Anal. Chem. 2003, 75, 3404-3410
Rationalization of Retention and Overloading Behavior of Basic Compounds in Reversed-Phase HPLC Using Low Ionic Strength Buffers Suitable for Mass Spectrometric Detection David V. McCalley*
Centre for Research in Biomedicine, University of the West of England, Frenchay, Bristol BS16 1QY, U.K.
The retention and overloading behavior of some basic (and acidic) compounds has been studied on different RPHPLC columns in buffers of varying ionic strength. Anomalous retention patterns of acids and bases were found on one phase in low-pH, volatile buffers such as formic acid, favored for mass spectrometric analysis. Unusual retention compared to that in higher ionic strength phosphate buffers is attributed to the presence of positively charged sites existing on this phase at low pH. Overloading of bases as well as acids is shown to be a function of mobile-phase ionic strength. This result is a logical consequence of previous suggestions that mutual repulsion of ions held on the hydrophobic surface of the stationary phase, rather than overload of silanols, is largely responsible for overloading on pure silica RP columns. Thus, overloading occurs much more readily in low ionic strength formic acid buffers. Appreciable loss of efficiency can occur in such buffers when only 50 ng of some bases is analyzed on a standard-sized column. The analysis of ionogenic compounds using RP columns continues to be an area of difficulty in HPLC. The behavior in particular of basic compounds, of which many pharmaceuticals and those of biomedical significance form an important group, is generally less predictable and more problematic than that of neutral species. For example, RP columns are much more easily overloaded by bases than neutrals, when they are analyzed in protonated form with mobile phases of low pH.1-3 Furthermore, basic compounds can suffer from peak tailing and poor efficiency on silica-based RP columns, due to detrimental ionic interactions with ionized column silanol groups.4 HPLC with mass spectrometric (MS) detection is becoming increasingly popular, due to advantages over HPLC-UV in specificity for qualitative analysis, universality of the detection system, and sometimes also increased sensitivity of the technique. However, transfer of methods using phosphate buffers favored in HPLC-UV to low ionic strength volatile buffers favored in MS * Fax: 117 3442904. E-mail:
[email protected]. (1) McCalley, D. V. J. Chromatogr., A 1998, 793, 31-46. (2) Buckenmaier, S. M. C.; McCalley, D. V.; Euerby, M. R. Anal. Chem. 2002, 74, 4672-4681. (3) Eble, J. E.; Grob, R. L.; Antle, P. E.; Snyder, L. R. J. Chromatogr. 1987, 384, 45-79. (4) McCalley, D. V. LCGC 1999, 17, 440-456.
3404 Analytical Chemistry, Vol. 75, No. 14, July 15, 2003
detection is not always straightforward. For example, we have noted exclusion effects at low pH, leading to very low or even negative retention factor (k) values for some bases at low pH when formic acid is used with some RP columns.5 Consequently, substantially different separations can be obtained for bases using formic acid instead of phosphate buffers of the same molar concentration at the same acid pH. Furthermore, peak shapes of bases were shown to be somewhat inferior to those obtained in phosphate buffers when formic acid is used at low pH. We previously were unable to rationalize the differences obtained in these buffers. Although alternative approaches exist, low pH is often favored as a first choice for the analysis of bases, because silanols are largely un-ionized, leading to better peak shapes than can be obtained at neutral pH. Low pH is also the method of choice for the analysis of peptides and small proteins using RP chromatography,6 in conjunction with mass spectrometric detection. Involatile buffers can give ion source contamination and loss of performance. In addition to a preference for volatile buffers, buffers of low concentration are also superior in MS because they tend to lead to less signal suppression and loss of sensitivity.7,8 We have shown experimentally that a likely mechanism of overloading of bases at low pH is ionic repulsion between protonated species held on the hydrophobic surface of the stationary phase, at least on inert “type B” silica-ODS phases, which have a minimum number of acidic silanol groups 2. The idea that overloading of bases might be due to these mutual repulsion effects was first proposed by Snyder and co-workers, although overloading