Investigation of Anion Retention and Cation Exclusion Effects for

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Anal. Chem. 2007, 79, 5382-5391

Investigation of Anion Retention and Cation Exclusion Effects for Several C18 Stationary Phases Eric Loeser* and Patrick Drumm

Chemical and Analytical Development, Novartis Pharmaceuticals, East Hanover, New Jersey 07936

When mobile-phase salt content is increased, cationic analytes often show increased retention. This effect is generally attributed to chaotropic or ion pairing effects. However, a cation exclusion mechanism could explain the same effects. In this study, experimental conditions were manipulated to enhance cation exclusion effects and reduce chaotropic/ion pairing effects by using (1) low ionic strength mobile phases to reduce electrostatic screening, (2) a buffer anion (dihydrogen phosphate) that exhibits minimal chaotropic/ion pairing effects, and (3) columns that show evidence of a weak positive charge. Urea was used as neutral void marker and glycinamide (in protonated form) as cationic void marker. It was assumed the difference in retention volumes between void markers would reflect an “excluded volume”, inaccessible to cationic analytes. As ionic strength was lowered, it appeared as much as 80% of the pore volume became inaccessible to the glycinamide cation at the lowest ionic strength tested (1.4 mM). Three model cationic analytes showed retention loss approximately proportional to the excluded volume as ionic strength was decreased. This suggests that, under certain conditions, cation exclusion may become the dominant mechanism in mediating the retention of cationic analytes as the mobile-phase salt content is varied. Cationic analyte retention on C18 stationary phases has been studied in detail. Studies have shown significant retention increases of cationic analytes when salt content of the mobile phase was systematically increased at constant pH.1-3 Such retention enhancement is typically attributed to ion pairing or chaotropic effects. According to the classical ion pairing mechanism, it is assumed the anion component of the salt facilitates retention of the cationic analyte through formation of a neutral ion pair. According to the chaotropic mechanism, the driving force is the disruption of solvation, which facilitates analyte adsorption onto the hydrophobic stationary phase. While either of these mechanisms provides a satisfactory explanation for the general retention * To whom correspondence should be addressed. E-mail: [email protected]. (1) Gritti, F.; G. Guiochon, G. J. Chromatogr., A 2004, 63-75, 1041. (2) Dai, J.; Carr, P. W. J. Chromatogr., A 2005, 1072, 169-184. (3) Jones, A.; Lobrutto, R.; Kazakevich, Y. J. Chromatogr., A 2002, 964, 179187.

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trends, it is conceivable that an alternative mechanism, cation exclusion, may also contribute to the observed effects. If the stationary phase is positively charged, the cationic analyte will be subjected to a repulsive force which will impede entry into the pores of the column. In such a case, adding salt to the mobile phase will reduce the repulsive force between analyte and stationary-phase surface, due to the well-known electrostatic screening effect, which occurs as ionic strength increases.4 This can be expected to reduce the exclusion effect and increase retention by allowing the analyte greater access to the stationary phase within the pores.5,6 Thus, a cation exclusion process offers an explanation for increased retention normally attributed to ion pairing or chaotropic effects, but the mechanism is different. In order for cation exclusion to occur, it is necessary for the stationary-phase surface to have a positive charge. Recently, evidence for the existence of a positive charge on a commonly used commercially available C18 column (Symmetry) has been reported. Studies have shown significant retention of an inorganic anion and exclusion of various cationic analytes when the mobilephase pH is ∼3.7,8 Electrokinetic studies have also suggested a positive charge for Symmetry C18 as well as other C18 stationary phases at pH 3.9 These observations raise the possibility that cationic exclusion may play a significant role in the elution behavior of cationic analytes on certain C18 stationary phases, in addition to ion pairing and chaotropic effects. Differentiating between retention enhancements caused by ion pairing or chaotropic effects on the one hand, and reduction of exclusion effects due to electrostatic screening on the other, is a difficult problem. This is because in order to vary the ionic strength to study electrostatic screening effects, one must add salt to the mobile phase. Thus, some type of anion will be present and it cannot be known for certain if the resulting changes arise from ion pairing or chaotropic effects or from electrostatic screening effects. In principle, a cationic compound having negligible affinity for the stationary phase could be useful for (4) Stahlberg, J. J. Chromatogr., A 1999, 855, 3-55. (5) Jandera, P.; Buncekova, S.; Halamana, M.; Novotna, K.; Nepras, M. J. Chromatogr., A 2004, 1059, 61-72. (6) Neddermeyer, P. A.; Rogers, L. B. Anal. Chem. 1968, 40, 755-762. (7) Mendez, A.; Bosch, E.; Roses, M.; Neue, U. D. J. Chromatogr., A 2003, 986, 33-44. (8) McCalley, D. V. J. Sep. Sci. 2003, 26, 187-200. (9) Loeser, E. J. Chromatogr. Sci., in press. 10.1021/ac0704816 CCC: $37.00

