Characterization and applications of reversed-phase column selectivity based on the hydrophobic-subtraction model

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Journal of Chromatography A, 1191 (2008) 2–20

Characterization and applications of reversed-phase column selectivity based on the hydrophobic-subtraction model D.H. Marchand a , L.R. Snyder b , J.W. Dolan b,∗ a

University of Wisconsin-River Falls, River Falls, WI 54022-5001, USA b LC Resources, 15664 SE Woodland Hts., Amity, OR 97101, USA Available online 1 November 2007

Abstract A total of 371 reversed-phase columns have now been characterized in terms of selectivity, based on five solute–column interactions (the hydrophobic-subtraction model). The present study illustrates the use of these data for interpreting peak-tailing and column stability. New insights are also provided concerning column selectivity as a function of ligand and silica type, and the selection of columns for orthogonal separations is re-examined. Some suggestions for the quality control of reversed-phase columns during manufacture are offered. © 2007 Elsevier B.V. All rights reserved. Keywords: Column selectivity; Peak tailing; Orthogonal separation; Column stability; Hydrophobic-subtraction model

1. Introduction The retention characteristics and selectivity of columns used for reversed-phase chromatography (RPC) have been the subject of many investigations and hundreds of publications (for a partial listing, see [1–19] and accompanying references). These past studies have been motivated by a need for a better understanding of the RPC retention process, as well as the practical application of this knowledge in various ways; for example, • as an aid in understanding and solving various problems that may be related to column selectivity, - peak tailing (Section 4.1), - column degradation (Section 4.2), • the selection of columns of equivalent selectivity (replacement columns, Section 4.3.1), • the selection of columns of very different selectivity (orthogonal separations, Sections 4.3.2 and 4.4.3), - importance of ligand type (C8 , C18 , phenyl, etc.) and other properties of the column packing in affecting selectivity (Section 4.4), ∗

Corresponding author. Tel.: +1 971 241 0946. E-mail addresses: [email protected] (D.H. Marchand), [email protected] (L.R. Snyder), [email protected] (J.W. Dolan). 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.10.079

• improved selectivity-testing procedures for different production lots of RPC columns (quality control, Section 4.5). In the present article we will examine a recently developed model as a means for pursuing the above and related goals. The examples presented are in each case preliminary illustrations of a general approach, rather than representing final answers. 2. The hydrophobic-subtraction model [20,21] The selectivity of most RPC columns appears to be determined by five column characteristics: (i) (ii) (iii) (iv) (v)

hydrophobicity (H), steric resistance (S*), hydrogen-bond (H-B) acidity (A), H-B basicity (B), cation-exchange capacity (C).

Experimental retention data for 90 type-B alkylsilica columns (C3 –C18 ) and 150 different solutes were used to derive and evaluate the following relationship:   k = η H − σ  S∗ + β A + α B + κ C (1) log a = log kEB (v) (i) (ii) (iii) (iv) here, α is a separation factor; k and kEB are values of the retention factor for a given solute and the reference compound ethylben-

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zene (EB), respectively. Quantities η , σ  , β , α , and κ refer to certain properties of the solute molecule: hydrophobicity (η ), “bulkiness” (σ  ), H-B basicity (β ), H-B acidity (α ), and effective ionic charge (κ ). Corresponding column parameters H, S*, etc. are of primary practical interest and are defined above. The application of Eq. (1) for solute retention with type-B alkylsilica columns has been shown to be quite accurate; values of α from initial studies were predicted with an average standard deviation SD of ±1% (n = 2733). Values of the solute parameters η , σ  , etc. are consistent with relevant properties of the solute molecule, and column parameters H, S*, etc. can be reconciled with such column properties as ligand length and concentration, pore diameter, and the presence or absence of end-capping. Eq. (1) therefore appears to represent a fundamental description of reversed-phase retention and column selectivity. For the purpose of comparing columns of similar selectivity (Section 4.3.1), values of H, S*, A, and B can be regarded as essentially independent of mobile phase composition and temperature; values of C increase with mobile phase pH, due to the increasing ionization of silanol groups present in the column packing. Values of C can also vary with the type and amount of various ionic additives in the eluent, to the extent that these affect silanol ionization. 3. Experimental The sample and experimental conditions used in the present study were described in [22] (“18-solute procedure”). The sample consisted of a t0 -marker (thiourea) plus 12 neutral solutes, two rather strong bases (amitriptyline, nortriptyline), a weak base (4-n-hexylaniline), a quaternary ammonium compound (berberine), and two weak acids (n-butylbenzoic acid, mefenamic acid); 0.5 ␮g of each solute was injected. The mobile phase was 50% acetonitrile/pH 2.8 buffer; the temperature was 35 ◦ C, with a flow rate of 2.0 mL/min. In almost all cases, the alkylsilica columns had dimensions of 150 mm × 4.6 mm, and were packed with 5-␮m particles. At the present time, 223 typeB and 60 type-A alkylsilica columns have been characterized (see Sections 4.1 and 4.1.1 below for a definition of these two types of column); for values of H, S*, etc. for these and other columns, contact one of the authors. 3.1. Peak asymmetry measurements Values of peak asymmetry (As ) were of interest for ionized and ionizable solutes: the quaternary ammonium compound berberine and the five acidic or basic solutes in the above sample. Values of As were measured at 10% of the peak maximum [23] for all 19 solutes and for 80 alkylsilica columns (values of As were recorded or retained for only some of the 371 columns so far studied). Early peaks in the chromatogram exhibited some tailing, because of extra-column peak broadening. The following approximate expedient was used to carry out a (usually minor) correction for As values of the six ionized or ionizable solutes. Fig. 1 is a plot of As as a function of retention time tR for a type-A, alkylsilica column. Values of As for neutral solutes are shown as open circles, while values of As for ionized or ionizable

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Fig. 1. Correction of values of As for extra-column peak-broadening (partial chromatogram). See text for details.

solutes are indicated by closed circles. The latter solutes include amitriptyline (Ami+ ) and berberine (Ber+ ); data for nortriptyline (Nor+ ) are not shown in Fig. 1, and n-butylbenzoic acid (BBA), mefenamic acid (MFA), and 4-n-hexylaniline (4-HA) leave the column after 3 min. For each column studied, a smooth curve was drawn through data for the neutral solutes (open circles in Fig. 1). For the ionic solutes, excess values of As were determined as shown in Fig. 1 (value of δAs for Ami+ ). Values of As corrected for extracolumn effects were then calculated as δAs + 1. Finally, values of As shown in Fig. 5 (bases) and 6 (acids) are averages of corrected values for Nor+ , Ami+ and Ber+ (two protonated bases and a quaternary-ammonium compound), or BBA and MFA (two unprotonated acids), respectively. Additional experiments of peak-tailing as a function of sample weight were carried out in a second laboratory, using equipment (Shimadzu LC-10 HPLC) that was less subject to extra-column peak-broadening. Values of k for acidic and basic solutes from the latter study (Section 4.1.3) covered the range 1.8 < k < 7. In order to evaluate extra-column peak tailing over this range in k, we obtained values of As for the neutral solute propylparaben as a function of %B and k. For values of k ≥ 1.5, the average value of As was 1.0 ± 0.1; there was therefore no need to correct values of As reported in Section 4.1.3. 4. Results and discussion Dozens of different procedures have been reported which purport to describe column selectivity by means of retention measurements for selected test-solutes; e.g., see [14–19] and references therein. To be widely useful, a characterization procedure for measuring column selectivity should meet the following requirements: • all contributions to column selectivity should be accounted for, • each column parameter should correspond to a specific solute–column interaction; e.g., electrostatic interaction as represented by C and term v of Eq. (1) • selectivity data (e.g., values of H, S*, etc.) should be available for a significant fraction of the 600 + RPC columns that are commercially available,

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• different laboratories should be able to reproduce measurements of selectivity for a given column; e.g., values of H, S*, etc., • column selectivity data obtained for one set of experimental conditions should be applicable for the use of any mobile phase or temperature. At the present time, the hydrophobic-subtraction model and Eq. (1) appear to come closest to meeting the above requirements [19]. Thus, the accuracy of Eq. (1) when applied to type-B alkylsilica columns (±1% in α) suggests that all contributions to sample retention and selectivity are taken into account by Eq. (1) and related values of H, S*, etc. As noted above, each of the solute and column parameters of Eq. (1) are consistent with solute molecular structure and column properties, suggesting that each term of Eq. (1) corresponds to a specific solute–column interaction. At present, we have values of H, S*, etc. for 371 different RPC columns, which represents a significant fraction of all such columns that are sold today. One 4-way study [22] has confirmed that very similar values of H, S*, etc. can be obtained by measurements in different laboratories for identical columns (previously unused columns from the same production lot). The experimental variability in values of H, S*, etc. from different laboratories is not significant for the purpose of comparing column selectivity. Finally, values of H, S*, A, and B for similar columns are not significantly affected by changes in experimental conditions [24]; values of C vary with pH, so values of C at both pH 2.8 and 7.0 are measured (values of C for intermediate values can be estimated by interpolation). Consequently, reported values of H, S*, etc. can be used to compare columns (Sections 4.3.1 and 4.3.2), regardless of separation conditions; however, each such comparison must involve the same conditions for the two columns. 4.1. Peak tailing of acidic or basic solutes Peak-tailing is frequently observed for protonated bases that are separated on type-A columns (manufactured from “typeA” silica that is contaminated by varying amounts of certain trace metals, especially Al[III] and Fe[III]). This tailing has been ascribed to the electrostatic attraction of protonated bases (BH+ ) and ionized silanols ( SiO− ) [25–28]: BH+ + SiO− ⇔ SiO−+ HB