of ionized silanols on “type A” phases (phases with a large number of acidic silanols) was also considered.3,9 In a complex theoretical study based on an electrostatically modified Langmuir adsorption isotherm, Ha¨gglund and Ståhlberg calculated overload profiles and compared them with a small number of experimental results for acidic compounds such as p-toluenesulfonate. Theoretical results broadly agreed with experiments showing that overloading effects increased in pH 3 sodium phosphate buffers as the ionic strength was reduced.10,11 In light (5) McCalley, D. V. J. Chromatogr., A 2003, 987, 17-28. (6) Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. Practical HPLC Method Development; Wiley: New York, 1997. (7) Temesi, D.; Law, B. LCGC (Int.) 1999, 12, 175-180. (8) Law, B.; Temesi, D. J. Chromatogr., B 2000, 748, 21-30. (9) Cox, G. B.; Snyder, L. R. J. Chromatogr. 1989, 483, 95-110. (10) Ha¨gglund, I.; Ståhlberg, J. J. Chromatogr., A 1997, 761, 3-11. (11) Ha¨gglund, I.; Ståhlberg, J. J. Chromatogr., A 1997, 761, 13-20. 10.1021/ac020715f CCC: $25.00
© 2003 American Chemical Society Published on Web 06/06/2003
of these results, we decided to study the overloading behavior of bases (as well as acids) as a function of ionic strength, since to our knowledge, such a study has not been carried out previously on these important compounds. We have included a study of the overloading effects in formic acid solutions commonly used in HPLC-MS, to determine whether it may contribute to the inferior peak shapes of bases found in this weakly acidic (low ionic strength) mobile phase.5 We have also extended our previous investigations of anomalous retention effects in low ionic strength mobile phases found on some stationary phases to include acidic as well as basic compounds, to understand their origin.5 EXPERIMENTAL SECTION An Agilent 1100 HPLC system with high-pressure binary mixing and Chemstation computer data processing was used in all experiments, together with UV detection at 215 nm, employing a 1-µL detector cell and small lengths of 0.01-cm-i.d. connection tubing to minimize extracolumn effects. Temperature control was achieved by immersing the column and injector in a thermostat water bath held at 30 °C. A 3 m × 0.5 mm i.d. length of stainless steel tubing connected between the pump and injector and also immersed in the bath was used to preheat the mobile phase before delivery to the injector and column. Column efficiency (N) was determined using the half-height and Dorsey-Foley procedure, the latter using the formula
Ndf ) 41.7[tr /w0.1]2/[As +1.25]
(?)
where tr is the retention time, w0.1 is the peak width at 10% of peak height, and As is the peak asymmetry factor.12 For determining the column saturation capacity (ws) for heavily overloaded peaks, which gave right-angled triangle shapes, we used the specific method advocated by Snyder and co-workers to calculate column efficiency.13 ws was then calculated from the formula
Wbase )
16t02(1 + k0)2 6t02k02wx + N0 ws
where Wbase is the peak width at base, wx is the sample mass of an overloaded peak, t0 is the column dead time, k0 is the retention factor, and N0 the column efficiency for a small sample.6 In principle, ws can then be calculated from only two injections, the first with a low sample mass and the second such that the peak is quite heavily overloaded. In practice, more injections are required to establish the point at which further reducing sample mass no longer gives peak shape improvement and to determine an appropriate sample mass to overload the column, where typically a right-angled triangle shape is produced.13 The asymmetry factor (As) was measured by dropping a perpendicular from the peak apex to the baseline and calculating the ratio of the widths of the rear and front sides of the peak at 10% of peak height. The United States Pharmacopoeia tailing factor was calculated in a similar fashion from the width of the peak divided by twice the width of the front side of the peak, at 5% of peak height. All (12) Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983, 55, 730-737. (13) Snyder, L. R.; Cox, G. B.; Antle, P. E. Chromatographia 1987, 24, 82-96.
Figure 1. Separation of acidic and basic test compounds on a Symmetry 100 column. Mobile phase A, acetonitrile-0.02 M phosphate buffer, pH 2.7 (28:72 v/v). Mobile phase B, acetonitrile-0.02 M formic acid, pH 2.7 (28:72 v/v). Flow rate, 1 cm3 min-1. Detection, UV at 215 nm. Column temperature, 30 °C.