© 2007 American Chemical Society Published on Web 05/27/2007

independently studying the amount of cation exclusion. It could be considered as a “cationic void volume marker”, to establish what fraction of the column volume, if any, is inaccessible to cationic analytes relative to neutral ones. This would allow the severity of the exclusion effect to be evaluated as ionic strength is varied. To test this approach, we used the glycinamide cation to investigate exclusion effects as a function of mobile-phase ionic strength. Experiments were run in which the ionic strength of the mobile phase was varied in the relatively low range of 1.425.5 mM while maintaining a constant pH of 3. Three commercially available C18 stationary phases were tested, based on evidence of an apparent positive surface charge under pH 3 conditions.7-9 The retention of both anionic and cationic analytes was evaluated at six different ionic strength values. To minimize the contribution from ion pairing or chaotropic effects, phosphate was used as the buffering agent due to its relatively low cation retention enhancement properties.10 Interestingly, as ionic strength was varied, the observed trends in retention of both cationic and anionic analytes suggested that all three of the C18 stationary phases are positively charged to varying degrees at pH 3, and when ionic strength was low, it appeared that cationic analytes were prevented from entering a large fraction of the pore volume within the column. EXPERIMENTAL SECTION HPLC Instrument. The chromatographic system consisted of an Alliance 2695 separations module and 996 photodiode array UV detector (Waters Corp., Milford, MA). Empower chromatography software (Waters) was used for instrument control, data acquisition, and data processing. The instrument was equipped with a column temperature control module and an in-line solvent degasser. The flow rate was 0.6 mL/min. The PDA spectrum was acquired in a wavelength range of 192-300 nm using a spectral bandwidth of 2.4 nm and a sampling rate of 5 Hz. Chromatograms were later extracted at suitable UV wavelengths from the electronically stored PDA data. HPLC Columns. Three different C18 type columns were used in this study, all of which are commercially produced. All columns were new. Dimensions were 3 × 150 mm (i.d. × L), with nominal particle diameter of 3 µm. One of the columns, Gemini (Phenomenex, Torrance, CA), contains a relatively new “hybrid”-type stationary phase designed for extended pH range stability.11 XterraMS (Waters) is an earlier generation hybrid stationary phase.12 Symmetry (Waters) is a widely used silica-based, end-capped C18 stationary phase. Various stationary-phase properties are shown in Table 1. Mobile-Phase Preparation and pH Measurements. Chemicals were reagent grade or better. Acetonitrile (MeCN) was HPLC grade, and water was purified using a Purelab Ultra system (Elga Labwater, Lowell, MA). Mobile phases were prepared in 33% MeCN, which was prepared by combining two volumes of water (10) Roberts, J. M.; Diaz, A. R.; Fortin, D. T.; Friedle, J. M.; Piper, S. D. Anal. Chem. 2002, 74, 4927-4932. (11) Loo, L.; McGinley, M. LC-GC 2005 (Feb), p. 78. (12) Cheng, Y-F.; Walter, T. H.; Lu, Z.; Iraneta, P.; Alden, B. A.; Gendreau, C.; Neue, U. D.; Grassi, J. M.; Carmody, J. L.; O’Gara, J. E.; Fisk, R. P. LC-GC 2000, 18, 1162-1172.

Table 1. Properties of the Three Columns Used in This Studya

column Gemini C18 Symmetry C18 Xterra-MS C18

part. diam (µm)

surf area (m2/g)

pore vol (mL/g)

pore diam (Å)

3.0 (3.0) 3.5 (3.53) 3.5

(400)

(1.06)

(340)

0.83b

(179)

(0.72)

100 (98) 100 (98) (130)

%C (14.0) (19.67) (15.62)

a Physical characterization data furnished by column manufacturers. Actual test results (where available) are shown in parentheses below the nominal values. Columns were 3.0 × 150 mm dimensions. b Calculated from the following formula:13 total pore volume (mL/g) ) (1/4)[d(nm)S(m2/g)] × 10-3.

Table 2. Compounds Used as Model Analytes

ID

analyte

charged group

charge at pH of mobile phase

B1 B2 B3 G+ U N1 N2 NO3Cl-

homophenylalanine ethyl ester diphenhydramine nortryptylamine glycinamide urea p-cresol propyl paraben nitrate chloride

1° amine 3° amine 2° amine 1° amine none none none inorganic acid inorganic acid

+1 +1 +1 +1 0 0 0 -1 -1

with one volume of MeCN. For buffered mobile phases, stock solutions of acid and salt components were first prepared in 33% MeCN, and then appropriate volumes of stock solutions were pipetted into volumetric flasks and diluted to final volume with 33% MeCN to achieve the desired concentrations. To eliminate the possibility of variations in retention due to slight changes in MeCN content, a large batch of 33% MeCN was prepared and all buffers were made from the same batch. All pH measurements were made using a model 340 pH meter (Corning, Acton, MA) equipped with an Accumet combination electrode (13-620-285, Fisher Scientific, Fair Lawn, NJ). Sample Preparation. The compounds used as model analytes are shown in Table 2. Stock solutions were prepared in 2:1 (v/v) water/MeCN at concentrations of 2.5 g/L and then diluted 1 part stock into 9 parts mobile phase prior to HPLC analysis. Injection volume was 2 µL (Mass load of 0.5 µg). All retention values (k) were calculated based on the mean of at least two injections, using the formula (tR - t0)/t0 unless stated otherwise. Preparation of Buffered Mobile Phases for a Range of [I] Values and Constant pH. In this study, a mobile-phase solvent content of 33% MeCN was used, which provided adequate retention for the model analytes. The following equation was used to determine the amounts of phosphoric acid and ammonium phosphate needed to produce a series of mobile phases where I was varied while holding pH constant at 3

[H +] )

- (Ka + XA) + x(Ka + XA)2 + 4Ka(HA) (1) 2

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Table 3. Amounts of Acid and Salt Used To Prepare Mobile Phases, Calculated Equilibrium Values of [A-], and pH from Eq 1, and Several Measured pH Valuesa H3PO4 (mM)

NH4+H2PO4(mM)

[A-]Eq (mM)

pH (calcd)

pH (measd)

22.0 15.1 8.71 4.48 2.89 2.07

25.5 16.8 8.74 3.41 1.40 0.37

26.5 17.8 9.74 4.41 2.39 1.36

3.00 3.00 3.00 3.00 3.00 3.00

2.98 2.99 3.00

a Using electrode calibrated with aqueous buffers and with correction factor applied, See text.