Peak tailing for acidic solutes has received less attention in the literature, but as will be seen below, can occur for both type-A and -B columns, even when the sample size is ≤0.5 ␮g. 4.1.1. Type-A vs. -B alkylsilica columns RPC column packings produced before 1990 were manufactured from type-A silica. Basic compounds often tail on these older columns [25–27], which has been attributed to ionic interaction as in Eq. (2), and the presence of more acidic silanols that are activated by metal-contamination of the silica. This problem encouraged the development of less-acidic packings made from higher-purity (“type-B”) silica, by means of the hydrolysis of “metal-free” tetraalkylsilicates. Since 1990, most newly introduced reversed-phase packings have been made from typeB silica. However, some metal contamination may be present in every silica, as a result of exposure to metals during silica manufacture. Consequently, the designation of a column on the basis of its date of commercialization (or claims by the manufacturer) as “type-B” may be imprecise, leading to uncertainty as to whether a so-called type-B column might be susceptible to tailing peaks or other problems associated with older, type-A columns. We will next consider two criteria for distinguishing “type-A” from “type-B” alkylsilica columns: • values of the column-selectivity parameters B (hydrogenbond basicity) and (especially) C (cation-exchange capacity), • peak tailing for 0.5-␮g samples of cationic solutes at low pH (for a given column, average values of As for Nor+ , Ami+ , and Ber+ ). 4.1.1.1. Values of B and C. A frequency-plot of the number of columns as a function of C-2.8 (value of C for a mobile-phase pH = 2.8) is shown in Fig. 2a for 85 type-B columns [30]; Fig. 2b shows a similar plot for 38 type-A columns [31]. The designation of “type-A” and “type-B” for these columns was made on the basis of either manufacturer claims or the date a given column was first sold. It is seen that 95% of the type-B columns have C-2.8 < 0.25, while only 11% of the type-A columns have

(2)

Peak-tailing of ionized solutes also occurs on newer type-B columns (made from purer silica) for larger samples, as a result of column overload (see Section 4.1.3 below and accompanying Fig. 7). For low-pH (≤3) mobile phases and 150 mm × 4.6 mm columns (as in the present study), peak-tailing for type-B columns can be observed for sample weights greater than 0.5 ␮g [28]; this has been attributed to ionic repulsion among retained solute molecules [29]. During our tests for the measurement of values of H, S*, etc., the weight of each injected solute was 0.5 ␮g, suggesting that any tailing observed cannot be attributed to column-overload of the latter kind.

Fig. 2. Frequency of columns with different values of C-2.8. (a) Type-B columns; (b) type-A columns. Data of [30,31].

D.H. Marchand et al. / J. Chromatogr. A 1191 (2008) 2–20

C-2.8 ≤ 0.25. Thus, a value of C-2.8 = 0.25 provides an approximate boundary between older (type-A) and newer (type-B) columns (vertical dotted line in Fig. 2). Values of B are also informative in this connection. Non-ionized carboxylic acids appear to interact with the trace metals present in type-A packings, as indicated by larger-than-expected values of the column parameter B (values of B are a measure of preferential retention of carboxylic acids, as a result of hydrogen bonding with basic sites in the stationary phase). In Fig. 3a, values of B are plotted vs. H for several type-B columns. An approximate correlation is noted (r2 = 0.61), with boundaries corresponding to ±2.5 SD indicated by dashed straight lines (same boundaries shown also in Fig. 3b). In Fig. 3b, a similar plot for several type-A columns is shown, where about 1/3 of the columns have larger values of B (data within the dashed circle) than would be expected for type-B columns. Larger values of B thus appear to provide an additional indication of type-A-like behavior. The largest value of B so far found for a known type-B column is 0.05 units, which allows

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Fig. 4. Classification of alkylsilica columns based on column basicity B and cation-exchange activity C-2.8. () type-A column; () type-B column. See text for details.

us to combine measurements of both B and C-2.8 for a slightly better classification of type-A from -B columns. This is illustrated in Fig. 4, where it is seen that 95% of type-B columns have C-2.8 < 0.25 and B < 0.05, while 95% of type-A columns fall outside these limits. While it appears that we can distinguish most type-A from -B columns as in Figs. 3 and 4, a small number of type-A and -B columns overlap in these plots; part of this overlap may be attributable to varying concentrations of trace metals that are incorporated into the silica during the manufacturing process. Another consideration is the presence or absence of column end-capping (which is usually, but not always, disclosed by the manufacturer). Thus, it was noted previously [20] for a non-end-capped Symmetry C18 column that the value of C-2.8 was increased by 0.24 units vs. an end-capped column. Similarly, the Prontosil 300-5-C30 column is available in endcapped and non-end-capped versions, where C-2.8 is increased by 0.10 units for the latter. While non-end-capped columns will thus appear more “type-A-like”, most alkylsilica columns sold today are end-capped.

Fig. 3. Excess column basicity (values of B) for type-A columns (b) vs. type-B columns (a) [20]. See text for details.

4.1.1.2. Peak tailing of cationic (basic) solutes. Fig. 5 is a plot of average values of As vs. C-2.8 for the three cationic (basic) solutes and 124 type-A and -B alkylsilica columns (0.5-␮g samples; different columns vs. those reported in Fig. 2). For columns with C-2.8 < 0.25 (i.e., type-B columns), the average value of As = 1.0 ± 0.2. Note that tailing tends to increase for ionized bases or cations as the value of C-2.8 increases, as summarized in Table 1 (however, a few columns with C-2.8 0.25 do not exhibit significant peak tailing for basic solutes). These results are consistent with the above “definition” of type-B columns (C-2.8 < 0.25), in that excessive peak tailing is associated with type-A columns. We also see in Table 1 that the solute 4-HA tails as a function of C-2.8 in similar degree as for Ami+ and Nor+ . However, while retained molecules of the latter two solutes are fully ionized, this is not the case for 4-HA, which is estimated to

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Fig. 5. Peak-tailing for cationic solutes as a function of C-2.8 (both type-A and -B columns). Values of As for each column are averages of values for berberine, nortiptyline and amitryptiline.

be 71% ionized in the mobile phase [24]. Since the retention of non-protonated vs. protonated solutes is preferred by a factor of 10 or more [32], retained 4-HA is presumed to be predominantly non-ionized. Note that the present study uses a mobile phase of 50% ACN/30 mM phosphate buffer (pH 2.8). The relatively high buffer concentration (30 mM) for experiments reported here tends to improve peak symmetry, while the use of ACN as B-solvent yields less symmetrical peaks compared to the use of methanol or tetrahydrofuran [28]. Therefore, other mobilephase conditions may result in either improved or worsened peak symmetry for basic solutes, compared to data reported here. Fig. 6. Peak-tailing of non-ionized carboxylic acids as a function of excess column basicity (δB, as defined in Fig. 4). (a) Type-B columns; (b) type-A columns. Values of As for each column are averages for 4-n-butylbenzoic acid plus mefenamic acid. See text for details.

4.1.2. Peak tailing of non-ionized acidic solutes Although peak tailing in the past has been associated mainly with protonated basic solutes, we have observed that acidic solutes can also tail when either type-A or -B columns are used. Because the interaction of acidic solutes with basic column sites is measured by the column parameter B, we might anticipate a correlation between values of B and peak tailing. More specifically, the extent of peak tailing for acids appears to correlate approximately with excess values of B (δB, measured as shown in Fig. 3b), or δB = B − 0.131 + 0.141H

increased metal contamination of the silica; however, values of δB do not correlate with values of C-2.8 (r2 = 0.04), apparently because metal contaminants affect values of B and C-2.8 differently. For type-B columns (Fig. 6a), no significant tailing of BBA or MFA occurs when δB ≤ 0.00; also, As ≤ 2 for 80% of all type-B columns (acidic solutes). Tailing of acids at pH = 2.8 can occur with type-A columns, regardless of the value of δB (Fig. 6b). Note that BBA and MFA are only 1–2% ionized in the mobile phase, so their retention factors k correspond to the retention of the undissociated acid in each case. The data of Figs. 5 and 6 are summarized in Table 1.

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Eq. (3) applies for any column; values of δB ≈ 0 for type-B alkylsilica columns. Positive values of δB may correspond to

Table 1 Average peak tailing as a function of the value of C-2.8 for the ionized or ionizable solutes of the present study Value of C-2.8

Average As Ami+

C < 0.25 0.25 ≤ C < 0.5 0.5 ≤ C < 1 C≥1

1.1 1.7 2.7 3.8

± ± ± ±

Nor+ 0.1 0.7 1.1 3.0

Data for 123 type-A and -B columns.