measurements were made using the Chemstation. Phosphate buffer was prepared from KH2PO4, adjusting to the required pH with concentrated H3PO4. Formate buffer was prepared by weighing the appropriate quantity of pure acid. pH was measured in all cases before addition of organic solvent. pH changes caused by increase in ionic strength due to addition of KCl were at most 0.04 pH unit and were ignored.14 All results were the mean of at least duplicate injections. Column void volume was measured by injection of uracil. Columns were equilibrated by passage of at least 25 column volumes of mobile phase. However, using Symmetry 100, the retention of bases was found to drift downward, and that of acids to drift upward, as previously noted by Snyder and co-workers.15 We observed this effect particularly with low ionic strength mobile phases. For this phase, columns were equilibrated typically with 75 volumes of mobile phase in order to achieve stable retention factors. The columns were all previously unused bonded silica-ODS phases. They comprised Symmetry 100 (surface area (SA) 341 m2 g-1, % C 19.9, pore diameter 9 nm), Symmetry 300 (SA 112 m2 g-1, % C 8.5, pore diameter 24 nm), Discovery C18 (SA 194 m2 g-1, % C 12.6, pore diameter 19 nm), Chromolith, 10 cm × 0.46 cm i.d. (monolithic column, SA 300 m2 g-1, % C 18.0, mesopore diameter 19 nm). All had 5-µm particle size and were 25 cm × 0.46 cm i.d. except where stated. Acetonitrile (far-UV grade), phosphate salts, and formic acid were obtained from Fisher Scientific (Loughborough, U.K.) and test solutes from Sigma-Aldrich (Poole, U.K.). Acetonitrile is highly flammable and toxic. It needs handling in a fume hood using appropriate protective equipment. Solvent wastes require specialist disposal (e.g., high-temperature incineration). RESULTS AND DISCUSSION Retention Differences of Acids and Bases in Formic Acid and Phosphate Buffers at Low pH. Figure 1 shows chromatograms of two strong acids (benzenesulfonic and naphthalene(14) Perrin, D. D.; Dempsey, B. Buffers for pH and metal ion control; Chapman and Hall: London, 1974. (15) Marchand, D. H.; Williams, L. A.; Dolan, J. W.; Snyder, L. R. J. Chromatogr., A, in press.
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Table 1. Values of k for Acidic and Basic Compounds on Four Different Phases Using Acetonitrile-Phosphate or Acetonitrile-Formate Buffer (28:72, v/v, Both 0.02 M, pH 2.7) BSA
2-NSA
diphen
nortrip
column
phosph
form.
phosph
form.
phosph
form.
phosph
form.
Symmetry 100 Symmetry 300 Discovery C18 Chromolith Performance
0.25 0.11 0.09 0.06
3.1 0.53 0.38 0.13
1.26 0.62 0.58 0.36
9.6 1.9 1.2 0.54
4.5 3.3 3.8 2.5
0.58 3.7 2.4 1.6
12.1 8.8 10.1 6.5
2.0 9.9 6.7 4.4
sulfonic) and two strong bases (diphenhydramine and nortriptyline) using phosphate and formic acid buffers (0.02 M, pH 2.7) mixed with acetonitrile (72:28, v/v) on a Symmetry 100 C18 column. These compounds are all expected to be in the ionic form at the pH of the mobile phase (pKa values of BSA, 2-naphthalenesulfonic acid (2-NSA), diphenhydramine (diphen), and nortriptyline (nortrip) 0.88, 1.00, 9.0, and 10.0, respectively.16,17 Clearly, surprising and substantial differences in retention exist dependent on choice of buffer. Using formic acid, the acids elute after the bases; however, using phosphate buffer of the same molar concentration and pH, the bases elute after the acids. This reversal in the retention of acids and bases when formic acid is used instead of phosphate is shown only for the Symmetry 100 phase. Table 1 lists k for the four compounds using four different RP columns tested with the same mobile phases. For Symmetry 100, k for bases decreases by a factor of 6-8 times, whereas k for acids increases by a factor of 7-12 times when a change from phosphate to formate is made. For Discovery C18 and Chromolith, smaller changes are shown, e.g., for Discovery; k for bases decreases by a factor of ∼1.5, whereas k for acids increases by 2-4 times. For Symmetry 300, the increase in retention for acids is rather similar to Discovery and Chromolith; however, for this phase, the retention of bases increases slightly in formate buffer. Rose´s and co-workers, including researchers from Waters, the manufacturer of Symmetry columns, used lithium nitrate as a probe and mass spectrometric detection to investigate the presence of ionized groups on Symmetry 100 and a number of other Waters phases.18 It was shown that, using a pH value of 6.0 (measured in the organic solvent-water combination), NO3elutes just after Li + since no cation exchange was found for this column in the pH range 3-6. However, at acidic pH values (pH 3.4), NO3- very surprisingly eluted several minutes after Li+, indicating that NO3- is retained by ion exchange. The anion exchange process was confirmed by the authors using measurements of the MS chromatograms of the buffer anions as well as the solute ions. Furthermore, the authors demonstrated a sigmoidal dependence of the retention of NO3- on Symmetry C18, which is typical of anion exchange, yielding an estimate of the pKa value of the sites around 5.4. This behavior could not be demonstrated on the other phases studied. The presence of anion exchange sites was attributed to the presence of residues of the particular base catalyst used in the proprietary bonding process. A likely explanation of our results on Symmetry 100 is that protonated bases undergo repulsion from protonated stationary(16) Sparc on-line calculator, UGA, 2001; http://ibmlc2.chem.uga.edu/sparc/ index.cfm. (17) McCalley, D. V. J. Chromatogr., A 1996, 738, 169-179. (18) Mende´z, A.; Bosch, E.; Rose´s, M.; Neue, U. D. J. Chromatogr., A 2003, 986, 33-44.
