
Figure 4. Variation of the buffer capacity of the studied buffers with swpH and addition of acetonitrile. Acetonitrile percentages are indicated in the figure.
The first and last terms of eq 20 describe the buffer capacity of strong acids and bases, and the central terms, the buffer capacity of the different weak acid/conjugate base pairs. There are as many central terms as acid/base buffer pairs, i.e., acidity constants. Each one gives a bell-shaped plot of β versus pH. When these acidity constants are different enough, the buffer capacity of each acid/base pair is independent of the ones of the other acid/base pairs, and the maximum buffer capacity does not depend on pKa values. The maximum1,13 is reached when [H+] ) Ka (or pH ) pKa) for which
βmax ) 0.576c
(21)
This is the case for acetic, ammonium, and phosphoric acids. The pKa1 value of the last one is very low and the contribution of [H+] to eq 20 must be taken into account too. For these systems, the addition of acetonitrile decreases c by dilution and the buffer capacity decreases in the same ratio. That is to say, the buffer capacity of a buffer in 50% acetonitrile is half the buffer capacity in water. But this is not the same when the different pKa values of the acid/base system are close, such as in phthalic and citric acids. The buffer capacity at a given pH depends on two or more acid/ base systems and on the pKa values of these systems in addition to buffer concentration. For instance, a 0.05 mol‚kg-1 potassium
phthalate solution is recommended by the IUPAC as a primary standard for pH calibration in water and in acetonitrile-water mixtures (as well in other solvents).2-4 The buffer capacity is given by the minimum in the valley between the two peaks for phthalic acid in Figure 4, and this minimum depends on the distance between the two pKa values. From eq 20, it can be calculated that then
β ) 4.606c
[
]
xKa1Ka2 (xKa1 + xKa2)2
(22)
Figure 1 shows that as acetonitrile is added the difference between the two pKa values increases and therefore the minimum of the valley in Figure 4 decreases in addition to the decrease by dilution. The buffer capacity of this buffer in pure water have a reasonable value, but Figure 4 shows that in 60% acetonitrile the solution is almost not buffered. Despite IUPAC recommendations, we do not recommend this buffer for calibration of electrode systems in high acetonitrile percentages. A buffer prepared by an equimolar mixture of hydrogenphthalate and phthalate would be more advisable. The same effect is observed for solutions of dihydrogencitrate or hydrogencitrate although to a minor degree because Figure 1 shows that the variation of the difference between the pKa values Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
3815
Table 4. Comparison of Experimentally Measured swpH Values of Buffers with s wpH Values Calculated from Buffer Composition or Estimated by the Equations Developed in This Work a s wpH
% MeCN 0 10 20 30 40 50 60
exp calc est exp calc est exp calc est exp calc est exp calc est exp calc est
2.29 2.36 2.36 2.37 2.44 2.46 2.45 2.55 2.55 2.53 2.65 2.61 2.62 2.74 2.70 2.70 2.83 2.66 2.78
3.00 3.07 3.11 3.12 3.20 3.24 3.24 3.36 3.39 3.37 3.50 3.49 3.49 3.62 3.65 3.61 3.75 3.68 3.73
4.00 4.11 4.14 4.16 4.28 4.30 4.32 4.47 4.49 4.48 4.65 4.62 4.63 4.80 4.83 4.79 4.94 4.93 4.95
5.00 5.18 5.16 5.18 5.38 5.33 5.35 5.58 5.54 5.53 5.76 5.69 5.70 5.91 5.92 5.88 6.05 6.05 6.05
6.00 6.20 6.18 6.20 6.42 6.38 6.40 6.65 6.61 6.60 6.81 6.79 6.80 6.94 7.04 7.00 7.04 7.19 7.20
7.00 7.21 7.15 7.18 7.44 7.33 7.35 7.62 7.54 7.53 7.77 7.69 7.70 7.90 7.91 7.88 8.03 8.02 8.05
8.00 8.14 8.15 8.18 8.36 8.33 8.35 8.51 8.53 8.53 8.63 8.69 8.70 8.78 8.91 8.88 8.86 9.01 9.05
9.00 8.97 8.97 8.94 8.89 8.89 8.88 8.78 8.84 8.82 8.71 8.73 8.76 8.57 8.70 8.70 8.44 8.59 8.64
10.00 9.96 9.98 9.94 9.88 9.91 9.89 9.78 9.87 9.83 9.69 9.79 9.77 9.59 9.73 9.72 9.45 9.62 9.66
a exp, experimentally measured s pH; calc, s pH calculated from the buffer composition and the pK values of buffer components at the given a w w percentage of acetonitrile; est, swpH estimated from eqs 15 and 18 and the parameters of Table 3.