1.0 1.2 1.3 2.5

± ± ± ±

Ber+ 0.2 0.4 0.5 2.9

1.0 1.2 4.2 8.6

± ± ± ±

BBA 0.1 0.3 3.2 11.2

1.4 2.9 3.1 4.6

± ± ± ±

MFA 1.2 3.4 3.7 2.4

1.6 3.1 3.3 6.4

± ± ± ±

4-HA 1.6 3.5 3.4 4.3

1.1 1.3 1.7 2.9

± ± ± ±

0.3 0.4 0.9 2.3

D.H. Marchand et al. / J. Chromatogr. A 1191 (2008) 2–20

The tailing of basic or cationic solutes on a given column appears to be solute specific. Thus, values of As for different bases (Ami+ , Nor+ , Ber+ , or 4-HA) correlate poorly (r2 values of 0.00–0.27). Similarly, the correlation of As values for cations vs. acids is also poor (r2 = 0.01–0.19). McCalley and Brereton [33] and others [34] have called attention to similar differences in peak tailing for various combinations of columns and basic solutes. Thus, when peak tailing occurs for a given base on a specific column, other acids or bases may tail to a greater or lesser extent on that column. On the other hand, As values for the two acids BBA and MFA are highly correlated: As (MFA) = 1.05 As (BBA); r2 = 0.93. Thus if an acid tails on a given column, other acids appear likely to also tail. The latter, very tentative, conclusion is based on only two acidic solutes; however, their molecular structures are quite different. When tailing of acids or bases is encountered with low-pH mobile phases, the substitution of a column with C-2.8 < 0.25 (bases), or C-2.8 < 0.25 and δB < 0.00 (acids), should result in more symmetrical peaks. For mobile phases of higher pH, one qualitative study [20] found that the tailing of strongly basic solutes at pH 6.0 was “low” for columns with C-6.0 < 0.1, “moderate” for C-6.0 ≈ 0.5, and “high” for C-6.0 ≈ 1. Thus, it appears that columns with C > 0.25 should be avoided for the separation of cationic solutes at any pH. 4.1.3. Peak tailing vs. sample weight Peak tailing of protonated bases separated on type-B columns has been studied exhaustively by McCalley [28]. For low-pH mobile phases, it is now believed that electrostatic repulsion between retained, ionized molecules leads to premature overloading of the column [29], so that peak tailing is then observed for sample weights >0.5 ␮g and 150 mm × 4.6 mm-diameter columns (tailing for smaller samples can occur for low-ionicstrength mobile phases [35]). An example of such behavior is shown in Fig. 7 for the retention of Nor+ on a type-B column. As the sample size increases beyond 0.5 ␮g, peak tailing (values of As ) increases, while the plate number N decreases. At the same time, the retention factor k exhibits a more gradual decrease with increasing sample size. The separation of non-ionic compounds exhibits a similar behavior as in Fig. 7, except that column overload (with increase in As , and decrease in N and k) occurs for sample weights that are two orders-ofmagnitude larger (≥50 ␮g) – because electrostatic repulsion is absent. We have carried out experiments as in Fig. 7 for three typeA and two type-B columns, each of which exhibits significant tailing of acids and/or bases in our column-test procedure (0.5␮g sample). The selectivity characteristics of these five columns are summarized in Table 2. For type-A column “A” of Table 2, Fig. 8 plots values of N and As as a function of sample weight for the two protonated bases (a, Nor+ ; b, Ami+ ) and two nonionized acids (c, BBA; d, MFA); Fig. 9 provides corresponding plots of the retention factor k vs. sample weight for column “A”. Fig. 10 shows examples of peak shape as a function of sample size for MFA and Ami+ (column “A”). Note that each example of Fig. 10 spans an identical time (1.2 min), so that relative peak widths can be compared visually.

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Fig. 7. Elution behavior of nortriptyline (Nor+ ) as a function of sample weight. Conditions: 250 mm × 4.6 mm Inertsil ODS-2 column (type-B); mobile phase is 55% methanol/phosphate buffer (pH 3.0); 30 ◦ C; 1.0 mL/min. Figure is adapted from [36].

Numerical values of N, As , and k as a function of sample size are summarized in Table 3 for column “A”. A similar study of McCalley for different type-B columns (28% acetonitrile/pH 3 buffer as mobile phase, 0.2-␮g sample) resulted in an average value of N = 14,500 for nortriptyline [37] – a value which is similar to that for neutral solutes of similar molecular weight. The 250-mm columns of the latter study suggest a value of N = (150/250) × 14,500 ≈ 8700 for the 150-mm type-B columns used in the present study. The latter value of N is seen to be much larger than the values observed in Table 3 for Ami+ (1274–1377) or Nor+ (2136–2191), when the sample weight is 0.5-␮g or less. The behavior of the bases Nor+ and Ami+ with type-A column “A” can be summarized as follows: • Plots of N, As , and k vs. sample weight (Figs. 8 and 9) are similar in form as for type-B columns in Fig. 7 (a decrease in N and increase in As , plus a more gradual decrease in k, when the sample weight exceeds 0.5 ␮g). Table 2 Selectivity characteristics of five columns which were used for the further study of peak tailing at low pH Column

H

S*

A

B

Type-A A (C18 ) B (C8 ) C (C18 )

0.97 0.82 1.01

−0.05 −0.04 −0.07

0.28 −0.07 0.24

−0.05 0.14 0.12

0.87 0.45 2.04

Type-B D (C8 ) E (C18 )

0.84 0.96

−0.03 −0.02

−0.10 −0.13

0.03 0.01

−0.09 0.09

Conditions the same as described in Section 3.

C-2.8

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Table 3 Effects of sample weight on separation for column “A” of Table 2 As

Nor+ Ami+ Ber+ BBA MFA a

N

k

0.003 ␮g

0.5 ␮g

15 ␮g

0.003 ␮g

0.5 ␮g

15 ␮g

0.003 ␮g

0.5 ␮g

15 ␮g

2.6 2.7 –a 2.4 2.3

3.2 4.0 1.7 2.8 3.4

8.5 9.5 7.8 1.4 1.3

2136 1274 –a 2649 2348

2191 1377 977 6610 6169

442 364 469 5231 8479

2.38 3.10 2.55 3.56 6.87

2.29 2.93 2.43 3.35 6.64

1.83 2.30 1.89 3.28 6.47

Value not determined because of limit of detection.

• Values of N for sample weights <0.5 ␮g are 4- to 6-fold smaller than expected for type-B columns, and do not vary significantly with sample weight for sample weights of 0.003–0.5 ␮g. • Average values of As for sample weights <0.5 ␮g (As = 2.8 ± 0.3) are much greater than the average values found in our study for type-B columns (As = 1.0 ± 0.2), and do not appear to vary significantly with sample weight (see especially Fig. 8a and b). For the non-ionized acids BBA and MFA, we have observed peak tailing with type-B columns and 0.5-␮g samples – especially for columns with values of δB > 0.00 (Fig. 6a). Peak tailing

of acids is more common for type-A columns, and can occur for any value of δB (Fig. 6b). Figs. 8c,d and 9 show plots of As , N, and k as a function of sample size for BBA and MFA (type-A column “A”). The form of these latter plots for acidic solutes and type-A columns is in each case quite different from that observed for protonated bases on type-A columns (Figs. 8a,b, Fig. 9; Table 3), and can be summarized as follows for column “A” (and similarly for other columns which exhibit tailing peaks for small weights of acidic solutes; see Table 4): • Plots of N, As , and k for acidic solutes vs. sample weight (Figs. 8 and 9) show an increase in N and decrease in As when

Fig. 8. Plate number N and peak asymmetry As as a function of sample weight for bases (a, Nor+ ; b, Ami+ ) and acids (c, BBA; d, MFA). Column “A” from Table 3; other conditions as in Section 3.

D.H. Marchand et al. / J. Chromatogr. A 1191 (2008) 2–20

Fig. 9. Retention factors k as a function of sample weight for bases (Nor+ ; Ami+ ) and acids (BBA; MFA). Column “A” from Table 3; other conditions as in Section 3.

the sample weight exceeds 0.01 ␮g; values of k increase as sample size decreases below 0.5 ␮g. • Values of N for type-A columns and 15-␮g samples are nearnormal (N = 5200–8500). • Values of As for type-A columns and sample weights >0.5 ␮g (Table 3; As = 1.3, 1.4) are similar to values found for neutral solutes and type-B columns (As ≈ 1.0); values of As for acidic solutes appear to reach a maximum value of As = 3.5–4.0 for a 0.1-␮g sample. Table 4 Effects of sample weight on separation for columns B-E of Table 3 As

N

0.003 ␮g

0.5 ␮g

15 ␮g

0.003 ␮g

0.5 ␮g

15 ␮g

Column B Nor+ Ami+ BBA MFA 4-HA

1.7 1.3 2.3 2 1.2

1.9 2.2 6.8 9 1.3

5 5.8 3.4 4.5 1.5

4845 5051 1651 798 7813

4901 4703 2051 1132 8119

1231 1078 7480 8240 8527

Column C Nor+ Ami+ BBA MFA 4-HA

–a –a –a –a –a

4.1 5.2 4.7 3.7 4.2

14.3 14.6 5.8 5.2 4.9

Column D Nor+ Ami+ BBA MFA 4-HA

1.3 1.3 1.9 1.4 0.9

1.4 1.4 2.8 4 0.9

3.3 3.6 1.6 1.6 0.4

5405 5771 4510 4252 9471

5286 5440 7442 6654 9896

1600 1459 10030 10615 4102

Column E Nor+ Ami+ BBA MFA 4-HA

1.3 1.1 2 1.8 1

1.3 1.3 4 3.4 1

3.5 3.8 1.7 2.5 0.5

10765 10887 7339 6424 13695

10588 10711 9781 10509 13810

3446 3173 14245 13913 10375

a

–a –a –a –a –a

Value not determined because of limit of detection.