3406 Analytical Chemistry, Vol. 75, No. 14, July 15, 2003
Table 2. Effect of Increasing Ionic Strength on k of Acids and Bases, Symmetry 100a mobile phase
BSA
2-NSA
diphen
nortrip
0.004M phosph 0.004 M phosph +0.008 M KCl 0.004 M phosph + 0.016 M KCl 0.004 M phosph, pH 2.7
0.57 0.26 0.19 0.57
2.2 1.2 1.1 2.3
2.8 4.9 5.3 2.9
7.8 13.1 14.2 8.1
a Starting mobile phase, acetonitrile-0.004 M phosphate buffer, pH 3.1 (28:72, v/v).
phase groups when low ionic strength buffers such as formic acid are used. Thus, some parts of the hydrophobic stationary phase may not be accessible to the bases. For some basic solutes on Symmetry 100, total exclusion occurs in formic acid, resulting in negative k values.5 Formic acid has a pKa of 3.75, and from Ostwald’s dilution law, a 0.02 M solution can be calculated to be only ∼9% ionized. The ionic strength of this solution is less than one-tenth that of a 0.02 M phosphate buffer of the same pH prepared as described in the Experimental Section. The greater concentration of ions in the phosphate buffer reduces the repulsion between protonated basic solute and protonated column groups, allowing increased hydrophobic interaction to occur. At the same time, the higher concentration of anion in the phosphate buffer competes with negatively charged solutes for column anion exchange sites, giving reduced retention of the acidic solutes. Table 2 shows k obtained when 0.02 M phosphate buffer is diluted five times (resulting in a slightly higher pH of 3.1) and for subsequent spiking of this buffer with KCl to increase ionic strength. Clearly, the more dilute phosphate buffer (0.004 M) gives considerably reduced k for bases and increased k for acids compared with 0.02 M phosphate buffer, giving k values more similar to those obtained with formic acid. We did not reduce the phosphate buffer strength further due to the resultant low buffer capacity of such a mobile phase. As the ionic strength is increased by addition of KCl, k for both acids and bases returns to values similar to those obtained with 0.02 M phosphate buffer. This experiment proves that the anomalous retention pattern in formic acid is largely due to an ionic strength effect, rather than being due to some peculiar property of formic acid. However, other effects could contribute to the retention differences, such as Hofmeister series or “salting out” effects, which tend to increase hydrophobic retention of compounds. Buffer ions such as phosphate and formate could also adsorb to different extents on the stationary phase, giving different “ion pair” effects.19 Note that (19) Roberts, J. M.; Diaz, A. R.; Fortin, D. T.; Friedle, J. M.; Piper, S. D. Anal. Chem. 2002, 74, 4927-4932.
Table 3. Effect of Increasing Ionic Strength on k of Acids and Bases, Discovery C18a mobile phase
BSA
2-NSA
diphen
nortrip
0.004 M phosph 0.02 M phosph 0.02 M phosph + 0.02 M KCl 0.02 M phosph + 0.04 M KCl 0.02 M phosph + 0.06 M KCl
0.15 0.09 0.07 0.07 0.06
0.72 0.58 0.55 0.53 0.52
3.5 3.8 4.4 4.5 4.4
9.5 10.1 11.7 12.0 11.7
a
Mobile phase, acetonitrile-phosphate buffer, pH 2.7 (28:72, v/v).