of citric acid with addition of acetonitrile is much smaller than that for phthalic acid. This buffer system provides good continuous buffer capacity for aqueous wwpH values below 7, and we recommend it for RP HPLC applications. Notice that the addition of acetonitrile produces a shift in the plots of Figure 4 toward higher swpH values for the neutral acids and toward lower swpH values for the cationic acids (neutral bases) because of the swpKa variation of the different compound buffers (see Figure 1). For the common RP HPLC buffers, the major factor that decreases buffer capacity is the decrease of buffer concentration when the organic modifier is added. This should be taken into account in practical work, especially when working with a gradient elution that may come to high organic modifier concentrations or when one works isocratically with high organic solvent contents. Experimental Evaluation of the Model. The developed model has been evaluated by preparation of several buffers at round pH values and experimental measurement of the swpH of these buffers. At each pH value, the 0.01 mol‚L-1 aqueous buffer with the highest buffer capacity between all studied systems (phthalic buffers excluded) was selected. The most acidic buffer studied was a 0.01 mol‚L-1 solution of phosphoric acid with a w w wpH value of 2.29. Buffers at wpH values of 4.00, 5.00, and 6.00 were prepared from citric acid. Sodium dihydrogenphosphate buffers cover wwpH 7.00 and 8.00. The most basic buffers were prepared from ammonia (wwpH 9.00 and 10.00). Acetonitrilewater buffers were prepared from these buffers by addition of acetonitrile to obtain 10, 20, 30, 40, 50, and 60% of acetonitrile (v/v) mixtures. The swpH of all these buffered solutions were measured and the measured pH values compared with those calculated from the buffer composition and with those estimated by application of eqs 15 and 18. The results obtained are presented in Table 4. The agreement between the pH calculated from the buffer composition and that experimentally measured is very good for 3816 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
the phosphoric and citric buffers. As expected, the differences between both pH values increase with the percentage of acetonitrile added. Buffers prepared from ammonia give experimental pH values somewhat lower than those calculated from the buffer composition and the pKa value of ammonium. The average of the differences between experimental and calculated pH for the acetonitrile-water buffers is -0.01 ( 0.07 (mean ( SD), which shows that the accuracy and precision achieved are very good. The comparison between the measured pH values and those estimated from eqs 15 and 18 and the parameters of Table 3 follows the same trends indicated in the previous paragraph for the calculated pH values. In fact, the average of the differences between experimental and estimated pH is -0.02 ( 0.07, which shows that the precision and accuracy of this method are as good as those of the pH calculation from buffer composition. The advantage of the method is that the knowledge of the swpKa value of the buffer components and the exact composition of this buffer are not required. The parameters of Table 3 allow an easy estimation of the pH variation of buffers prepared from the acids and bases listed in the Table with an accuracy of (0.1 pH unit for most compositions and with a maximum of (0.2 pH unit for high acetonitrile percentages. This is indeed the maximum precision that can be achieved for pH measurements in nonaqueous and mixed solvents. Estimation of the Degree of Ionization and Prediction of RP HPLC Retention. Retention in reversed-phase liquid chromatography is strongly dependent on the degree of ionization of the analyte.8,10,29-37 In general, ionized compounds are poorly retained in hydrophobic stationary phases, whereas nonionized (29) Lopes Marques, R. M.; Schoenmakers, P. J. Chromatogr. 1992, 592, 157182. (30) Schoenmakers, P. J.; Tijssen, R. J. Chromatogr., A 1993, 656, 577-590. (31) Lewis, J. A.; Lommen, D. C.; Raddatz, W. D.; Dolan, J. W.; Snyder, L. R.; Molna´r, I. J. Chromatogr. 1992, 592, 183-195. (32) Lewis, J. A.; Dolan, J. W.; Snyder, L. R.; Molnar, I. J. Chromatogr. 1992, 592, 197-208. (33) Horva´th, C.; Melander, W.; Molna´r, I. Anal. Chem. 1977, 49, 142-154.
