High-performance liquid chromatography (advances in packing materials)

High-Performance Liquid Chromatography (Advances in Packing Materials) David J. Anderson

Department of Chemistry, Cleveland State University, Cleveland, Ohio 44 1 15

The previous review of high-performance liquid chromatography (HPLC) in clinical chemistry appearing in the applications review of Analytical Chemistry (01) covered the area of HPLC separation methodologies, specifically reviewing the topic of direct injection techniques, which are techniques that allow direct injection of protein-containing samples (such as serum) onto HPLC columns. In this review, advances in packing materials are reviewed, specifically covering advances in base supports and reversed-phase packing materials that seek to address the problems encountered with conventional silica-based packing materials. These problems include limited stability, adverse chromatography resulting from active sites on the silica surface, and lack of reproducibility. In addition to this, the development of packing materials that are tailored for high-speed analysis of macromolecules is reviewed. The time period for the present review is the last six years (publication date 1989 to Chemical Abstracts date October 1, 1994), with significant articles prior to this also referenced. This review is limited to articles that characterize packing materials that have been introduced commercially during the review period. It is also limited to those packing materials that are high performance (<l@pm diameter). In this review, plate heights are reported as reduced plate heights, which is the plate height divided by the particle diameter. Use of reduced plate heights allows direct comparison of data for packing materials of different sizes. Lower values for reduced plate height indicate better efficiency, the best columns having values between 2 and 3.

GENERAL REVIEWS Several books have been published that extensively review

various aspects of HPLC packing materials that are designed for biomacromolecules (02, 03) and general applications (04). Review articles have also been published covering HPLC column considerations in biotechnology (Os) and for proteins and peptides (06). Features of commercially available columns, packing materials, and accessories that are newly introduced at the Pittsburgh Conference are tabulated and summarized each year in issues 3 and 4 (March and April) of LC-GC (07-018). Besides these summaries, listings characterizing HPLC packing materials available from various manufacturers have been p u b lished (04, 05, 019). A review of the transmission electron microscopic characterization of the pore structure within wide- pore silica and high-performance polymer packing materials has been published (020).

BASE SUPPORTS The base support is the core material supporting the stationary

phase (in some cases the base support also serves as the stationary phase). A packing material consists of both the support and the stationary phase. Silica-based supports continue to occupy prominence in HPLC usage, having decided advantages in several aspects in comparison to other support materials. There are, however, problems with silica-based supports, which have led to the development of other support materials. The different base supports used in commercially available HPLC packing materials are discussed below.

High-Performance Silica Supports. For the most part, silica supports are still superior to other supports in terms of efficiency, rigidity, and permanence of physical structure. Silica’s rigidness allows for fast flow rates and, thus, decreased analysis times. In addition, silica supports have greater versatility (in terms of types of stationary phases and characteristics of the support structure) and have more extensive quality assessment and control, due to their longer history of commercial development. Several reviews have been published which describe characteristics of silica-based supports in general (021) and in the separation of biomacromolecules in particular (022-024); with a tabulation of the characteristics of various commercially available native (021) and bonded (022) silica packing materials published. Reviews of the effect of silica structure on reversed-phase chromatography have been published (025, 026).

Even with these advantages, however, there are several major problems with silica-based supports. These problems are severe peak tailing in the chromatography of basic compounds, limited pH stability, and intra- and intercompany variability of performance for identical chemistry columns.

Extensive reviews have been written detailing the nature of the strong adsorption sites on silica and the various approaches to minimizing the presence and/or effect of these sites (027- 029). These strong adsorption sites are responsible for the adverse chromatography of basic compounds seen for silica-based reversed-phase packing materials and are due to the presence of strongly reactive silanols (note, this is only a small percentage of the unreacted silanols that are present on the reversed-phase silica packing material), trace metals, or both. The most common strategy for chromatographing basic compounds on silica supports is to add amine modifiers (such as triethylamine) to the mobile phase to block the silanol sites. Low pH, high ionic strength, or ion-pairing reagentcontaining mobile phases are additional strategies. New reversed-phase silica packing materials (base-deactivated packing materials) have been developed which improve the chromatography of basic compounds without resorting to the mobile-phase moditications mentioned above. This type of packing material is discussed below in a separate section. Another approach is to use altemative-based supports (alumina, polymer, porous graphite), which are also described in this review.