where XA and HA are the initial amounts of salt and acid and Ka is the dissociation constant of the acid.14 To compensate for organic solvent influence, pKa values obtained in the actual water/ solvent mixture were used for the calculation. A pKa value of 2.90 was used based on interpolation of literature values.15 The amounts of acid and salt used in the mobile phases are shown in Table 3. The pH of several mobile phases was measured to check the prediction of eq 1, using a pH meter calibrated with aqueous buffers. It has been shown that, for a typical glass pH electrode, the slope of the mV versus pH calibration curve is not significantly affected by organic solvent, but the intercept of the calibration curve will be shifted slightly.15 Therefore, a correction factor is necessary due to the effect of the organic solvent on the intercept of the calibration curve. We experimentally obtained a correction factor by measuring solutions of strong acid, which were assumed to be fully dissociated. Three different strong acids (perchloric, hydrochloric, and nitric acids) of 1 mM concentration in 33% MeCN gave pH readings of 2.83, 2.82, and 2.82, respectively, indicating that a correction factor of 0.18 is appropriate when using the pH meter, which had been calibrated with aqueous buffers to measure solutions in 33% MeCN medium. When this correction factor was applied, good agreement was obtained between the measured pH of the mobile phases and the target pH value predicted by eq 1 (Table 3). RESULTS AND DISCUSSION Retention of Anions and Exclusion of Cations on C18 Columns at pH 3. As observed by others,7 our experiments showed significant retention of nitrate ion on Symmetry C18 at pH 3, as well as with the Gemini C18 column. Xterra-MS also showed retention of nitrate, but to a lesser degree. Example chromatograms are shown in Figure 1, which shows the substantial k values obtained with Gemini and Symmetry columns. We evaluated the retention data by plotting k as a function of reciprocal anion concentration in the mobile phase. Linearity of such plots is often used as evidence to support an ion-exchange interac(13) Halpaap, H. J. Chromatogr. 1973, 78, 63-75. (14) Skoog, D. A.; West, D. M.; Holler, F. J. Fundamentals of Analytical Chemistry, 5th ed.; Saunders: New York, 1988; pp 193-195. (15) Espinosa, S.; Bosch, E.; Roses, M. Anal. Chem. 2002, 74, 3809-3818.

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Figure 1. Chromatograms showing significant retention of nitrate anion on Gemini C18 (left) and Symmetry C18 (right). Mobile phases were pH 3 ammonium phosphate in 33% MeCN. From bottom to top, concentration of buffer anion (H2PO4-) in mobile phase (equivalent to I) was 9.74, 4.41, 2.39, and 1.36 mM. Detection wavelength was 217 nm. Table 4. Linear Regression Analysis of Plots of Nitrate Retention Factors k vs 1/[A-]a column

slope

intercept

r2

Gemini Symmetry Xterra-MS

3.01 ( 0.23 2.18 ( 0.09 0.24 ( 0.04

-0.05 ( 0.10 0.09 ( 0.04 -0.01 ( 0.02

0.999 >0.999 0.998

a Where [A-] is the buffer anion concentration in the mobile phase (in mM units). The 95% confidence limits are shown.

tion,16,17 based on a simple stoichiometric retention model, which leads to the well-known equation

k ) K[A-]-1

(2)

where [A-] is concentration of competing anion in the mobile phase and K is a combined constant that reflects both the ionexchange equilibrium constant and the ion-exchange capacity of the column.18 When the k values were plotted in this matter (Table 4), excellent coefficients of linearity (r2) were obtained from linear regression analysis, supporting the view of an ion-exchange-type interaction. The relative steepness of the slopes obtained for nitrate ion (reflecting the combined constant K in eq 2) follows the trend Gemini ∼ Symmetry > Xterra-MS, where the slope for XterraMS was only ∼10% compared to Symmetry. We also evaluated chloride ion retention for Gemini and Symmetry columns (data not shown), and in both cases, slopes were ∼80% of the slope obtained for nitrate and r2 values were >0.997. In order to probe cation exclusion effects, we used glycinamide as a polar model analyte, assumed to have a positive charge under the pH 3 conditions and assumed to have negligible adsorption onto the hydrophobic C18 stationary-phase surface in the 33% (16) Cox, G. B.; Stout, R. W. J. Chromatogr. 1987, 384, 315-336. (17) Roses, M.; Oumada, F.; Bosch, E. J. Chromatogr., A 2001, 910, 187-194. (18) Skoog, D. A.; Leary, J. J. Principles of Instrumental Analysis, 4th ed.; Saunders, New York, 1992; pp 654-655.

Figure 3. Simplified schematic diagram of hypothetical column, divided into discreet interstitial and porous regions, having respective volumes (VI) and (VP). Bulk mobile phase is driven by pressure through interstitial domain from left to right (white arrows) and diffuses into and out of porous domain (vertical black arrows). All of the stationary phase surface area (A) is within the porous domain.

Figure 2. Chromatograms (UV 193 nm) showing effect of mobilephase ionic strength on exclusion of glycinamide cation (G+) and retention of chloride anion (Cl-). I values for the mobile phases are (starting from top) 26.5, 4.41, and 1.36 mM. Bottom trace shows neutral void marker urea (U). Left panel is Gemini C18 column; right panel is Symmetry C18.