–a 172 345 653 300

–a 57 3990 2116 147

9

These trends appear to continue for larger sample weights; for a 30-␮g sample of BBA, for example, As = 1.3 and N = 7400, compared with values of As = 1.4 and N = 6610 for a 15-␮g sample. However, for a sample size of >50 ␮g, values of As for all solutes are expected to increase, with a decrease in N. Values of As and N as a function of sample weight are summarized in Table 4 for the remaining four columns of Table 2. For type-A columns “B” and “C,” similar trends are seen as in Table 3 for column “A”. Column “C,” which has a very large value of C-2.8 = 2.0, shows exceptional tailing (As = 3.7–14.6) for both acids and bases. For basic solutes and sample weights ≤0.5 ␮g, type-B columns “D” and “E” exhibit larger values of N (avg. N = 8100) and near-symmetrical peaks (As = 0.9–1.4), with changes in values of N and As for larger samples that are similar to those of Fig. 7. The weak base 4-HA exhibited fronting (As = 0.4–0.5) for larger samples and type-B columns. 4.1.4. Origins of peak tailing for acids and bases (sample weights ≤0.5 μg) Peak tailing as a function of sample size might arise for reasons other than column overload (neutral solutes) or ionic repulsion (ionized solutes): • strong retention sites present in low concentration, with consequent overloading of these sites – even for sample-weights <0.5 ␮g, • slow sorption–desorption kinetics (“kinetic tailing”) of the retained sample molecule, regardless of sample size. Either of the above two possibilities might arise as a result of the strong interaction of acids or bases with ionized silanols or trace-metal impurities in the column packing. For peak-tailing of protonated bases or quaternary ammonium compounds on type-A column “A,” our results are consistent with the presence of a single, homogenous set of strong sites (presumably ionized silanols) which exhibit kinetic tailing. For small samples (0.003–0.5 ␮g), values of N, As , and k are essentially constant, but the peaks are wider (smaller N) and tail more (larger As ) than when type-B columns are used. The latter results are inconsistent with an overloading of strong sites present in lower concentration, instead suggesting that we are dealing here with kinetic tailing. When sample size exceeds 0.5 ␮g for these protonatedbases, ionic repulsion would be expected to have a similar effect on values of N, As , and k for both type-A and -B columns; this is observed, which provides further evidence in support of McCalley’s theory of electrostatic repulsion [29]. Peak shapes in Fig. 10 corroborate the latter interpretation. Thus, the Ami+ peak for a 15-␮g sample shows a “right-triangle” shape (possibly with a minor amount of kinetic tailing) that is characteristic of column overload due to electrostatic repulsion, while the 0.003 and 0.5␮g peaks exhibit exponential tailing (as expected for “kinetic” tailing). The corresponding behavior of non-ionized acids that tail for sample weights ≤15 ␮g (either type-A or -B columns) suggests a 2-site model for retention. A small concentration of strongly-retentive, possibly tailing, sites appears to exist, along with a larger concentration of less-retentive sites which are free

10

D.H. Marchand et al. / J. Chromatogr. A 1191 (2008) 2–20

Fig. 10. Peak shape as a function of sample weight for mefenamic acid (MFA) and amitriptyline (column A, conditions given in Section 3).

from kinetic tailing. Thus, for larger samples, most of the solute molecules are retained on the weaker sites, with resulting nearsymmetrical peaks, near-normal values of N, and constant values of k. As sample size decreases, relatively more of the solute molecules are retained on (overloaded) stronger sites, so that N decreases, As increases, and values of k increase. Values of N, As , and k vs. sample weight over the range 0.003–0.5 ␮g suggest a possible combination of overloaded strong sites and kinetic tailing, but further work is required to work out the details. 4.2. Column degradation and changes in selectivity It is well known that RPC columns degrade with continued use, due to (a) loss of the bonded phase when a low-pH mobile phase is used, or (b) dissolution of the underlying silica at higher pH [38,39]. The loss of bonded phase is accompanied by an increase in accessible silanols, so that (typically) the reten-

tion of neutral solutes decreases (lower H), and the retention of protonated bases increases (higher C) as the column ages. For columns that have been end-capped, the end-capping is lost first at low pH (short alkyl chains are less stable than longer ligands [40]). Table 5a summarizes changes in values of H, S*, etc. (δH, δS*, etc.) for eight different type-B C18 columns that were tested when new, stored for 2–27 months in acetonitrile, then tested again; each of these columns was end-capped. Columns A1 and A2 are made by company “A”, B1 and B2 by company “B”, and C1-4 by company “C.” Original values of H, S*, etc. prior to storage and re-testing are shown in Table 5b. There is little change in values of H, S*, or B from initial to final testing (small values of δH, δS*, or δB in Table 5a), but large, consistent increases in values of A (avg. δA = 0.04) and C-2.8 (avg. δ[C2.8] = 0.12), suggesting an increase in number and/or strength of non-ionized (A) and ionized (C) silanols due to a loss of bonded phase. Average, combined values of δA plus δ(C-2.8) vary with

D.H. Marchand et al. / J. Chromatogr. A 1191 (2008) 2–20

11

Table 5 Changes in column selectivity (column degradation) with time Columna

Timeb

Change in column parameters with timec

(a) Changes in column parameters A1 27 A2 27 B1 15 B2 15 C1 2 C2 2 C3 2 C4 2 Average Standard deviation

␦H

␦S*

␦A

␦B

␦(C-2.8)

␦(C-7.0)

0.000 0.006 0.003 0.000 0.002 −0.002 0.000 0.004

−0.007 −0.003 −0.004 −0.014 −0.006 −0.015 0.016 0.004

0.026 0.002 0.022 0.081 0.099 0.055 0.014 0.045

0.001 0.006 0.003 0.007 0.005 −0.019 0.005 −0.013

0.005 0.008 0.070 0.149 0.013 0.103 0.350 0.234

0.032 0.028 −0.045 −0.009

5 2 2 2 −1 −2 −2 −1

0.002 0.003

−0.003 0.010

0.043 0.034

−0.001 0.010

0.117 0.124

0.002 0.036

1 2

H (b) Original column parameter values A1 0.94 A2 0.97 B1 1.03 B2 0.86 C1 0.98 C2 0.78 C3 1.08 C4 0.94 H (c) Correlations δA δ(C-2.8)

r2

for δH, δS*, etc. vs. H, S*, etc. 0.2 0.06

␦ kEB (%)

S*

A

B

C-2.8

C-7.0

0.02 0.02 0.03 0.02 −0.02 0.08 −0.07 −0.06

0.07 0.11 0.07 −0.04 −0.04 −0.17 0.07 0.03

0.02 0.01 −0.01 0.01 0.01 0.04 −0.04 0.00

0.23 0.16 −0.12 −0.13 0.18 −0.06 −0.32 −0.08

0.25 0.17 −0.14 0.00 0.19 0.49 0.12 0.18

S*

A

B

C-2.8

C-7.0

0.00 0.41

0.48 0.00

0.14 0.45

0.02 0.77

0.01 0.01

Use-conditions defined in text; type-B columns. a Column designations refer to manufacturer (A, B, C) and different column types supplied by that manufacturer (1–4). b Time (months) during which columns were stored between initial and final testing. c Change in values of H, S*, etc. from initial to final tests.

the column source: company A, 0.01; company B, 0.08; and company C, 0.11. Since these values increase inversely with the storage time (27, 15, and 2 months, respectively), this suggests that the time of storage in acetonitrile contributes little to column degradation – an observation that is consistent with the experience of other workers (U. D. Neue, J. J. Kirkland, unpublished studies). It also appears that columns A-1 and -2 are the most stable, while columns C-1 to -4 are the least stable. This suggests that different, proprietary manufacturing processes (including the silica source) may affect column instability as summarized in Table 5a. Correlations r2 of values of δA and δ(C-2.8) vs. H, S*, etc. are summarized in Table 5c. The most pronounced correlation is for values of δ(C-2.8) vs. C-2.8: δ(C-2.8) = 0.11 − 0.57C-2.8

(r = 0.77) 2

(4)

That is, as C-2.8 increases, δ(C-2.8) decreases. Differences in average values of C-2.8 among the three column manufacturers of Table 5 can be explained by one (or possibly both) of two possibilities: (1) the use of a more acidic silica by company A, or (2) a more thorough end-capping by company C. That is, values of C-2.8 will increase with silica acidity, and decrease

with more complete end-capping. If differences in the extent of end-capping are the major factor, it would be reasonable that δ(C-2.8) decreases with a decrease in end-capping (consistent with the data of Table 5 and Eq. (4)), since with less end-capping, there will be less loss of end-capping – other factors equal. This would also suggest that column end-capping is more complete for company C, and less complete for company A. In this connection it would be interesting to measure the extent of end-capping for the columns of Table 5, but we have not done this. The next most significant correlation of Table 5c is the dependence of values of δA on A: δA = 0.05 − 0.26A

(r 2 = 0.48)

(5)

The same argument as above for Eq. (4) can be made for Eq. (5), since a decrease in end-capping presumably also leads to an increase in non-ionized silanols and values of A. Thus, a reduction in column end-capping leads to less loss in endcapping, and a slower increase in the number of non-ionized silanols (and values of A) as a result of column use. However, it is surprising that there is no correlation of values of δA and δ(C2.8) (r2 = 0.02). This suggests that the original concentrations and production (during use) of non-ionized and ionized silanols

12

D.H. Marchand et al. / J. Chromatogr. A 1191 (2008) 2–20

are uncorrelated, an observation which (if true) merits further investigation. The only other correlation of any significance in Table 5c is that of δ(C-2.8) vs. S*: δ(C-2.8) = 0.05 − 0.26S ∗

(r 2 = 0.41)

Two columns with small values of Fs possess similar selectivity and should therefore provide similar separations of a given sample when other separation conditions are the same; two columns with large values of Fs will be very different in selectivity.