Another disadvantage of silica supports is their pH limitation, limited to a pH range of 2-8. This is due to the dissolution of silica at alkaline pHs [which dramatically increases at pHs above 9 (above the normal solubility of 100 ppm at pH 2-8)l and to the hydrolysis of bonded phases (022, 023). Other base supports (polymer, alumina, porous graphite), polymer-mod~ed silica, and ultrapure silica have been developed to extend the usable range of mobile-phase pH. These alternatives to conventional silica packing materials are reviewed in separate sections below.

Quality control remains a significant issue with silica-based packing materials (as well as with other base support packing materials). Significant differences in retention properties exist for the same type of packing materials from different manufacturers. Differences also exist for different lots of the same packing material from the same manufacturer. J. T. Baker Co. has published an extensive study characterizing its wide-pore silica

Analytical Chemistry, Vol. 67, No. 12, June 15, 1995 475R

packing materials, including lot-to-lot variability (030). In this study the percent variability range [calculated as 100(high - low)/ mean] and CVs [in parentheses, percent coefficient of variation, calculated as 100(standard deviation) /mean] for ligand density was 18.2 ( E = 9, CV = 5.4%), and 16.3% (n = 7, CV = 5.8%) on different lots of CIS and cation-exchange wide-pore silica packing materials, respectively (each made from trifunctional silanes giving a polymeric coverage). There was also variability in the physical characteristics in different lots of the base wide-pore silica support (n = 5) with percent variability ranges (and CVs) of 8.6 (CV = 3.7%), 16.9 (CV = 7.8%), and 13.9% (CV = 5.6%) for the pore diameter (30 nm), pore volume, and surface area, respectively. Reproducibility data for a high-stability c8 silica packing material (Zorbax SB-Cg, Rockland Technologies) showed a capacity factor variation of only 3.5% for the chromatography of toluene and N,iV’- dimethylaniline on 19 different lots produced over a 2-year period (031). To address the lot-to-lot reproducibility problems, some manufacturers guarantee the separation of specific compounds through stringent quality control measures. However, chromatographic performance changes with time with conventional silica packing materials, and thus reproducibility is even an issue for different runs on one column (032, 033).

High-Performance Polymer Supports. Types. Reviews have been published on polymer-based HPLC supports (034- 036), two of which describe various packing materials that are commercially available from various manufacturers (034, 035). Commercially available polymer-based supports are classified as either hydrophilic or hydrophobic. Reviews published for some of these supports are indicated below. The hydrophobic supports include poly(styrene-divinylbenzene) (PS-DVB) (039, divinylbenzene (DVB) (which has 5 10% styrene content), and poly(alky1 methacrylate). The hydrophilic supports include poly(hydroxy- alkyl methacrylate or acrylate) (PHAM) (038, 039) [Zhydroxy- ethyl methacrylate copolymerized with ethylene dimethacrylate (HEMA) (039) is a common example of this support] and poly- (vinyl alcohol) (PVA).

Polymer reversed-phase packing materials include unmodified DVB, E-DVB, and poly(alky1 methacrylate) or PS-DVB, PHAM, HEMA, and PVA that have been modified with the usual reversed- phase alkyl ligands. Bioanalytical size-exclusion, affinity, and ion- exchange chromatographies require a hydrophilic surface on the support, in order to prevent mixed-mode retention mechanisms for smaller analytes or nonspecific adsorption of macroanalytes. Thus, supports used in these three modes are either hydrophilic supports or are hydrophobic supports that are hydrophilically modified. Unmodified PS-DVB and DVB supports are utilized extensively in nonaqueous size-exclusion chromatography, such as is used in polymer analysis. Polymer-based supports are used sparingly in normal-phase chromatography, in which the use of inorganic supports dominates. Given below is a summary of the characteristics of polymer supports with respect to the nature of the pores, efficiency, selectivity, degree of inertness to the adsorption of proteins and other compounds, reproducibility, and general comments about operating conditions.

Operational Characteristics. The major advantage of polymer- based packing material in comparison to silica is its increased range of pH stability. The ranges of pH stability are stated to be 1-13 for PS-DVB packing materials and 2-12 for HEMA (039) and other PHAM (038) packing materials. A CIS PVA packing material (Asahipak ODP-50) was immersed for 48 h in 50 mM

sodium phosphate/acetonitrile (90:lO) at pHs 2, 10, and 13 at 50 “C, showing no weight or efficiency loss at pHs 2 and 10, while showing a 1% decrease in weight and a 12% loss in retention time (calculated from a figure in the paper) at pH 13 (040). The manufacturer’s catalog rates the pH stability of its PVA packing materials to be from either 2 to 9 or 2 to 12 for size-exclusion, 2 to 13 for reversed-phase, and 2 to 12 for ion-exchange packing materials (‘91-’92 Technical Data Compendium, Asahi Chemical).