the small change in pH from 2.7 to 3.1 appears to have little effect on the retention of the acids and bases (Table 2). This observation is not surprising considering the pKa values of the probe compounds (see above), which would predict little change in the ionization state of the compounds over this pH range; also the acid used in pH adjustment contributes relatively little to the total ionic strength. The within-batch and batch-to-batch reproducibility specifically of Symmetry 100 C18 columns (and in other publications, of C18 stationary phases from other leading manufacturers) was studied in great detail by Kele and Guiochon.20 The batch-to-batch reproducibility of retention for 27 out of 30 components tested was measured as below 1.4% RSD, which was reported as “most impressive” by the authors. This good within-batch and batch-tobatch reproducibility of modern silica C18 phases has recently been reaffirmed in a publication by Stella et al.21 Furthermore, we have observed these unusual retention properties of Symmetry 100 both on the present column and a similar one made from a different batch of the material.5 Thus, we conclude that the behavior of Symmetry 100 columns reported here is representative of the typical performance of this phase. Table 3 shows a similar study of the effect of ionic strength on the retention of the test compounds for Discovery C18 but using rather higher buffer concentration and ionic strength. As buffer ionic strength increases, small drops in the retention of acids together with small increases in the retention of bases occur, although as before, these changes level out at higher buffer ionic strength. Although changes are in the same direction as found for Symmetry 100, the magnitude is considerably less. For example, when changing from 0.02 to 0.004 M phosphate buffer, pH 2.7, k for diphenhydramine drops by almost 40% on Symmetry 100 (see Tables 1 and 2) but only ∼8% for Discovery (Table 3). It is conceivable that some positively charged groups exist also on Discovery C18 (and Chromolith, which from Table 1 shows some similarity in behavior to Discovery) at low pH. However, the small increases in retention of the bases as ionic strength increases could instead be attributed to Hofmeister or salting-out effects, or ion pair effects that would increase hydrophobic retention of the bases.19 Clearly the finding that the retention of protonated bases increases with addition of salt is quite different from the behavior of older silicas, where retention decreases under similar conditions. Over the last 10 years, manufacturers have introduced ODS phases based on very high purity silica substrates (type B silicas). In contrast, older silicas (type A silicas) have significant (20) Kele, M.; Guiochon, G. J. Chromatogr., A 1999, 830, 55-79. (21) Stella, C.; Seuret, P.; Rudaz, S.; Tchapla, A.; Gauverit, J.-Y.; Lanteri, P.; Veuthey, J.-L. Chromatographia 2002, 56, 665-671.
concentrations of metal impurities that can enhance the acidity of neighboring silanol groups.4 These acidic silanol groups can remain ionized even at quite low pH and thus strongly retain protonated bases by cation exchange.22 Retention factors for the bases diphenhydramine and nortriptyline on the large-pore Symmetry 300 phase are similar in phosphate and formate buffers (Table 1). Unusually, in comparison with the behavior of the other phases, the retention of both acids and bases is higher in formic acid than in phosphate. It is possible that protonated solutes might not be excluded from some parts of the stationary phase in a large-pore packing, even if positively charged stationary phase sites existed. However, the higher retention of the acids BSA and 2-NSA in formic acid may again be indicative of the presence of such sites, with the negatively charged acids having greater retention in this low ionic strength buffer. We considered the possibility of making physical measurements (such as of ζ potential) on the packing materials in the different mobile phases studied in this work. However, physical measurements have in general proved to be much less sensitive in detecting changes in chromatographic materials than chromatographic measurements themselves.23 It is also often very difficult to make such measurements under the exact conditions used for chromatography, which may greatly reduce their value. Effect of Sample Load on Column Performance in Formate and Phosphate Buffers. We noted previously5 that peak shapes for bases were considerably worse (for compounds not excluded from the stationary phase) using formate compared with phosphate buffers at low pH although we could not explain this observation. It is possible that competitive interactions of buffer K+ ions in the phosphate buffer occur with ionized (negatively charged) silanols, giving reduced tailing. A small number of highly acidic silanols could contribute to this tailing of the basic solutes, even if these sites do not contribute greatly to retention, as has been shown by the ionic strength experiments detailed above. An alternative explanation of the different peak shapes could be that the same column has different sample capacity in different buffers. Whereas we have previously reported varying sample capacities when using mobile phases of different pH,1,2 to our knowledge a change in sample capacity using different buffers at the same pH has not been reported previously. As discussed above, if mutual repulsion of protonated solutes is the major reason for overload at low pH on highly inert phases made from pure silica,2 then indeed it might be predicted that the ionic strength of the buffer could influence overload. Table 4 shows the column saturation capacities (ws, see above) measured for all four columns in the study, in both phosphate and formate buffers at pH 2.7. In almost all cases, right-angled triangle-shaped peaks were obtained as sample load was increased, allowing ready calculation of ws.6 For diphenhydramine on Symmetry 100, a distorted peak was obtained showing evidence of peak splitting. For all compounds on Chromolith using formic acid, peaks exhibited distortion and fronting even at low sample load; no results are shown in these cases. Results are not quoted for BSA due to the low k values often obtained with the specified mobile phases, which would reduce the accuracy of the calculation. The results show clearly (22) Cox, G. B.; Stout, R. W. J. Chromatogr. 1987, 384, 315-336. (23) Nawrocki, J. J. Chromatogr., A 1997, 656, 29-71.