(6)

That is, there is an increased loss in ionized silanols for smaller values of S* (less steric resistance to entry of solutes – or other mobile phase species). This might appear reasonable – since it suggests easier entry of a reactive species that can remove end-capping. But why is there not a similar correlation between values of δA and S*? If steric hindrance plays a role in the loss of bonded phase, it may not be very important and could be obscured by other factors. Values of δ(C-7.0) tend to be much smaller (avg. 0.002 ± 0.036) than values of δ(C-2.8) (0.117 ± 0.124), which may reflect the fact that at pH 7.0 a much larger number of silanols are ionized; consequently, any small increase in silanol concentration would have a relatively small effect on values of C-7.0 (since values of A and C are related to the logarithm of silanol concentration). Finally, the value of k for ethylbenzene (kEB ) shows an insignificant change for used columns (avg. δkEB = 1 ± 3%), which is in agreement with corresponding changes in column hydrophobicity (avg. δH = 0.002 ± 0.003). This supports a loss of end-capping groups (with generation of additional silanols), rather than a loss of C8 or C18 , because ligands contribute much more to the retention of non-polar solutes and values of H than end-capping. Because values of C-2.8 do not to change as a result of column-testing at pH 2.8, the major contribution to column deterioration in these studies seems attributable to the brief exposure of the columns to pH 7.0 mobile phase during the initial measurement of C-7.0 values. It is possible that any change in column selectivity as a result of (brief) exposure of the column to pH 7 mobile phase is short term, and may form part of the initial conditioning of new columns when used at higher pH. Similar short-term changes in the retention of ionized solutes, as a result of exposure of the column to pH 7, were noted previously [41]. The data of Table 5 constitute at most a preliminary study, and are presented primarily as an illustration of how column degradation might be interpreted in terms of changes in values of H, S*, etc. 4.3. Comparing column selectivity For any two alkylsilica columns, the difference in their selectivity can be described in terms of Eq. (1) by a function Fs [20] which is equal to the weighted distance between two columns 1 and 2 whose values of H, S*, etc. are plotted in 5-dimensional space: Fs = {[12.5(H2 − H1 )]2 + [100(S2∗ − S1∗ )]2 + [30(A2 − A1 )]2 1/2

+ [143(B2 − B1 )]2 + [83(C2 − C1 )]2 }

(7)

4.3.1. Selecting columns of very similar selectivity After a routine HPLC method has been developed, it is often applied in different laboratories for several months or years. There will then be a need for column replacement from time to time, due to the usual deterioration of the column with use. While in most cases there is no difficulty in obtaining a replacement column of the same kind (e.g., StableBond C18) that will provide equivalent separation, this is not always the case – especially for samples that are more difficult to separate. Furthermore, when a method is transferred to another laboratory or country, a specific column may not be available at the new site. In either case, it is helpful if an alternative replacement column (different source and/or kind) with similar selectivity can be identified quickly for use with the assay procedure. Eq. (7) can be used for this purpose; a value of Fs ≤ 3 signifies “equivalent” column selectivity in almost all cases, while a value of Fs ≤ 10 suggests that the two columns are adequately similar for the separation of many samples. The selection of an alternative, equivalent column (low values of Fs for the original and replacement columns) is facilitated by the use of appropriate computer software (see www.usp.org or one of the authors), combined with values of H, S*, etc. for a large number of commercial columns. The usefulness of this procedure for selecting a replacement column has been confirmed for several routine assay procedures that involved the use of different columns [42]. 4.3.2. Selecting “orthogonal” columns By “orthogonal” columns, we mean two columns with very different selectivity; that is, with large values of Fs from Eq. (7). There are various reasons for choosing an orthogonal column. First, when developing a reversed-phase separation [25], a large change in separation selectivity may be needed in order to improve the resolution of certain peaks in the chromatogram. Second, during method development for samples of initially unknown composition, there may be a concern that a minor component might be overlapped by a larger peak in the chromatogram – and therefore missed in the final analysis. Third, a similar situation may arise in the use of a routine procedure for future samples that might contain additional, unanticipated impurities. In either of the latter two cases, it is desirable to have available one or more “orthogonal” separations, for which selectivity is very different from that provided by the original assay procedure; if two peaks overlap in the routine separation, they are then more likely to be separated (and observable) in the orthogonal separation. The use of Eq. (7) for the latter two applications has been demonstrated for a dozen different routine assay procedures [43]. Fourth, orthogonal columns are required for the technique of thermally-tuned tandem-column optimization [44], in which two columns connected in series are operated at different temperatures in order to better control selectivity. Finally, orthogonal columns may be required for two-dimensional sep-

D.H. Marchand et al. / J. Chromatogr. A 1191 (2008) 2–20

13

Table 6 Summary of values of H, S*, etc. as a function of ligand and pore diameter Columnsa