Operational disadvantages of polymer-based packing materials compared to silica-based packing materials include lesser pressure tolerance, swelling/shrinkage changes that occur with a change of organic modifier content in the mobile phase, or both. In general, the DVB and PS-DVB supports have adequate pressure limitations for operation at high flow rates. On the other hand, the hydrophilic polymer supports are less rigid and thus cannot be used at the very high flow rate range [operation at normal HPLC flow rates (1 mWmin) is usually not a problem, however]. Pressure limits have been reported to be 6000 psi for DVB, at least 4500 psi for PS-DVB (depending on the pore size) (039, and 2900 psi for HEMA @IO series) (039) packing materials. PS-DVB packing materials usually require a minimum of 20% organic modifier to prevent swelling/shrinkage effects. DVB packing materials require only 10% organic modifier. Hydrophilic polymer packing materials do not require organic modfier and can be used with mobile phases varying from 0 to 100% organic content. Swelling/shrinkage of hydrophilic polymer packing materials does occur with a change in organic modifier concentration, however, to a much less extent than that seen for PS-DVB (041).

Pore Characteristics. Pore size is an important consideration in the chromatography of macromolecules. It is the basis of separation in size-exclusion chromatography. In other types of chromatography, the pore size must be large enough to ensure unrestricted movement of the macromolecule within the pores. The mean pore diameter should be 210 times the diameter of the molecule to ensure that diffusion in the pore will not be restricted (restricted diffusion results in increased band broaden- ing) (022). The diameter of a random coil or globular protein can be approximated from its molecular weight, as given in a published table (042).

Various pore sizes are available in polymer supports. For example, the diameters of the meso- and macropores present in a PS-DVB support available from one manufacturer are 100,300, 1000, and 4000 A (037). Meso- and macropores have a range of pore diameters of 20-500 and 2500 A, respectively (021). For any particular polymer support of specified meso- or macropore diameter, there is a distribution of pores of different sizes within that support. The distribution of pore sizes in silica supports usually spans one to three decades of the mean pore diameter (043) ; however, there are commercial silica packing materials that have a narrower pore size distribution (Kromasil, Eka Nobel).

Table 1 (020, 035, 044) shows the approximate distribution of pore sizes for several commercially available polymer packing materials and one silica packing material. The distribution of pore diameters is wider for the polymer packing materials than the silica packing material. In another work, the distribution of pore size was found to be a factor 3-5 times lower for silica packing materials in comparison to polymer packing materials (045). In addition to the meso- and macropores, polymer supports (unlike silica) have micropores, which are pores that are <20 A in

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Table 1. Pore Diameter Characteristics of Some Commercially Available Packing Materials Measured by Inverse SECa

pore diam range (A)

packing chem pore diam 50% peak 10% peak

makeup (peak in distribn) (A) distribn limits distribn limits ref

LiChrospher Si 500 silica Asahipak ODP-50 c]8 PVA TSK Cl8-4PW PLRP-S 300 PS -DVB TSK G4000PW PHAM

c I 8 PHAM

5 10 110 3 80 220 1000

3 10- 1000 2 10- 1800 020 68-310 54-2300 044 280-1200 66-2700 044 190- 1100 170-3000 044 440-2000 120-2700 035

a Approximated from figures given in the sited references; peak pore diameter and peak distribution limits are calculated for pore size distributions analogous to retention, half-height, and tenth-height times for chromatographic peaks. PVA, PS-DVB, and PHAM are defined in text.

diameter (021). The micropores in one polymer packing material (TSK G4000 PW, a PHAM packing material) were found to have a peak distribution of 11 A, a 50% peak distribution range of 8-19 A, and a 10% peak distribution range of 6-35 A, as determined by inverse SEC (approximated from figures contained in the paper) (035). This concurs with data characterizing micropores in a native PS-DVB packing material (PLRP-S 1000 A), which found a mean diameter of 12 A (046).