Analytical Chemistry, Vol. 75, No. 14, July 15, 2003
3407
Table 4. ws Values (mg) for Test Solutes Measured in Formic and Phosphate Buffersa 2-NSA
diphen
Table 5. Effect of Increasing Ionic Strength on ws (mg) on Symmetry 100a
nortrip
column
phosph
form.
phosph
form.
phosph
form.
Symmetry 100 Symmetry 300 Discovery C18 Chromolith Performanceb
1.5 1.0 1.0 0.6
0.8 0.2 0.3
5.6 3.9 5.1 1.8
0.5 0.6
6.2 4.3 5.8 1.8
1.4 0.5 0.7
a Mobile phase, acetonitrile-0.02 M phosphate buffer, pH 2.7 (28: 72, v/v) in each case. b Values for Chromolith normalized for 25-cm column length.
that the column saturation capacity is considerably reduced for bases in formic acid compared with phosphate buffer. Using phosphate buffer, ws is ∼4-9 times greater for nortriptyline on the three particulate columns, and ∼8 times greater for diphenhydramine on two of these phases, than the values in formic acid. The sample capacity for the acid 2-NSA is also increased in phosphate buffer by ∼2-5 times. These results suggest that the higher ionic strength of the phosphate buffer could reduce the mutual repulsion of protonated base held on the hydrophobic surface. Furthermore, since acids are also affected, a similar overloading mechanism is indicated for both acids and bases. Both of these results provide further evidence that mutual repulsion, rather than silanol overload, is largely responsible for loading behavior of protonated bases on inert, new-generation silica phases.2 Some further deductions from Table 4 are possible. Widepore Symmetry is more easily overloaded that the narrow-pore column. This result can be attributed to the lower surface area of the underlying silica (note that both columns have approximately the same ODS coverage in micromoles per square meter; manufacturer’s data). ws values for Chromolith are normalized to a 25-cm column length, so that they can be compared with those for the three particulate columns. Clearly, the capacity of the monolith is a factor of ∼3 times less than the standard-pore particulate phases, which can be attributed (although see caution below) to the sparser silica structure and, thus, the smaller mass of stationary phase in the column.24 Finally, it is somewhat intriguing that ws values for Symmetry 100 are rather similar to those for Discovery C18. If Symmetry has positive charges on the surface at low pH, it might be expected that sample capacity for protonated bases might be low, since the surface positive charge might contribute to mutual repulsion of the solutes. However, a direct comparison of any phases from different manufacturers that are based on totally different silica substrates is somewhat dangerous, considering that they differ in so many other ways. For instance, Discovery and Symmetry 100 have quite different surface areas (see Experimental Section). The question arises again as to whether the reduced capacity of the phases in formic acid is due rather to some peculiar property of formic acid or whether it is really due to the ionic strength effect proposed above. Thus, we investigated the variation in ws using phosphate buffers of different concentration, also adding KCl to increase the ionic strength (see Table 5). In 0.004 M phosphate buffer, Symmetry 100 shows low ws values, similar to (24) McCalley, D. V. J. Chromatogr., A 2002, 965, 51-64.
3408 Analytical Chemistry, Vol. 75, No. 14, July 15, 2003
mobile phase
2-NSA
diphen
nortrip
0.004 M phosphate 0.004 M phosphate + 0.008 M KCl 0.004 M phosphate + 0.016 M KCl
1.0 1.3 1.8
1.6 4.6 6.5
1.5 5.1 7.1
a Starting mobile phase, acetonitrile-0.004 M phosphate buffer, pH 3.1 (28:72, v/v).