MeCN mobile phases. Although inorganic ions can be used to study exclusion effects, the use of glycinamide allowed UV detection to be used. When the hydrochloride salt of glycinamide was injected, both the glycinamide cation and chloride anion were visible at 193-nm detection. This is shown in Figure 2 for the Gemini and Symmetry columns. In addition to retention of chloride, another important feature of Figure 2 is the significant exclusion of the positively charged glycinamide cation. The exclusion is largest at lowest I, where electrostatic screening of the buffer ions is minimal. As I increases, the increase in retention time of the glycinamide is consistent with increased electrostatic screening, thereby reducing the amount of exclusion from the pores.5,6 The anion retention and cation exclusion effects are both consistent with an overall positive charge on the stationary-phase surface. The overall difference in retention between urea and glycinamide appears small in Figure 2. However, this seemingly small difference in retention time suggests that a very significant amount of the pore volume has become inaccessible to the glycinamide cation. We explored the exclusion aspect further in the following sections. Cation Exclusion Mechanism. An interesting aspect of the chromatograms in Figure 2 is the elution of glycinamide with an earlier retention time than the neutral void marker urea. Calculating k for glycinamide according to (tR - t0)/t0 or (VR - V0)/V0 will obviously afford negative values, because retention time (tR) for the glycinamide peak is lower than for the urea peak used to determine t0. The simplest way to rationalize the negative k values is that the volume within the column which is accessible to the glycinamide cation is smaller compared to the volume accessible to the urea void marker. This is presumably due to a repulsive force from the positively charged stationary-phase surface, which prevents the glycinamide cation from entering a significant fraction of the pore volume that is accessible to the urea void marker. Before considering the relationship between ionic strength and exclusion, the exclusion phenomenon was first evaluated purely in terms of retention volume relationships. We considered an

Figure 4. Schematic diagram of hypothetical column showing excluded volume (VE), which is inaccessible to a charged analyte, due to a repulsive exclusion force.

excluded volume (VE), defined as the volume of bulk mobile phase within the pores that is not accessible to a charged analyte, but fully accessible to a neutral analyte. This is illustrated in Figure 3, which shows a schematic representation of the column. Although the diagram is simplified, it nevertheless is useful for visualizing volume relationships of the different column domains. Note that all of the stationary phase resides within the porous region of the column, but the mobile phase occupies both the porous and interstitial domains. For a charged analyte under low ionic strength conditions, the repulsive force will lead to the condition in Figure 4, where part of the pore network has become inaccessible. Note that the analyte will be blocked from reaching the stationary phase within the excluded region. Since essentially all stationary phase is within the pores, it follows that the fraction of inaccessible stationary phase will be the same as the fraction of inaccessible pore volume. We evaluated this model by using the glycinamide cation as a probe for determining the excluded volume (VE). The following assumptions were made: (1) The excluded volume (VE) is defined as a region of space within the pore volume that is accessible to neutral analytes but not to cationic analyte. (2) The neutral void marker (urea) can access all of the volume of the bulk mobile phase, but the glycinamide cation, on the other hand, is restricted from the excluded volume (VE). Hence, glycinamide acts as a “charged” void marker. (3) Any analyte having a positive charge will be subjected to the same exclusion force as glycinamide, and will therefore be restricted to the same region of the pore network. (4) All stationary phase is within the pore region. Therefore, when a certain fraction of the pore volume becomes inaccessible to cationic analytes, a proportional amount of stationary-phase surface area will also become inaccessible. Analytical Chemistry, Vol. 79, No. 14, July 15, 2007

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Based on the assumptions above, it follows that VE can be determined experimentally by comparing retention times of the neutral and charged void markers. However, in order to evaluate the relevance of the VE values, the accessible pore volume for neutral analytes under the experimental conditions must be known. Therefore, before addressing the case of a charged analyte being excluded, we first describe how the specifications of the C18 silica gel packing materials were used to estimate values of accessible pore volume (VP), based on well-known principles of silica-based C18 materials and several assumptions as described in the following section. Determination of Accessible Pore Volume of Columns. Column manufacturers typically provide pore volume and surface area specifications for the silica gel used for the columns. However, these measurements are generally taken before derivatization with C18 ligands. Under actual use, the accessible pore volume of C18 columns is considerably smaller than would be expected based on specifications of the underivatized silica. A significant part of the underivatized silica pore volume is occupied by the C18 ligands.19-21 In addition, the surface of C18 stationary phases may also contain a layer of adsorbed solvent, which is known to be of significant thickness in MeCN/water mobile phases.22,23 Thus, the accessible pore volume of the original silica skeleton is reduced both by the volume of C18 ligands and by the volume of adsorbed MeCN, the latter being dependent on mobile-phase composition. To calculate an accessible pore volume for the conditions used in the current study, several assumptions were made as follows. We assumed that the pore volume after attachment of C18 ligands (VPC18) was 50% of the underivatized silica pore volume, based on studies in which pore volumes of silica materials have been measured before and after C18 derivatization.19-21 It was also assumed that the surface area value after C18 derivatization (AC18) could be obtained from the surface area of underivatized silica by using a correction factor of 0.64, based on reported measurements of surface area before and after C18 derivatization.20,21 Based on these assumptions, the pore volume and surface area after derivatization were calculated from specific pore volume and surface areas in Table 1 by applying the correction factors and multiplying by the weight (w) of packing recovered from the columns after drying to constant weight (Table 5). To calculate the volume occupied by the adsorbed MeCN layer (VMeCN), we used the formula AC18 × T, where T is the layer thickness. Since the mobile phases all contained MeCN at 33% level and all three columns were C18 type, it was assumed that T was the same for all experiments and was not significantly affected by the ionic strength. The validity of the latter assumption is supported by the retention times of two neutral analytes, which did not change significantly as ionic strength was varied. The value for T was estimated based on reported literature values for other C18 (19) Rustamov, I.; Farca, T.; Ahmed, F.; Chan, F.; LoBrutto, R.; McNair, H. M.; Kazakevich, Y. V. J. Chromatogr., A 2001, 913, 49-63. (20) Szabo, Z.; Ohmacht, R.; Huck, C. W.; Stoggl, W. M.; Bonn, G. K. J. Sep. Sci. 2005, 28, 313-324. (21) Sands, B. W.; Kim, Y. S.; Bass, J. L. J. Chromatogr. 1986, 360, 353-369. (22) Kazakevich, Y. V.; LoBrutto, R.; Chan, F.; Patel, T. J. Chromatogr., A 2001, 913, 75-87. (23) Poplewska, I.; Antos, D. Chem. Eng. Sci. 2005, 60, 1411-1427.