Retention Selectiuity. The base support of polymer packing materials [PS-DVB, ClS(C1) PVA, clS(cI)-PHAM, and poly(alky1 methacrylate) were tested] show definite strong retention of rigid planar solutes (such as polynuclear aromatic hydrocarbons) and specific retention (although the retention is not as strong as the rigid planar solutes) of “flexible” aromatic compounds (these compounds do not contain fused aromatic rings) (047, 048). Although the aromatic composition of PS-DVB and the carbonyl composition of the other polymer supports have been implicated in a JC retention mechanism of aromatic compounds, it is now accepted that the micropores play an essential part in the selectivity of polymer supports, through a steric compatibility mechanism, in which a rigid, compact structure is required for the solute to be retained by the micropores (047,048). Capacity factors resulting from a phenyl group in the solute have been estimated to be 2.7, 1.6, and 1.4 for PS-DVB, poly(alky1 methacrylate), and CIS PVA, respectively, compared to a capacity factor of 0.94 for Cl8 silica (80% methanol mobile phase; calculated from data given in the paper) (047). It should be pointed out, however, that the C18 PHAM packing material showed a capacity factor of 0.47 (Le., reduced in comparison to CIX silica) (047). This compares with the much larger capacity factors of 5.0 and 4.7 for the polynuclear aromatic hydrocarbon pyrene on C1 PHAM and C1 PVA, respectively, compared to a capacity factor of 0.58 on C1 silica (80% methanol mobile phase) (048). It should be noted, however, that there is considerably less selectivity difference in comparing base polymer supports (even PS-DVB) with silica supports in the chromatography of aromatic compounds (either rigid planar or flexible) when either tetrahydrofuran (THF) or acetonitrile is used as an organic modiiier, probably explained by a diminished role of the micropores in the column’s selectivity, resulting from a swelling of the micropores in these solvents (047, 049).

Eficiency. The presence of micropores in polymer supports leads to unfavorable chromatographic characteristics for some low molecular weight analytes. Generally, polymer supports have decreased efficiencies compared to silica, the extent of which depends on the solute, the chromatographic conditions, and the characteristics of the micropore structure.

The efficiency characteristics of four different types of reversed- phase polymer packing materials and one reversed-phase silica packing material have been examined for 25 different solutes, using different mobile-phase organic modifiers (047). Several studies, including this one, have shown that the chromatography of rigid solutes (which are retained in unswollen micropores through a steric compatibility mechanism) gave poor efficiencies, depending on the mobile-phase organic m o d ~ e r . Poor efficiencies were noted for native PS-DVB packing materials in mobile phases containing alcohol or DMSO (049) and for CIS PHAM, C18 PVA, poly(alky1 methacrylate), and PS-DVB packing materials in mobile phases containing methanol (047). This study also showed another undesirable characteristic of polymer packing materials using methanol-containing mobile phases, in that all the polymer packing materials studied showed a substantial increase in plate height with increasing capacity factor (k? (unlike the performance of CIS silica packing materials) (047). On the other hand, this, along with another study, showed greatly improved efficiency for polymer packing materials using THF or acetonitrile mobile-phase modifiers, presumably because of the ability of these modifiers to cause swelling of the micropores (047, 049). The extent of improvement in column performance by changing mobile-phase modiiier from methanol to THF or acetonitrile, however, depends on the characteristics of the polymer support. The best performers were poly(alky1 methacrylate) (Shodex DE- 613) and CIS PHAM (TSK Cls-4PW) packing materials, which showed reduced plate heights for all 25 solutes of approximately 3-5 and 4-7.5, respectively, compared to a reduced plate height of 2.8-3.3 for the CIS silica column (using 40%THF mobile phase, at a flow rate of 1, mL/min) (047). In addition, there was no increase in reduced plate height with increased k‘ noted for either of these columns (047). The C18 PVA (Asahipak ODP-50) and the native PS-DVB (PLRP-S 300) packing materials showed reduced plate heights in the ranges 2.5-32 and 4-45 for the 25 solutes, respectively, which showed a distinctive trend for increased plate height for increasing k’ (047). Most of the higher reduced plate heights for these latter two columns resulted from rigid planar compounds (40% THF mobile phase at 1 mL/min) (047).