Table 6. Effect of Increasing Ionic Strength on ws (mg) on Discovery C18a mobile phase
2-NSA
diphen
nortrip
0.004 M phosphate 0.02 M phosphate 0.02 M phosphate + 0.02 M KCl 0.02 M phosphate + 0.04 M KCl 0.02 M phosphate + 0.06 M KCl
0.8 1.0 2.1 2.1 2.2
1.7 5.1 8.1 10.5 12.1
2.0 5.8 9.0 11.8 13.7
a
Mobile phases, acetonitrile-phosphate buffer, pH 2.7 (28:72, v/v).
those found with the same column using formic acid (compare Table 4). As the mobile phase is spiked with KCl, ws values increase for both bases and 2-NSA. The values of ws for 2-NSA, diphenhydramine, and nortriptyline in 0.004 M phosphate buffer spiked with 0.016 M KCl (1.8, 6.5, and 7.1 mg, respectively, Table 5) are similar to those found for the same column in 0.02 M phosphate buffer (1.5, 5.6, and 6.2 mg, respectively, Table 4). Since addition of KCl has no effect on buffer capacity, the buffer capacity of the former mobile phase is considerably lower than that of the latter. This indicates that no buffer overload has occurred in these measurements. Table 6 shows similar results for the Discovery column, including values of ws measured at rather higher ionic strength than for Symmetry. ws values are shown to increase considerably when changing from 0.004 M phosphate buffer to 0.02 M phosphate buffer to which 0.06 M KCl has been added. The increase is by a factor of ∼3 for 2-NSA and by a factor of 6-7 for diphenhydramine and nortriptyline. These experiments add weight to the proposal that the differences in ws values shown in Table 4 between formate and phosphate buffers are due principally to differences in ionic strength rather than being connected with particular properties of the individual buffers. Clearly, if ws values are much lower in buffers such as formic acid, deterioration in peak shape may be encountered at even lower levels than normally expected. For instance, as a “rule of thumb”, sample masses below ∼0.5 µg are recommended to avoid overloading for typical basic solutes on standard size columns, when used with phosphate buffers.25,26 Overloading may be somewhat more critical for monolithic or wide-pore columns. Figure 2 shows a plot of column efficiency (measured using the half-height method) against sample load for diphenhydramine analyzed on Discovery C18 using either acetonitrile-phosphate or acetonitrile-formate buffer (0.02 M, pH 2.7). Whereas sample masses of 0.5 µg and below have little effect on N using the phosphate buffer, 0.5 µg causes an ∼50% loss in efficiency when (25) Leach, D. C.; Stadalius, M. A.; Berus, J. S.; Snyder, L. R. LCGC (Int.) 1988, 1, 22-30. (26) McCalley, D. V.; Brereton, R. G. J. Chromatogr., A 1998, 828, 407-420.
Figure 2. Column efficiency (N) vs sample load: Discovery C18 effect of (a) diphenhydramine, (b) nortriptyline, and (c) 2-naphthalenesulfonic acid load on N. Mobile phases and other conditions as Figure 1.
the formate buffer is used. Note that k for diphenhydramine is smaller using formate buffer than phosphate buffer (Figure 2); i.e., a greater proportion of the injected solute will be in the mobile phase at any instant in the formate buffer. Thus, on retention considerations alone, peak shape deterioration is predicted to occur less readily in this case using formate buffer. Loss of efficiency of ∼10% occurs when sample masses higher than ∼0.05 µg (representing only 10 µL of a 5 ppm solution of the analyte!) are injected using formate buffer. This finding indicates that serious problems with peak shape may occur due to overloading in “mass spectrometry” friendly buffers. Previously, we compared column performance in phosphate and formic acid buffers used a fixed sample mass of 0.2 µg.5 Reduced column efficiency is now
shown to be due to overloading in the formate buffer, even with this rather small sample mass. Very similar load plots are shown in Figure 2 for nortriptyline and also for the acidic compound 2-NSA. The similar overloading behavior of acids and bases, which we demonstrated previously for purely polymeric phases but not for silica ODS phases,2 is indicative of a similar overloading mechanism for negatively and positively charged compounds. Note, however, that k for 2-NSA is greater in formate buffer than phosphate; this factor would tend to contribute to readier loss of efficiency in formate for this compound. However, ws values (Table 4), which take k into account, still indicate a considerably higher ws for 2-NSA in phosphate buffer. Analytical Chemistry, Vol. 75, No. 14, July 15, 2003
3409
Table 7. Column Efficiency and Asymmetry Measurements under Nonoverloading Conditionsa,b 2-NSA
diphenhydramine
nortriptyline
column
N
N (df)
As
Sf
N
N (df)
As
Sf
N
N (df)
As
Sf
Symmetry 100 Discovery C18 Symmetry 300 Chromolithb
13 500 20 200 12 700 20 700
6 610 12 500 8 700 10 100
2.1 1.4 1.5 1.9
1.7 1.2 1.4 1.6
19 000 20 100 16 100 25 900
15 900 15 300 10 400 13 000
1.2 1.3 1.6 1.7
1.1 1.1 1.4 1.7
19 400 19 900 16 100 22 800
16 600 15 400 11 800 3 200
1.2 1.3 1.5 3.2
1.1 1.1 1.3 2.6
a Mobile phase, acetonitrile-0.02 M phosphate buffer, pH 2.7 (28:72, v/v). b Values normalized to a 25-cm column length. S , U.S. Pharmacoepeia f tailing factor.