S*

A

0.41 0.60 0.53

−0.08 −0.12 −0.12

−0.08 −0.08 −0.19

C4 B

0.69 ±0.03

−0.03 ±0.03

C4W B

0.45 ±0.26

0.02 0.04 0.05

0.04 −0.08 0.06

0.66 0.81 0.36

1.2 2.8 0.7

1 1 1

−0.23 ±0.17

0.02 ±0.01

0.04 ±0.07

0.38 ±0.42

3.3 ±1.4

7

8

−0.06 ±0.07

−0.08 ±0.16

0.03 ±0.02

0.02 ±0.06

0.53 ±0.20

1.9 ±1.1

4

7

0.80

0.04

−0.25

0.00

−0.28

0.11

5.9

1

C5W B

0.69 ±0.05

0.00 ±0.03

−0.34 ±0.05

0.02 ±0.01

0.11 ±0.03

0.27 ±0.08

1.3 0.3

2

4

C8 B

0.84 ±0.06

0.00 ±0.04

−0.12 ±0.15

0.02 ±0.02

−0.03 ±0.18

0.25 ±0.39

5.4 ±1.6

41

12

C8W B

0.77 ±0.06

−0.02 ±0.04

−0.13 ±0.13

0.03 ±0.01

0.15 ±0.08

0.39 ±0.30

1.9 ±0.5

10

8

C18 B

0.99 ±0.08

0.01 ±0.04

−0.01 ±0.15

0.00 ±0.06

0.00 ±0.19

0.24 ±0.41

8.8 ±2.7

94

19

C18W B

0.95 ±0.05

0.01 ±0.03

−0.05 ±0.13

0.01 ±0.01

0.22 ±0.11

0.31 ±0.24

3.2 ±1.0

18

11

C30 B

1.05 ±0.10

−0.01 ±0.04

0.09 ±0.41

−0.02 ±0.04

−0.08 ±0.33

0.45 ±0.39

13.0 ±7.4

2

31

0.69

−0.02

−0.06

0.03

0.31

0.68

1.0

1

C8 A

0.44 ±0.37

0.02 ±0.03

0.12 ±0.17

0.02 ±0.03

0.15 ±0.16

0.42 ±0.16

4.6 ±4.7

7

21

C18 A

0.94 ±0.10

−0.05 ±0.06

0.14 ±0.15

0.01 ±0.05

0.79 ±0.47

1.18 ±0.61

6.4 ±1.8

44

67

C18W A

0.87 ±0.07

0.04 ±0.10

0.26 ±0.14

−0.09 ±0.19

0.48 ±0.17

1.05 ±0.27

2.0 ±0.2

4

32

C4W A

kEB

Fs c

C-7.0

C5 B

B

nb

C-2.8

C1 B C3 B C3W B

H

0.92

−0.13

0.57

0.00

0.51

1.79

6.9

1

C30W A

0.91 ±0.02

−0.08 ±0.03

0.22 ±0.20

0.02 ±0.01

0.34 ±0.06

1.06 ±0.55

3.4 ±0.9

3

9

Polar end-capped C18 B

0.90 ±0.11

−0.04 ±0.04

−0.02 ±0.21

0.02 ±0.03

−0.02 ±0.14

0.40 ±0.37

7.4 ±2.3

15

14

Embedded polar group

0.74 ±0.17

0.00 ±0.06

−0.22 ±0.37

0.12 ±0.10

−0.27 ±0.32

0.53 ±0.42

5.9 ±2.4

20

33

Phenyl B

0.63 ±0.08

−0.12 ±0.06

−0.20 ±0.15

0.02 ±0.02

0.13 ±0.10

0.68 ±0.37

2.7 ±1.1

17

20

Phenyl A

0.64 ±0.08

−0.16 ±0.00

−0.27 ±0.04

0.02 ±0.00

0.56 ±0.29

0.89 ±0.12

2.3 ±0.3

2

51

Cyano B

0.43 ±0.03

−0.09 ±0.03

−0.49 ±0.18

0.00 ±0.02

0.02 ±0.10

0.72 ±0.23

1.0 ±0.5

15

10

Cyano A

0.41 ±0.06

−0.14 ±0.04

−0.11 ±0.30

−0.01 ±0.02

0.57 ±0.18

1.25 ±0.10

0.8 ±0.6

2

18

Perfluorophenyl

0.65 ±0.10

−0.11 ±0.04

−0.25 ±0.06

0.01 ±0.04

0.40 ±0.42

0.96 ±0.55

4.3 ±2.0

4

36

Fluoroalkyl

0.66 ±0.05

−0.07 ±0.14

−0.11 ±0.30

0.03 ±0.01

0.87 ±0.23

1.18 ±0.34

3.7 ±0.4

2

25

“Cationic”d

0.57 ±0.17

0.04 ±0.06

−0.53 ±0.34

0.24 ±0.11

−2.33 ±0.91

−0.41 ±0.84

3.7 ±2.4

6

78

Zirconia base

0.97 ±0.35

0.01 ±0.21

−0.62 ±0.33

0.00 ±0.07

2.01 ±0.23

2.01 ±0.23

0.8 ±0.5

3

32

C30 A

14

D.H. Marchand et al. / J. Chromatogr. A 1191 (2008) 2–20

Table 6 (Continued ) Columnsa

H

S*

A

B

C-2.8

C-7.0

kEB

nb

Average valuese SDe Fs (i)e,f

0.83 ±0.19 2.4

−0.02 ±0.07 7.0

−0.10 ±0.27 8.1

0.02 ±0.07 10.0

0.14 ±0.52 43

0.53 ±0.56 47

5.5 ±3.3

371

Fs c

“W” refers to pore diameters ≥20 nm; “A” and “B” refer to type-A and -B silica, respectively; e.g., “C8W B” refers to a wide-pore, type-B C8 column. The number of columns of each type in our database. c A measure of the range in selectivities of columns within a given group; larger values of F mean that columns of a specified type or group are more different s from each other in terms of selectivity; see Section 4.4.1 for further details. d Group consists of following 6 columns: Hypersil Prism C18 RP, Zorbax Bonus RP, EC Nucleosil 100-5 Protect 1, Hypurity Advance, Prevail Select C18, Inertsil CN-3. e For all 371 columns (values are essentially the same for the 368 silica-base columns). f e.g., F for H = 12.5 × 0.19, where “12.5” is the coefficient for H in Eq. (7). s a

b

aration, where two different columns are used sequentially for the separation of a given sample. For further insight into the best choice of orthogonal columns for the above (or other) applications, see Section 4.4.3 below. 4.4. Column selectivity vs. ligand and silica type RPC columns are described by the manufacturer in terms of the nature of the ligand (C18 , C8 , phenyl, etc.), particle pore diameter, and (sometimes) silica type (A or B). A recurring question is: how well do these characteristics (alone) determine column selectivity, and differentiate columns of different selectivity? Values of H, S*, etc. allow a closer look at column selectivity as a function of the ligand and particle (Type-A or -B). 4.4.1. Ligand length and particle pore diameter The selectivity of an individual column can be described by its values of H, S*, etc. When columns of a given ligand type (C18 , C8 , phenyl, etc.) are considered as a group, the selectivity of the group can be described in terms of average values of H, S*, etc. for all columns within the group. Table 6 summarizes average values of H, S*, etc. for the various ligands that are represented in our study of 371 columns. Data for each ligand are further subdivided into narrow-pore (<20 nm) and wide-pore (≥20 nm) columns. The standard deviations of each parameter value for all columns within the group (SD[H], SD[S*], etc.) then define the range in selectivity of the column group. The quantity Fs in Table 6 (last column) is a related measure of the range in selectivity for each column type, calculated from Eq. (7) using SD(H) in place of H2 -H1 , SD(S*) in place of S*2 –S*1 , etc. The quantities SD(H), SD(S*), etc. are standard deviations of values of H, S*, etc. for columns within a given group (see Table 6). While values of C for the zirconia-base columns are extreme, exclusion of these (three) values has no significant effect on the average values of Table 6. 4.4.1.1. Alkylsilica columns. If ligand type were the primary determinant of column selectivity, average values of H, S*, etc. for each column group should be significantly different. Furthermore, values of Fs for two columns in different groups would generally be greater than Fs values for two columns within the same group. We could test this conjecture by comparing values

of Fs from Eq. (7) for every pair of columns in our database (0.5 × 3712 values of Fs ). A more convenient comparison, one that can be represented graphically, is suggested by the observation that the quantities B and especially C are the primary contributors to values of Fs , when Fs > 10 (Section 4.4.3). Consequently, a plot of values of B vs. C-2.8 for different column groups allows an approximate comparison of group selectivity (see related Fig. 1 in [45]). Such a plot for different type-B alkylsilica columns is shown in Fig. 11. The dashed ellipse in Fig. 11 for narrow-pore C18 columns represents a ±1 SD range in values of B and C-2.8 for this column group; other column groups in Fig. 11 exhibit a similar range in SD values of B and C-2.8 (dashed ellipses not shown). It is apparent from Fig. 11 that the selectivities of different type-B alkylsilica columns overlap extensively for different ligand lengths and pore diameters. That is, column selectivity is determined to only a limited extent by ligand length and pore diameter. For purposes of identifying column type, however, values of H and the retention factor for ethylbenzene (kEB ) together provide a reasonable differentiation of type-B alkylsil-

Fig. 11. A comparison of selectivities for different type-B alkylsilica columns as a function of values of B and C-2.8. The dashed ellipse indicates the 1-SD range in selectivities of each column type, illustrated in the figure for narrow-pore C18 columns. See text for details.

D.H. Marchand et al. / J. Chromatogr. A 1191 (2008) 2–20

15

Fig. 12. A comparison of column type for different type-B alkylsilica columns as a function of values of H and kEB . The dashed ellipse indicates the 1-SD range for each column type. See text for details.

ica columns – as illustrated in Fig. 12. Larger-pore columns will have less surface area, so it is expected that values of kEB will be smaller. Since the dashed ellipses correspond to a ±1-SD variation in values of H and kEB (i.e., only 67% of all columns in each group are contained within their ±1-SD elipses), there is appreciable overlap of columns in each group of Fig. 12. Thus, values of H and kEB are only approximate (but possibly the best available) indicators of ligand length and pore diameter for type-B alkylsilica columns. 4.4.1.2. Monolithic and silica-methylsilane hybrid columns. These are two, recently introduced, column types that are compositionally unique. A relevant question is: how different in selectivity are these column when compared to conventional RPC columns that carry the same ligand? Monolithic columns can be characterized as type-B alkylsilica, but they possess a completely different pore structure [46] compared to porous particles. Three monolithic columns are included in our present database: Chromolith RP18e (Merck), Onyx Monolithic C8, and Onyx Monolithic C18 (Phenomenex). If each of these columns is compared with porous-particle columns that have the same ligand (see average values of H, S*, etc. in Table 6), values of Fs (Eq. (7)) for the three monoliths are 9, 4, and 13, respectively. The latter values can be compared with differences in selectivity among conventional columns of the same type, defined by the quantity Fs – values of which are listed in the last column of Table 6 for each column type. For type-B C8 and C18 columns, values of Fs range from 8 to 19. That is, the selectivity of monolithic columns appears no more different from corresponding conventional columns, than the latter are from each other. For a fuller discussion of the quantity Fs , see Section 4.4.1 above. Silica-methylsilane hybrid columns have been introduced by Waters under the label XTerra MS C8 and C18 . Their selectivity also appears similar to that of conventional type-B C8 and C18 columns, respectively (Fs values of 10 and 12, respectively, compared to Fs values of 8–19 for other C8 and C18 columns [Table 6]). Two studies have shown that the silanols present on hybrid column-packings do not ionize for a mobile-

Fig. 13. A comparison of column selectivities for different column ligands as a function of values of B and C-2.8. The dashed ellipse indicates the 1-SD range in selectivities of each column type; narrow-bore, type-B columns. See text for details.

phase pH < 8 [47,48]. Average values of A, C-2.8, and C-7.0 for the hybrid columns differ from average values for conventional type-B C8 and C18 columns, respectively, by −0.15, 0.11, and −0.23 units. As expected, values of C-7.0 are significantly lower for the hybrid columns, although the somewhat higher values of C-2.8 are puzzling. However, values of C-2.8 for conventional C8 or C18 columns vary by ±0.18 and ±0.25 (1 SD), respectively; i.e., values of C-2.8 for the hybrid columns fall within the range of variability for conventional columns. 4.4.2. Other ligands Fig. 13 shows a similar plot (as in Fig. 11) of values of B and C-2.8 for narrow-pore, type-B columns of different ligand type. Corresponding values of Fs in each direction (x and y) are indicated on the sides of Fig. 13, showing that values of Fs for selected columns can exceed a value of Fs = 300. A previously unrecognized column type is noted in Table 6: the “cationic” group. “Cationic” columns have extremely low values of C-2.8 (<−1.0), which may be consistent with a positively charged silica surface at low pH (see Table 6 for examples of “cationic” columns). The “cationic” ( ), embedded-polar-group (), fluoroalkyl (♦) and zirconia-base () columns are each relatively distinct in terms of selectivity, but other column groups in Fig. 13 overlap extensively. Ligand type is again only roughly related to column selectivity. 4.4.3. Columns for orthogonal separation Previous workers have described the use of different columns combined with other changes in separation conditions (e.g., mobile phase pH), in order to achieve maximum changes in separation selectivity or “orthogonal” separation [49–53]. Section 4.3.2 above describes a variation on this approach [43], using columns of different selectivity that were selected on the basis of their values of Fs . By means of our data for 371 different RPC columns, it is now possible to reexamine the logic