Figure 5. Variation of the ionization of acid/base compounds with the addition of acetonitrile to aqueous buffers of wwpH 8.0. (A) H2PO4-/ HPO42- buffer; (B) NH4+/NH3 buffer. Compounds: (]) 3,5-dichlorophenol, (0) 2,4-dichlorophenol, (4) 2-nitrophenol, (×) 3-bromophenol, (9) 2,4,6-trimethylpyridine, and (b) N,N-dimethylbenzylamine.
compounds may be strongly retained. The ionization of an acid/ base compound in a particular RP HPLC buffer depends on the pH of the buffer and on the pKa of the compound. For a compound that has a unique acid/base equilibria ruled by the acidity constant Ka, the mole fraction (R) of each one of the two species that coexist in the mixture (HAz and Az-1) is
RHA ) RA )
[HAz] z
[HA ] + [A
z-1
[HA ] + [A
1 1 + 10pH-pKa
(23)
)
1 1 + 10pKa-pH
(24)
]
[Az-1] z
)
z-1
]
For a neutral acid (z ) 0), RA is its degree of ionization (R), whereas for a neutral base (z ) +1), R ) RHA. Since both the pH of the buffer and the pKa of the analyte change with the addition of acetonitrile, the degree of ionization changes as well.37 If the pH and pKa changes are close to linear, they can be described by eqs 14 and 15 and the difference between pH and pKa that determines the degree of ionization can be easily estimated from s wpH
- swpKa ) wwpH - wwpKa + (mpH - mpK)φMeCN
(25)
which shows that the variation of the degree of ionization of the analyte depends on the difference between the m values of buffer pH and analyte pK. Equation 18 and the parameters of Table 3 allow estimation of the mpH of the buffers studied here, and in previous works,19,20 we have reported the mpK values for several compounds. An illustrative example is given in Figure 5. An RP HPLC buffer of wwpH 8.00 with acceptable buffer capacity can be prepared from a hydrogenphosphate/phosphate buffer or from an ammonium/ (34) Bosch, E.; Bou, P.; Allemann, H.; Rose´s, M. Anal. Chem. 1996, 68, 36513657. (35) Rose´s, M.; Canals, I.; Allemann, H.; Siigur, K.; Bosch, E. Anal. Chem. 1996, 68, 4094-4100. (36) Rose´s, M.; Bolliet, D.; Poole, C. F. J. Chromatogr., A 1998, 829, 29-40. (37) Sy´kora, D.; Tesarˇova´, E.; Popl, M. J. Chromatogr., A 1997, 758, 37-51.
ammonia buffer. The mpH values for these two buffers are 1.75 and -0.61, respectively. We consider the ionization of the following compounds: 2-nitrophenol, 2,4-dichlorophenol, 3,5-dichlorophenol, 3-bromophenol, 2,4,6-trimethylpyridine, and N,N-dimethylbenzylamine that have a wwpKa value close to 8 (6.64, 7.37, 8.14, 9.09, 7.49, and 8.93, respectively16,17). The corresponding mpK values are 3.42, 3.82, 2.85, 2.97, -2.30, and -2.08.16,17 Figure 5 shows that addition of acetonitrile decreases the ionization of all compounds in both buffers, but the decrease is different. In the phosphate buffer, the ionization of neutral acids (phenols) decreases because the pKa of the phenols increases to a larger degree than the pH of the buffer (mpK > mpH > 0). However, the ionization of neutral bases decreases much more because the pH of the buffer increases (mpH > 0) and the pKa of the base decreases (mpK < 0). This fact has been already reported in the literature for RP HPLC separations. Sy´kora et al.37 studied the effect of mobilephase pH with methanol-phosphate buffers in the retention of 19 neutral bases in several columns. They observed apparent shifts of the retention versus pH plots (wwpH) toward pH values more acidic than the wwpKa value of the base (∼2.5 pH units of difference for 60% methanol). They demonstrated that the shifts were a combination of the two individual shifts caused by the change of dissociation of the phosphate buffer (which produces a mobile-phase pH change) and by the change of the pKa of the compound with addition of methanol to the aqueous buffer solution. McCalley38-40 studied the protonation of bases in methanolwater, acetonitrile-water, and tetrahydrofuran-water and concluded that half-protonation is obtained at aqueous pH values of the phosphate buffer much lower than the aqueous wwpKa value of the base. The same effect was observed by Neue et al.41 for a phosphate buffer of wwpH 7.00 in 65% (v/v) methanol. At mobilephase pH 7.0 and for pKa values of bases (amitriptyline, doxepin, propanolol) close to 9, the bases should be completely protonated. (38) McCalley, D. V. LC-GC Eur. 1999, 638-650. (39) McCalley, D. V. J. Chromatogr., A 1994, 664, 139-147. (40) McCalley, D. V. J. Chromatogr., A 1995, 708, 185-194. (41) Neue, U. D.; Serowik, E.; Iraneta, P.; Alden, B. A.; Walter, T. H. J. Chromatogr., A 1999, 849, 87-100.