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Table 5. Calculation of Maximum Accessible Pore Volume (VP)a V column

w (g)

VPC18 (mL)

Gemini C18 Symmetry C18 Xterra-MSC18

0.60 0.62 0.61

0.318 0.257 0.268

a

AC18 (m2)

MeCN

(mL)

VP max (mL)

154 135 70

0.099 0.095 0.049

0.219 0.162 0.219

For definition of terms, see text.

columns.24 The layer thickness has been reported to reach a maximum of ∼8.5 Å at 40% MeCN content. Since the mobile phases used in the current study contained only 33% MeCN, we reduced the value of T by roughly 20% and assumed a layer thickness of 7 Å for the 33% MeCN mobile phases employed in the current study. Based on these assumptions, the maximum accessible pore volume (VP) was obtained by subtracting VMeCN from VPC18 as shown in Table 5 for each of the three columns. We then considered the case of a cationic analyte being subjected to an ion exclusion phenomenon as described in the following section. Determination of Excluded Volume. As stated previously, we assume that exclusion occurs because the charged analyte is not able to gain complete access to the maximum accessible pore volume, through the action of an exclusion force caused by electrostatic repulsion between analyte and stationary phase. As I decreases and electrostatic screening is reduced, the repulsive force becomes greater, and an increasingly larger region of space within the pore network becomes inaccessible to the analyte, defined as the excluded volume (VE). It is further assumed that the exclusion effect occurs only within the pore network, and the interstitial region of the column (VI) is not involved. Figure 4 shows how the volume of exclusion VE is related to the volume of the other column domains. Note that this diagram represents only one particular ionic strength. As ionic strength is varied, VE will change. Neutral analytes still have complete access to VP, while charged analytes will be restricted to the pore region defined by VP - VE. To experimentally determine VE, it is assumed that both urea and glycinamide cation have negligible adsorption to the stationary phase under the 33% MeCN conditions. This means that their elution volumes will indicate the total volume of mobile phase within the column that they can access. In a sense, urea and glycinamide can be thought of as neutral and charged void markers, respectively. For urea, no exclusion force exists, and the retention volume is

VU ) VP + VI

(3)

For glycinamide, the exclusion force prevents access to the region VE, and the overall retention volume of glycinamide (VG) is therefore smaller than VU by the amount VE. Therefore, the volume (24) Kazakevich, Y. V.; LoBrutto, R.; Vivilecchia, R. J. Chromatogr., A 2005, 1064, 9-18.

Table 6. Excluded Volume (VE) Based on Difference between Retention of Glycinamide Cation Void Marker and Neutral Void Marker (Urea) as [I] of Mobile Phase Is Varieda column

VU (mL)

[I] (mM) Gemini Symmetry Xterra a

VE 26.5

0.606 0.530 0.583

17.8

0.088 0.053 0.052

0.096 0.058 0.055

9.74

4.41

2.39

1.36

0.113 0.074 0.060

0.141 0.094 0.072

0.161 0.110 0.087

0.173 0.122 0.101

Retention volume of urea (VU) is also shown for each column.

VE can be determined from experimental values of VU and VG according to

VE ) VU - VG

(4)

Table 6 shows values of VE as [I] was varied from 25.5 to 1.4 mM, calculated with eq 4 and experimental VU and VG values. Note that, as I decreases, VE increases significantly. For Gemini and Symmetry, VE values begin to approach the accessible pore volume (VP), reaching ∼75-80% of VP at the lowest ionic strength value, and for Xterra-MS, the VE value was 46% of VP. This illustrates how serious the problem of exclusion becomes when I is low. This is important because virtually all of the stationary phase is located within the pores of the column. This can easily be shown by calculating the theoretical surface area of uniform spheres having a 3.5-µm diameter typical of HPLC packings, using an estimated density of 2.2 g/cm3.25 The surface area in this case is ∼0.4 m2/g. This is less than 1% of the 200-400 m2/g values for typical C18 columns, a clear indication that essentially all of the surface area resides within the pore network of the silica. If all cationic analytes are excluded to the same extent as the glycinamide cation, and are therefore unable to reach the majority of the stationary phase within the pores, this will undoubtedly cause the retaining ability and resolving power of the column to be significantly reduced. We further explored the validity of this model by evaluating the retention of three monocationic analytes of varying hydrophobicity as described in the following section. Effect of I on Elution Behavior of Moderately Retained Cationic Analytes. The elution behavior of three positively charged protonated basic analytes (B1, B2, B3) and two neutral analytes was also evaluated as a function of I, summarized in Figure 5. In all cases, k for cationic analytes decreased as I was decreased. One could argue that the retention change was due to ion pairing or chaotropic effects from the anions in the buffer. These effects cannot be ruled out as a contributing factor because, as I was increased, the concentration of anions in the mobile phase also increased. However, attributing such large retention changes solely to ion pairing or chaotropic effects seems unreasonable when considering the relatively low inherent retention enhancement of phosphate ions compared to other acid anions.10 Furthermore, for the most polar analyte B1, k was negative at the lowest I value for Gemini and Symmetry, indicating that the analyte was (25) Unger, K. K.; Jilge, G.; Kinkel, J. N.; Hearn, M. T. W. J. Chromatogr. 1986, 359, 61-72.