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of prior attempts at designing and evaluating orthogonal separation. Average values of H, S*, etc. and SD values for the entire column set are reported at the bottom of Table 6. The average contribution of individual parameters H, S*, etc. to Fs (see Eq. (7)) is given by the value of SD times each weighting factor in Eq. (7). For H, the contribution is 0.19 × 12.5, or a value of 2.4; we will refer to the latter quantity as Fs (H), with similar contributions from column parameters S*, A, B, and C defined as Fs (S*), Fs (A), Fs (B), and Fs (C). Values Fs (i) of the latter quantities are given at the bottom of Table 6, which show that the contribution to Fs from C (Fs [C] = 43–47) far outweighs contributions from the remaining four parameters H, S*, A, and B (Fs [i] = 2.4–10), especially since the form of Eq. (7) results in a <10% contribution to Fs from any parameter whose value of Fs (i) is less than 1/3 as large as the largest value of Fs (i) ≡ Fs (C). A good example of the dominance of C in determining values of Fs is provided by Fig. 14. For the two columns shown (YMC PackPro C18 RS and Capcell C18 AG12), a value of Fs = 60 can be calculated, corresponding to an estimated average change in separation factors α of about 2-fold (quite large!). This suggests

a very different selectivity for these two columns. While their values of C-2.8 are quite different (−0.18 for the YMC column, and 0.54 for the Capcell column), values of H, S*, A, and B for the two columns are fairly similar. Consequently we can expect that cations will be relatively more retained on the Capcell column (Fig. 14b), compared to the YMC column (Fig. 14a) – but the relative retention of other compounds in this sample should be similar. As can be seen from the separations of Fig. 14a and b, this is just what we find. Fig. 14c shows a plot of values of log k for the YMC vs. Capcell columns; a good correlation (solid line, r2 = 0.997) is noted for the non-cations, but values of k for the cations (dashed line) are about 4-fold greater on the Capcell column – relative to values of k for other solutes in the sample. Thus, the large value of Fs for this example is deceptive, signifying little change in relative retention – except for the sample cations. It should be noted that large changes in C affect the retention of both ionized bases BH+ and acids R COO− ; however, the carboxylic acids in Fig. 14 are all non-ionized. The foregoing example challenges the use of values of Fs alone for the selection of orthogonal columns, as described in Section 4.3.2. For any two separations which differ in conditions, it similarly raises doubts about any comparison of values of k for two separations (as in Fig. 14c), where the sole measure of “orthogonality” is the overall correlation r2 [49–53]. Other workers have also noted this problem [49,52], suggesting that plots as in Fig. 14c should be inspected visually, in order to identify systems as in Fig. 14c that are of limited orthogonality. Thus, prior attempts at “orthogonal” separation appear to provide limited assurance that the relative retention of solutes other than acids or bases will be significantly altered. Consider also that just as protonated bases have a disproportionate effect on values of Fs for two columns, the retention of ionizable solutes is selectively affected by changes in mobile-phase pH – and to a lesser extent, by changes in temperature, solvent strength (%B), or gradient time [54,55]. That is, other experimental conditions can duplicate the ability of two columns to provide differences in retention for ionizable vs. non-ionizable solutes, making column selectivity due to differences in C somewhat redundant. The data of Table 7 provide further insight into the factors that affect relative retention for different sample compounds. For each set of data in Table 7, values of Fs have been calculated for the use of only one of the five column parameters; i.e., values Table 7 Contributions of each column parameter to maximum values of Fs at pH 2.8.

Fig. 14. Separation on two columns that differ mainly in their values of C. Sample: (1) N,N-dimethylacetamide; (2) N,N-diethylacetamide; (3) pnitrophenol; (4) 5,5-diphenylhydantoin; (5) acetophenone; (6) benzonitrile; (7) 5-phenylpentanol; (8) anisole; (9) n-butylbenzoic acid; (10) toluene; (11) cis-chalcone; (12) mefenamic acid; (13) trans-chalcone; (14) ethylbenzene. Conditions, see Section 3. (a and b) Chromatograms for YMC Pack Pro C18 RS and CAPCELL C18 AG120 columns; (c) plot of log k for YMC Pack Pro C18 RS vs. CAPCELL C18 AG120 columns. See text for details.

Contribution

Groupa

Columnb

Fs (H) Fs (S*) Fs (A) Fs (B) Fs (C)

7 2 18 36 364

12 47 65 108 437c , 450d

a Value of F for two column groups (as in [b]) when only a single s column parameter is used to calculate Fs ; e.g., for column parameter H, Fs = {[12.5(H2 –H1 )]2 }1/2 ≡ 12.5 (H2 –H1 ). b Same as (a), except the two most different columns are compared; i.e., for column parameter H, the two columns with largest and smallest values of H. c Silica-base columns only. d All columns (including Zr-base).

D.H. Marchand et al. / J. Chromatogr. A 1191 (2008) 2–20

of Fs (H), Fs (S*), etc. In each case, maximum values of Fs (i) are shown. For example, in the case of the parameter Fs (H), values of H from the various column groups of Table 6 were used to calculate values of Fs for each pair of (average) columns. The largest resulting value of Fs ≡ Fs (H) = 7 was obtained for C18 B columns vs. cyano B columns. To determine the largest value of Fs (H) for individual columns, data for all 371 columns were sorted according to values of H, from which the smallest and largest values could be obtained (H = 0.32, Discovery HS PEG; H = 1.28, ZirChrom PBD). Consequently, the maximum value of Fs (H) = 12.5 × (1.28–0.32) = 12. A similar procedure was used to obtain the remaining values of Table 7. Ideally, two orthogonal columns would have contributions Fs (i) from each column parameter that approach the maximum values of the last column of Table 7 (however this is most unlikely, based on present column data). Various means can be considered for an improved selection of columns that are orthogonal for all solutes, whether ionized or not. Several column-pairs with large values of Fs might be selected first, then further compared by means of Eq. (7) with the last term (C2 –C1 ) omitted; i.e., values of Fs (−C). Columnpairs with large values of both Fs and Fs (−C) can be examined further for comparable contributions to Fs (−C) from S*, A, and B (values of Fs [H] play at most a very minor role in affecting orthogonality). Because of the large change in column selectivity associated with values of C, even moderate differences in C will usually suffice for significant changes in relative retention for protonated bases. Finally, Zhao and Carr have noted an additional requirement for column orthogonality [56], which applies to Eq. (7) as follows: if the ratios H1 /H2 , S*1 /S*2 , etc. for two columns are constant (despite large differences in H1 and H2 , S*1 and S*2 , etc), the two columns are equivalent in terms of selectivity. That is, two solutes that are overlapped on one of the two columns will be overlapped on the other. A further analysis of means for maximizing column orthogonality, based on the above considerations, will be reserved for a later time. 4.4.4. Other (usually minor) contributions to column selectivity Eq. (1) provides a reasonable description of selectivity for most RPC columns and samples, but an exception exists for phenyl, cyano, and polymeric alkylsilica columns. For these columns and certain samples, three additional columnselectivity properties must be considered, aside from properties i − v of Section 2 (H, S*, etc): (vi) π-electron activity (P) (cyano and phenyl columns only), (vii) dipole moment (D) (cyano columns only), (viii) shape selectivity αTNN/BaP (C30 and polymeric C18 phases only). Phenyl columns can undergo π–π interactions with solute molecules such as polycyclic aromatic hydrocarbons, polynitro aromatics, etc., while cyano columns allow both π–π and dipole–dipole interactions [57]; dipole–dipole interactions favor the selective retention of aliphatic solutes that are substituted