Finally, Figure 2 shows that providing a sufficiently small amount of acidic or basic compound (e.g., 0.01 µg) is injected, the column efficiency is approximately the same in both formate and phosphate buffers. For the basic compounds, this observation seems to confirm that very few ionized silanols are present at this low pH on Discovery C18, otherwise a greater deactivating effect might have been expected due to the presence of K+ ions in the phosphate buffer leading to better peak shape. Also, the result appears to discount the possibility that a small number of ionized silanols exist, which could contribute to peak tailing but not appreciably to retention. Table 7 shows column performance parameters in phosphate buffer (0.02 M, pH 2.7) for 2-NSA, diphenhydramine, and nortriptyline on all four columns using a sample load that does not cause any peak shape deterioration in this mobile phase (0.05 µg). Symmetry 100 and Discovery give rather good peak shapes for the bases with relatively little tailing (asymmetry factors 1.3 or less), indicating low silanol interactions under the experimental conditions. Indeed, it is possible that peak shape problems for basic compounds on some highly inert stationary phases with low-pH mobile phases may be almost entirely due to phase overloading. Nevertheless, the wide-pore Symmetry phase shows greater evidence of the presence of ionized silanols, giving somewhat tailing peaks for bases even for these low sample masses. It is conceivable that fewer possibilities for silanol association in large-pore phases are the reason for this increased activity,15 resulting in more acidic ionized silanols that can interact with protonated bases. The Chromolith phase shows peak tailing for nortriptyline, as found previously.5 Thus, the column efficiency for nortriptyline using the Dorsey-Foley procedure, which takes peak tailing into account, is considerably less than for the other columns, although the half-height efficiency is better for both bases. However, some of this tailing is also found for neutral compounds (which gave asymmetry factors of 1.61.8; results not shown), so it is difficult to draw firm conclusions about the presence of ionized silanols on this phase. A major advantage of this latter phase is the relatively small deterioration in column efficiency that occurs with flow rate,24 a benefit that we have not exploited in the present study. For analysis of the (27) Atkins, P. W.; de Paula, J. Atkins’ Physical Chemistry, 7th ed.; Oxford University Press: Oxford, U.K., 2002.
3410 Analytical Chemistry, Vol. 75, No. 14, July 15, 2003
acid 2-NSA, Symmetry 100 gives the greatest tailing and lowest Dorsey-Foley efficiency, as would be expected if positively charged sites were present on the surface of this phase. CONCLUSIONS It has been shown that column capacity in RP chromatography for basic as well as acidic compounds at low pH decreases as the ionic strength of the mobile phase decreases. This result has important consequences for separation of pharmaceuticals, but also possibly peptides and proteins, when analyzed by HPLCMS using volatile organic buffers such as formic acid. Overloading effects can easily degrade the efficiency of a column and can be observed in some cases when as little as 50 ng of substance is analyzed on a 25 cm × 0.46 cm i.d. column using 0.02 M formic acid. This result can be attributed to the increased mutual repulsion effect in low ionic strength buffers of protonated cations held on the surface. These observations add further weight to the proposal that mutual repulsion of ions is the cause of overload rather than overload of ionized silanols. This proposal apparently holds at least on new-generation RP-HPLC columns made from pure silica, which appear to show little indication of ionized silanols on the surface at the low pH used in our study (pH 2.7). The mutual repulsion hypothesis has some similarity to the theory of Derjaguin, Landau, Verwey, and Overbeek, which describes the Coulombic repulsive and van der Waals attractive forces that exist between colloids.27 Positively charged sites, which exist on at least one popular C18 stationary phase, can lead to major differences in the elution patterns of acids and bases when low ionic strength buffers are utilized for HPLC-MS, compared with results obtained in phosphate buffers. Retention of acid anions may be greatly increased, whereas retention of protonated bases may decrease due to repulsion from some areas of the stationary phase. These effects are masked in phosphate buffers of the usual concentration that are employed for HPLC-UV analysis.
Received for review November 19, 2002. Accepted April 24, 2003. AC020715F