Figure 5. Effect of I on retention of cationic analytes B1 (squares), B2 (diamonds), and B3 (triangles) for Gemini (left), Symmetry (center), and Xterra-MS (right) C18 stationary phases using pH 3 (ammonium phosphate) buffered mobile phases with 33% MeCN (compounds identified in Table 2). Also shown are retention factors for neutral analytes N1 (white circles) and N2 (black circles).

being excluded from at least some of the pore volume. These affects are further illustrated in Figure 6. In addition to the drastic reduction in retention for the three cationic analytes as I was decreased, the elution of analyte B1 at an earlier retention time than a neutral void marker is also evident at the lowest ionic strength. This indicates that, at the lowest I value, the volume within the column that is accessible to B1 has become smaller than the volume accessible to a neutral void marker. The simplest way to explain the apparent exclusion effect is that the sign of the charge on both the column and analyte is positive and that the repulsive force between the analyte and column decreases the ability of the analyte to enter the pores of the stationary phase. As I was increased, a screening effect occurred, which presumably reduced the repulsion and allowed increased access of the analyte into the pore network of the column,5,6 thereby causing a relatively large retention increase. Exclusion of cations has also been reported by others for the Symmetry C18 stationary phase.8 Losses in retention of cationic analytes occurred for XterraMS as I decreased, but to a smaller degree. This is reasonable considering the lower retention of anions observed for XterraMS (Table 4) compared to the Gemini and Symmetry columns. It is also noteworthy that the retention of cations appeared to reach a plateau for the Xterra-MS at the highest I values in the range of ∼20-25 mM, whereas for the Gemini/Symmetry columns the retention never appeared to reach a plateau even at the highest I value. This suggests that an even further increase of I would be necessary to fully screen the electrostatic interactions between the cationic analytes and the Gemini/Symmetry stationary phases. Interestingly, while Xterra-MS afforded greater retention for compounds B1, B2, and B3 compared to Gemini/Symmetry, the opposite occurred for the neutral analytes. As shown in Figure 5, two different neutral analytes were both more strongly retained for Gemini/Symmetry compared to Xterra-MS by 32-35%. This again suggests that, for the Gemini and Symmetry stationary phases, the maximum possible retention of cationic analytes was not reached, even with the highest I value (26.5 mM) used in this study. Relationship between VE and Elution Volumes of Cationic Analytes. To further evaluate the experimental data in terms of Analytical Chemistry, Vol. 79, No. 14, July 15, 2007

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Figure 6. Severe loss of retention with decreasing I for three cationic analytes B1, B2, and B3 (compounds identified in Table 2). Left panel shows chromatograms from Gemini C18 column, right panel from Symmetry C18 column. Mobile phases were 33% MeCN with pH 3 ammonium phosphate buffers. Ionic strengths were (from bottom to top) 26.5, 9.74, 4.41, and 1.36 mM. Detection wavelength was 262 nm. Top chromatogram (193-nm detection) is neutral void marker urea (U) to illustrate exclusion of B1 at lowest ionic strength value.

the exclusion mechanism illustrated in Figure 4, a simple retention volume model was derived starting with the following equation describing adsorption chromatography26

VR ) V0 + V0D(A/VM)

(5)

where VR is the observed retention volume of the analyte, V0 is the hold-up volume of the column, D is the distribution constant, and (A/VM) is the phase ratio where A is surface area of stationary phase and VM is volume of mobile phase. In our case, we make the assumption that any adsorbed solvent layer is an immobile part of the stationary phase, and the outer surface of the solvent layer (in contact with the bulk mobile phase) is considered as the boundary between mobile phase and stationary phase. This allows the volume of stationary phase to be neglected in eq 5, thereby making V0 and VM the same and leading to the simplification

VR ) VM + DA

(6)

When exclusion of a cationic analyte occurs, the observed retention volume (VR) will become smaller. To account for the exclusion, the two terms VM and DA require different adjustment factors. This is because the pore network of the column is where exclusion occurs, and the pores contain nearly all of the surface area (Figure 4). Thus, when a cationic analyte is blocked from some fraction of the pore network, but still has full access to all of the interstitial volume, the accessible surface area will decline proportionally faster than the accessible volume, because the overall accessible volume contains a large component VI that is unaffected by exclusion. Based on the Figure 4 relationships, the volume will shrink according to (VI + VP) - VE, but the effective surface area will shrink more rapidly according to VP - VE. (26) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography; John Wiley and Sons, Inc.: New York, 1974; pp 25-35.

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Excluded volume terms can be introduced to the right side of eq 6 as follows

VR ) (VM - VE) + DA

[

]

VP - VE VP

(7)

where VP is the pore volume, and the factor in brackets is the fraction of “nonexcluded” stationary phase. The correction factor uses VP instead of VM, because essentially all of the surface area is within the porous domain of the column as previously described, so it is assumed that accessible stationary-phase surface area will shrink proportionally to the accessible pore volume. With rearrangement one obtains

VR - (VM - VE) ) DA -

[ ] DAVE VP

(8)

The quantity VM can be experimentally obtained from a neutral void marker such as urea (VU). VE is defined previously (eq 4) using the retention volume of glycinamide (VG). Based on these simple relationships, eq 8 can be rearranged to

[ ]

VR - VG ) DA - VE

DA VP

(9)

Equation 9 shows an interesting linear relationship between the quantity (VR - VG) and the excluded volume VE. By obtaining experimental retention volumes and plotting (VR - VG) against VE, one can potentially obtain values for DA and VP. An interesting hypothetical case is when the quantity (VR - VG) approaches zero. By setting the left side of eq 9 equal to zero, one obtains

VP ) VE

(10)

which indicates that the plots should theoretically intersect the x-axis at the value corresponding to the pore volume VP. Figure 7 shows the data obtained for the three analytes with the Gemini