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
3817
Figure 6. Elution of a mixture of ionizable compounds with a 60% acetonitrile mobile phase prepared from aqueous buffers of wwpH 8.0. (A) H2PO4-/HPO42- buffer; (B) NH4+/NH3 buffer. Compounds: 2-nitrophenol (1), 2,4,6-trimethylpyridine (2), 3-bromophenol (3), and N,Ndimethylbenzylamine (4). Chromatograms for the individual compounds in each mobile phase are also given.
In this instance, a small variation of the pH of the mobile phase should not influence ionization of the bases and therefore the retention relative to the neutral compound acenaphthene should not change. However, Neue noticed that the “apparent” pKa values of the bases (caused by combination of the phosphate buffer pH increase and base pKa decrease with the addition of organic modifier) were found to be around 6.5-7, i.e., around the wwpH value of the buffer. Therefore, in fact, the bases were more or less half-protonated (R ≈ 0.5) and small variation of pH caused appreciable variation of ionization and retention. In the ammonia buffer, the decrease of ionization is the contrary. The ionization of bases decreases slightly because the pK of the bases decreases more than the pH of the buffer with addition of acetonitrile (mpK < mpH < 0). The ionization of acids decreases much faster because the pKa of the acid increases when the pH of the buffer decreases (mpK > 0 > mpH). For instance, in 60% acetonitrile, 2-nitrophenol is highly ionized and N,N-dimethylbenzylamine almost not ionized in the phosphate buffer, but 2-nitrophenol is poorly ionized and N,N-dimethylbenzylamine quite ionized in the ammonia buffer. The different variation of the degree of ionization of ionizable compounds with addition of acetonitrile to aqueous buffers of the same pH value, but prepared from different buffer components, may lead to significant differences in RP HPLC retention. This has been tested for a mixture of 2-nitrophenol, 3-bromophenol, 2,4,6-trimethylpyridine, and N,N-dimethylbenzylamine eluted with a mobile phase with a 60% acetonitrile prepared from the aqueous phosphate or ammonia buffers at wwpH 8.00. The chromatograms obtained are presented in Figure 6. Figure 5 shows that 3-bromophenol and 2,4,6-trimethylpyridine are almost ionized in both buffers and thus their retention time must be the same with both mobile phases. N,N-Dimethylbenzylamine is almost not ionized in the phosphate buffer and in fact it shows the largest retention with this mobile phase (Figure 6A), but it is quite ionized in the ammonia buffer and thus it shows the lowest retention (Figure 6B). For 2-nitrophenol, the reverse behavior is observed. It is 3818 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
highly ionized in the phosphate buffer where it shows the lowest retention (Figure 6A), but it is poorly ionized in the ammonia buffer where it shows the largest retention (Figure 6B). CONCLUSIONS This study points out that the pH and buffer capacity of RP HPLC aqueous buffers change with the addition of organic modifier (acetonitrile). The buffer capacity of the buffer decreases mostly by dilution of the buffer concentration, although the variation of the pKa values of polyprotic acids used as buffer components may decrease buffer capacity too. The variation of the pH value of the buffer depends on the particular buffer components and composition. This pH change may influence the RP HPLC separation of acid/base compounds. A model has been proposed to allow an accurate prediction of this pH change for buffers in acetonitrile-water mixtures (phosphate, acetate, citrate, and ammonia buffers). The model can be used to choose what aqueous buffer will be the best to prepare an acetonitrile-water buffer with a required pH value. It has also been used to predict the change in the degree of ionization of acid/base compounds caused by the addition of acetonitrile to a particular aqueous buffer in order to prepare an RP HPLC mobile phase. The relative retention of the different compounds can be assayed from this degree of ionization. ACKNOWLEDGMENT We are thankful for joint financial support from the MCYT of the Spanish Government and FEDER of EU (projects BQU2001-2882 and BQU2001-3226) and from the Catalan Government (Grant 2001SGR00055). S.E. was supported by a grant from the Catalan Government (1998FI 00639). We are also grateful to the anonimous reviewers of this paper for helpful comments. Received for review January 8, 2002. Accepted April 29, 2002. AC020012Y