17

with functional groups that have large dipole moments (e.g., nitro, cyano). If we wish to take advantage of both of the latter solute–column interactions as a means of further increasing column orthogonality, this can be achieved by the use of a cyano column. “Shape selectivity” [58] represents an additional possible contribution to column selectivity, which is only partly recognized by the column parameter S* [20]. Polymeric stationary phases or C30 columns are capable of unique separations of certain compounds (polycyclic aromatic hydrocarbons, carotenes, steroids, etc.) – especially isomers. Depending on the nature of the sample to be separated, the use of a cyano, C30 , or polymeric column may be advantageous for increased orthogonality. For the separation of most samples, we believe that the above three column-selectivity properties are generally of minor importance, although π–π interactions are considered by others to be more important [59]. If it is known that samples of interest contain compounds likely to be strongly affected by these particular column properties, then additional consideration (beyond that of Section 4.4.3) should be given to the final selection of orthogonal columns. 4.5. Quality control (QC) during column manufacture Because manufacturers are concerned about possible differences in selectivity among different batches of a particular column (e.g., Symmetry C18), various test procedures are used to ensure lot-to-lot uniformity and column equivalency. With continuing improvements in both column manufacture and testing, significant batch-to-batch variations in selectivity are today less common [60,61]. However, column reproducibility is still a major (if overstated) concern for users [62]. Two requirements for an “ideal” test of column reproducibility are (a) all five column-selectivity properties (as measured by values of H, S*, etc.) must be monitored, and (b) required measurements of solute k-values must be sufficiently reproducible. In addition, the test procedure should be convenient and inexpensive. The selection of appropriate test solutes can be guided by values of the selectivity parameters η , σ  , etc. of Eq. (1), as tabulated in two publications [63,64]. Thus, four test solutes (A–D) are needed, each of which has a relatively large value of σ  , β , α , or κ . If values of S*, A, B or C change from batch-to batch, this will result in a change in k for the corresponding test solute (there is no need to measure actual values, or change in values, of S*, A, B, or C). Two additional solutes (E, F) with small values of σ  , β , α , and κ , and different values of η make a total of six test solutes in the test-solute mixture. Values of η in [63,64] can also be used to select solutes that can fit within a convenient retention range, preferably 1 ≤ k ≤ 5 (log k is approximately proportional to η [20], so the range in values of η should not exceed about log 5 = 0.7). An example of such a test-solute mixture is shown in Table 8. Final values of k can be controlled by varying the composition (%B) of the mobile phase. If testing for column selectivity is carried out with a smaller number of test solutes (commonly the case), the relative importance of each of the above test solutes can be inferred from values of Fs (i) shown at the bottom of Table 6; i.e., amitriptyline (first), 3-cyanobenzoic acid (second), etc.

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D.H. Marchand et al. / J. Chromatogr. A 1191 (2008) 2–20

Table 8 An example of a test mixture for monitoring column selectivity in different production batches Solute

η

σ

β

α

κ

1-Nitropropane 1-Nitroethanea Prednisone N,N-Diethylacetamide 3-Cyanobenzoic acid Amitriptyline

−0.8 −1.0 −1.2 −1.5 −1.2 −1.1

0.0 0.0 1.0 0.3 −0.1 0.0

0.0 0.0 0.1 0.5 0.0 0.0

0.0 0.0 0.0 0.1 0.9 0.3

0.0 0.0 0.0 0.1 0.0 0.8

Experimental conditions as in Section 3 and [22], except that %B must be adjusted to give k ≥ 1 for all peaks. If peak overlap occurs, an alternate testsolute selection must be made. Values in bold measure individual values of S* , A, B, and C. a Values estimated from other homologs of the nitroalkanes.

Small lot-to-lot variations in column surface area are common, but do not affect column selectivity and are therefore of less concern. The effect of variations in surface area on measurements of selectivity can be corrected for by the use of k-value ratios, as in Eq. (1). The results of Section 4.2 and [41] should also be noted, when tests for column reproducibility are being considered, i.e., the column must be properly equilibrated prior to testing. There are additional considerations in the selection of a final procedure for column QC, but these are well known to column manufacturers. 5. Conclusions Column testing according to Eq. (1) has now been completed for 371 different reversed-phase columns; additional columns will continue to be added to our database, when these are introduced by the manufacturer. Available column-selectivity parameters H, S*, etc. are examined in this paper for various possible practical applications. The following examples are intended as examples, rather than completed research projects. Older, more acidic, type-A alkylsilica columns can be differentiated with 95% certainty from more recent, less acidic type-B columns by means of the column parameters B (hydrogen-bond basicity) and especially C-2.8 (cation-exchange capacity at pH 2.8). A value of C-2.8 < 0.25 can be used for distinguishing most type-B from type-A columns. Type-B columns show excellent peak symmetry for the separation of basic compounds at low pH (for a sample weight ≤0.5 ␮g), whereas many (but not all) type-A columns exhibit significant peak-tailing. The degree of tailing at low pH for basic samples and type-A columns (for a sample weight ≤0.5 ␮g) tends to increase for larger values of C-2.8, and appears to result from kinetic rather than equilibrium effects (slow sorption–desorption, rather than overloading strong sites). Non-ionized carboxylic acids can exhibit peak tailing for sample weights <0.5 ␮g with both type-A and -B columns. TypeB columns give symmetrical peaks for acidic solutes when the “excess” column basicity δB < 0.0, but some type-B columns with larger values of δB exhibit peak tailing. Type-A columns can exhibit peak tailing for acidic solutes, regardless of their values of δB. When tailing of acids or bases is encountered for small sample-weights and low-pH mobile phases, the substitution of a

column with C-2.8 < 0.25 (bases), or C-2.8 < 0.25 and δB < 0.00 (acids), should result in more symmetrical peaks. Whereas peak tailing increases for basic solutes when the sample size exceeds 0.5 ␮g, the reverse is true for acidic solutes that tail for small sample weights. For sample weights ≤15 ␮g, peak tailing for acidic solutes tends to increase as sample weight decreases (for columns that exhibit tailing peaks for small weights of acidic solutes). The tailing of acidic solutes appears to result from a small concentration of strong sites in the column packing, in addition to a larger concentration of weaker sites. Column selectivity (stability) was studied as a function of usage: storage in acetonitrile for 2–27 months, accompanied by brief exposure to mobile phases of pH 2.8 and 7.0. For eight different type-B columns, there was no apparent effect on column selectivity as a result of storage in acetonitrile or limited use of the column at pH 2.8. There was an increase in silanol activity (as measured by an increase in values of A and C-2.8, corresponding to non-ionized and ionized silanols, respectively) which is apparently the result of a brief exposure of the column to pH 7.0 during testing; this may be part of the normal conditioning (“break-in”) for new columns. Changes in A correlate negatively with values of A (r2 = 0.48), while changes in C-2.8 correlate negatively with increase in C-2.8 (r2 = 0.77). These and other results are consistent with a loss in column end-capping during exposure to pH 2.8, accompanied by differing amounts of end-capping for the original columns; however, differences in silica acidity may also be a factor that affects changes in the column with use. Column stability was also found to correlate with the source (manufacturer) of the columns, which may mean differing extents of end-capping by each manufacturer (or other differences in the manufacturing process). The extent to which column selectivity is determined by ligand type and particle pore diameter was also examined. While these column characteristics are significant contributors to column selectivity, other factors appear to be of comparable importance. Previous workers have adopted different approaches for the selection of “orthogonal” columns (those which yield large differences in selectivity). We conclude that prior attempts at identifying orthogonal columns or separation conditions need to be re-evaluated, since they may provide limited assurance that most compound-pairs that are unseparated with one set of conditions are likely to be separated with the orthogonal conditions. Column selectivity is commonly tested by the manufacturer for different batches of each column type, using retention data from appropriate test solutes. We have briefly examined how such tests might be selected for a more complete characterization or improved QC of different column batches. The relative importance of individual test solutes in this regard can be ranked. Nomenclature

A A Ami+ As

“type-A” column made from metal-containing silica column hydrogen-bond acidity protonated amitriptyline peak asymmetry function

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B B BBA Ber+ C C-2.8 C-7.0 D Fs Fs (A) Fs (B) Fs (C) Fs (H) Fs (S*) Fs

“type-B” column made from pure silica column hydrogen-bond basicity 4-n-butylbenzoic acid berberine (a quaternary ammonium compound) column cation exchange capacity value of C for pH = 2.8 value of C for pH = 7.0 measure of column dipolarity column comparison function (Eq. (7)) value of Fs , considering only A2 -A1 term of Eq. (7) value of Fs , considering only B2 -B1 term of Eq. (7) value of Fs , considering only C2 -C1 term of Eq. (7) value of Fs , considering only H2 -H1 term of Eq. (7) value of Fs , considering only S*2 -S*1 term of Eq. (7) value of Fs for a column, relative to an average column of the same type (e.g., wide-pore, type-B C18 ) H column hydrophobicity 4-HA 3-n-hexylaniline H-B hydrogen-bond k retention factor kEB value of k for ethylbenzene MFA mefenamic acid Nor+ protonated nortriptyline P measure of column π-activity r correlation coefficient S* steric resistance to insertion of bulky solute molecules into the stationary phase SD standard deviation SD[H], SD[S*], etc. SD value for values of each column parameter H, S*, etc. (see next-to-last row of Table 2)

Greek letters α selectivity factor; ratio of k-values for two solutes α solute hydrogen-bond acidity αTBN/BaP ratio of k-values for tetrabenzonaphthalene/ benzo(a)pyrene [58] β solute hydrogen-bond basicity δA change in A as a result of column storage or use ␦As corrected value of As -1; see example of Fig. 1b δB “excess” value of B; see Fig. 3b and Eq. (3); also, change in B as a result of column storage or use (Table 5) δC change in C as a result of column storage or use δ(C-2.8) change in C-2.8 as a result of column storage or use δH change in H as a result of column storage or use δS* change in S* as a result of column storage or use η solute hydrophobicity κ charge on solute molecule (positive for cations, negative for anions) σ steric resistance of solute molecule to penetration into stationary phase

Acknowledgement We wish to thank Professors David McCalley (University of the West of England) and Peter Carr (University of Minnesota)

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