Figure 7. Example plots of VR - VG vs VE for three positively charged analytes as ionic strength is decreased from 26.5 to 1.36 mM for Gemini C18 column. The three compounds are B1 (squares), B2 (diamonds), and B3 (triangles). For each analyte, six different data points are shown corresponding to the six different mobile phase ionic strengths of 26.5, 17.8, 9.74, 4.41, 2.39, and 1.36 mM. Lines are obtained by linear regression analysis. Also shown (on x-axis) is calculated accessible pore volume VP to illustrate proximity to x-intercepts obtained by extrapolation. Table 7. Regression Analysis of (VR - VG) vs VE Plots (Example Shown in Figure 7) for Analytes B1, B2, and B3 for Three Different C18 Columns column Gemini Symmetry Xterra-MS

analyte B1 B2 B3 B1 B2 B3 B1 B2 B3

slope

y-int

r2

x-int

-3.31 -9.24 -19.5 -3.24 -9.11 -19.7 -3.32 -10.1 -21.7

0.643 1.79 3.79 0.450 1.27 2.74 0.589 1.75 3.75

0.995 0.998 0.999 0.995 0.995 0.996 0.995 0.997 0.996

0.194 0.194 0.194 0.139 0.139 0.140 0.177 0.172 0.173

column and plotted in this fashion. Results of regression analysis are shown for all three columns in Table 7. Correlation coefficients suggest approximately linear behavior, although for the Symmetry and Gemini columns a slight concave upward curved appearance is evident. Slopes obtained for each particular analyte are nearly identical from one column to another, suggesting that the values of D are all similar (Table 7). This is reasonable considering that all are C18 stationary phases, so analyte adsorption should be similar. For each column, extrapolations for all three analytes converge to a single value at the x-intercept. This can be interpreted to mean that all three analytes are subject to the same general exclusion effect through a common mechanism, which is related to the exclusion volume. Although only three different cationic analytes were evaluated, the data suggest that any compound having a single positive charge will exhibit the same behavior. Equation 10 suggests that the x-intercept of eq 9 corresponds to VP. The experimental x-intercept values (Table 7) are somewhat smaller than the calculated pore volumes for each of the columns (VP values in Table 5), with x-intercept values ranging from 89% of VP (Gemini) to 79% (Xterra-MS) of VP. The less than perfect agreement may be due to oversimplifications in either the derivation of eq 9 (such as the assumption of constant D as I is varied) or the calculation of VP values (due to inaccuracies in the

correction factors used for C18 ligand and adsorbed solvent volumes). Nevertheless, the consistency of x-intercept values obtained for each column seems to suggest that the excluded volume VE at each particular I value represents a well-defined region within the pore network that becomes inaccessible to all cationic analytes. It also suggests that the retention loss at low ionic strength is not caused by a change in the distribution coefficient, but rather by restriction of the analyte from the pore region. In addition to retention loss, we also noted a general loss in column efficiency toward the cationic analytes as I was lowered. Although a detailed discussion of column efficiency is beyond the scope of this report, the observed decline in efficiency (based on reduced plate counts) is consistent with the work of other investigators. It has been reported that, in addition to loss of retention, increased peak widths and reduced loading capacity were observed for cationic analytes as ionic strength was lowered, for several C18 columns with phosphate or formate buffers at similar pH to the current study.27,28 The observed loss in efficiency with decreasing I is another indication that the cationic analytes are no longer able to access all of the stationary phase and is consistent with an ion exclusion mechanism. Effect of Ionic Strength on Exclusion Volume. The previous sections showed a general increase in VE as I was decreased. However, VE does not vary with I in a simple linear relationship. This issue is discussed in a recent report, which describes the exclusion of a negatively charged analyte from a negatively charged silica surface.29 These investigators studied the exclusion effect as ionic strength was varied. A detailed study was conducted using LC packings of several different pore diameters (dP). The results were rationalized according to electrical double layer (EDL) theory. The Debye screening length (κ-1) was used as a measure of EDL thickness. It was shown that the dP/κ-1 ratio was a useful quantity to predict the severity of exclusion. When I was increased to the point where the dP/κ-1 ratio was ∼20 (i.e., dP ∼20 times κ-1), exclusion appeared to reach a minimum. When I was lowered to the point where the dP/κ-1 ratio was ∼1 (i.e., dP about the same as κ-1), exclusion reached a maximum, and it was assumed that the analyte could access only the interstitial volume when the dP/κ-1 ratio was less than 1. To determine if the same phenomenon was occurring in the current study, we calculated κ-1 values for the mobile-phase buffers according to the following equation for κ2 (which has been simplified for buffer salts of monovalent cation and anion)4

[

κ2 ) F 2

]

1000 × 2I 0RT

(11)

and using a value of 67 for the dielectric constant () of 2:1 (v/v) water/MeCN.9 The values obtained for κ-1 are shown in Table 8. Note that, at the lowest ionic strength used, the κ-1 value is approaching dP for the Gemini/Symmetry columns, meaning that the dP/κ-1 ratio is approaching 1. This coincides with the approach of complete exclusion for the Gemini/Symmetry columns, based on the significant VE values (Table 6) obtained for these columns (27) McCalley, D. V. Anal. Chem. 2003, 75, 3404-3410. (28) McCalley, D. V. Anal. Chem. 2006, 78, 2532-2538. (29) Nischang, I.; Chen, G.; Tallarek, U. J. Chromatogr., A 2006, 1109, 32-50.

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Table 8. Calculated Debye Screening Length Parameter (K-1) and Calculated Average Length of Pore Channel Per Buffer Ion (LP) as Function of Ia [I] (mM) κ-1 LP (dP ) 80 Å) LP (dP ) 110 Å)

26.5

17.8

9.74

4.41

2.39

1.36

17 6.2 3.3

21 9.3 4.9

28 17 9.0

42 37 20

57 69 37

76 121 64

a Calculated lengths are in units of Å. κ-1 values are for solvent of 2:1 (v/v) water/MeCN. LP has been calculated for two different dP values.

(approaching 75-80% of VP as shown in Table 5) at the lowest I value. Thus, as reported by others for anion exclusion for silica columns,29 the dP/κ-1 ratio appears to give a similar prediction of the approach toward complete exclusion of cations from the Gemini/Symmetry columns. For the Xterra-MS column, the pore diameter is larger than the other columns. This may explain why VE values were less significant for this column, since the dP/κ-1 ratio is larger for this column than for the Gemini/Symmetry columns. The general trend toward reduced exclusion of cationic analytes with increasing dP has also been observed by others for C18 columns. It was reported that a much lower amount of exclusion toward cations was observed when comparing a larger pore diameter Symmetry 300 column to Symmetry 100, using mobile phases of pH similar to those used in the current study.8 We also examined the density of ions in the mobile phase, in particular how many ions were located on average within the pore channel as a function of pore length. This was done by first calculating the average volume occupied per buffer ion (VIon) in solution according to

VIon )

1 2I × NA

(12)

where NA is the Avogadro constant and the factor of 2 is used so that both positive and negative ions will be included in the calculation (assuming anion and cation are both monovalent). Based on the VIon values, a theoretical length of pore channel per ion (LP) was then the calculated according to

LP )

VIon π(dP/2)2

(13)

where dP is the diameter of pore channel. The quantity LP represents the distance between ions within a hypothetical pore channel of uniform diameter in which it is assumed the ions are evenly distributed along the longitudinal axis of the pore channel. Calculations were made using dP values of 80 (for Gemini/ Symmetry columns) and 110 Å (for Xterra-MS column), which are 20 Å less than the values in Table 1 based on approximate diameter reductions expected after C18 derivatization.19,20 The values of LP show a trend similar to the κ-1 values, becoming much larger as I is decreased (Table 8). The relatively large LP values at lowest I illustrate how sparse the population of ions becomes in the pore channels as I is reduced. Note that, for the narrower 5390

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pores of Gemini/Symmetry, LP actually exceeds the dP at the lowest I value. Also, it is important to note that the LP values refer to total ions in solution. If one considers the ions of only one charge (+ or -), the LP values in Table 8 will be doubled. Based on the limited number of ions within the pore channels at the lowest I, it is conceivable that even a very weak surface charge could render the pore channel inaccessible to a charged analyte, due to the almost complete absence of any buffer ions to screen the repulsive interaction between surface and analyte. CONCLUSIONS Studies were conducted in which the ionic strength of the mobile phase was varied as the pH was held constant at 3. Several cationic and anionic analytes were injected under each mobilephase condition. By observing trends in retention as I was varied, indications of electrostatic interactions between analyte and stationary phase became apparent. All three stationary phases (Gemini, Symmetry, and Xterra-MS) showed evidence of a positive charge, based on retention of inorganic anions. Retention of nitrate was significant at the lowest ionic strength, and plots of k versus [I]-1 showed linear behavior for both nitrate and chloride anions. The columns also showed exclusion toward cationic analytes. The glycinamide cation was used to probe the exclusion effect and was treated as a “charged” void marker. As I was decreased, glycinamide eluted faster than a neutral void marker (urea). Based on the difference in retention times between glycinamide and urea, the volume of mobile phase within the column, which presumably became inaccessible to positively charged analytes (VE), could be estimated. To judge the severity of the apparent excluded volumes, estimates of the pore volume (VP) accessible to neutral analytes were calculated, applying correction factors for the volumes assumed to be occupied by C18 ligands and adsorbed solvent layer. The excluded volumes were in some cases up to 80% of the accessible pore volume at the lowest ionic strength mobile phase (1.4 mM). When subtracting the retention volume of the “charged” void marker glycinamide (VG) from the retention volumes of three moncationic analytes having different polarities, the resulting corrected retention volumes (VR - VG) showed a linear relationship with VE. The proportional loss in retention of several different cationic analytes, all having the same positive charge, suggests that even cationic compounds that appear to exhibit satisfactory retention may nevertheless be restricted from accessing a significant fraction of the stationary phase. Also, since the resolving power and loading capacity of the column can both be expected to decrease as more and more of the stationary-phase surface area becomes inaccessible, the exclusion behavior also offers an explanation as to why the cationic analytes showed a significant reduction in plate counts as I was decreased. As reported by others,29 the dP/κ-1 ratio appears to be a useful parameter for predicting the onset of total exclusion from the pores. While a large body of scientific data supports the important role of chaotropic and ion pairing effects in the RP-LC retention of cationic analytes, the results of this study suggest that, under certain circumstances, the additional mechanism of ion exclusion also becomes significant. However, it is unlikely that effects of ion exclusion will be observable unless the experimental conditions are deliberately manipulated to enhance ion exclusion effects, while simultaneously minimizing ion pairing or chaotropic effects.

In this study, the ion exclusion effect was augmented by utilizing stationary phases having an apparent positively charged surface character and also by keeping the ionic strength of the mobile phase low to reduce electrostatic screening effects. At the same time, chaotropic or ion pairing effects were attenuated by using buffers in which the anion (dihydrogen phosphate) is known for its poor cation retention enhancement properties. Arguably, the results suggest that, under this particular set of conditions, it is possible that ion exclusion effects are of comparable or even greater significance than chaotropic or ion pairing effects. Design-

ing experiments that can discriminate between retention enhancements as opposed to exclusion remains a significant challenge. ACKNOWLEDGMENT We thank Yuri Kazakevich (Seton Hall University) and Edward Paul (Richard Stockton College) for helpful discussions. Received for April 23, 2007.

review

March

8,

2007.

Accepted

AC0704816

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