In many cases, the limiting factor for the efficiency of polymer packing materials is the solute’s mass-transfer characteristics into and out of the micropores, with slow mass transfer noted for rigid molecules. The scale of rigidity in decreasing order is polynuclear aromatic hydrocarbons > flexible aromatic compounds > cycloal- kanes > linear alkanes (035). Less rigid compounds are for the most part unaffected by the micropore structure, as evidenced by an average reduced plate height of 3.8 obtained for a series of


alcohols on a cl,3 PVA column in 80% methanol mobile phase at a linear velocity of 3.5 cm/min (041). However, it should be noted that poor chromatographic performance was obtained for the same alcohols on a native PS-DVB packing material, showing a drastic increase in reduced plate height with increased k' (reduced plate height of 35 at k' = 15) (041).

Another issue affecting plate heights for polymer packing materials is flow rate. The optimum performance of polymer packing materials matches the optimum performance of silica packing material; however, the optimum flow rate is less for polymer packing materials. Thus, operation at the higher flow rates normally used with the silica-based packing materials leads to the comparatively poorer efficiencies for the polymer-based packing materials. As an example, a Cg PHAM packing material gave reduced plate heights ca. 3 for all polynuclear aromatic hydrocarbons and other rigid molecules, at a flow rate of 0.2 mL/ min (linear velocity of 0.3 mm/s) (035).

A CIS PS-DVB packing material was found to have better efficiencies than the native PS-DVB packing material (050). Plate heights for macromolecules such as proteins should be comparable for silica- and polymer-based supports since these molecules are excluded from the micropores (037). However, resolution of proteins in size-exclusion chromatography was lower for a PHAM packing material in comparison to a silica-based packing material (051).

Assessment of Inertness. HPLC supports used in bioanalytical reversed-phase, normal-phase, ion-exchange, size-exclusion, and affinity chromatography must be sufiiciently hydrophilic in order that the predominant interaction of the solute is with the immobilized ligand (or no interaction, as is the case for size exclusion) and not the support itself. As discussed below, most of the polymer supports are not inert, showing adsorptive properties to different types of molecules.

(a) PVA. Hydrophobic interaction on native PVA packing materials has been observed for human serum albumin (0.1 M Na2SO4/30 mM sodium phosphate pH 7 mobile phase) (052), nucleosides and nucleic acid bases (053), and nonionic surfactants (054). In another study, the performance of various sizeexclusion packing materials (native PVA, hydrophilically coated silica, agarose) was assessed for the chromatography of positively charged chymotrypsinogen and negatively charged ovalbumin using different concentrations of salt in the mobile phase (055). Results showed that the native PVA had less electrostatic retention at low concentrations of salt and a greater hydrophobic retention at high concentrations of salt (> 1 M NaCl), in comparison to the other packing materials (055). The hydrophobic retention on native PVA was most dramatic for the positively charged chymotrypsinogen (did not completely elute at mobile-phase concentrations of > 1 M NaCl), with only a small increase in retention noted for the negatively charged ovalbumin at 2 2 M NaCl (055). Riboflavin (056) and the compounds diketogulonic acid, dehy- droascorbic acid, and ascorbic acid (057) have been shown to reversibly adsorb to native PVA packing materials. Adsorption effects have been studied for peptides and amino acids at various mobile-phase conditions (058). Hydrophobic, ion-exchange, and ion-exclusion mechanisms have been demonstrated in the chromatography of peptides on native PVA packing materials (059).

(b) P M . Hydrophobic interaction has also been demonstrated on native HEMA packing materials (060, 061). Native HEMA packing materials contain a small amount of unreacted

double bonds which can hydrolyze to form carboxylate groups in strong alkaline conditions (039). Modifications of HEMA have been made to make it more hydrophilic, including (1) immobiliza- tion of saccharides on HEMA and (2) reaction of HEMA with epichlorohydrin, followed by hydrolysis (039). Carboxylate groups are not formed in alkaline solution with this latter modification (039). Hydrophobic interaction has also been demonstrated on other native PHAM packing materials (038, 062). Hydrophobic retention of recombinant bovine somatotropin was noted for a TSK PW sizeexclusion packing material (PHAM), with a rapid increase in elution time for mobile phases containing >0.4 M ammonium hydrogen carbonate and a loss of recovery noted at concentrations of >0.7 M (063). The TSK PW series packing materials (€'HAM) contain ionic charges, either carboxyl groups or positive charges (for G2000PW and oligo-PW), necessitating increased ionic strength of the mobile phase (038, 064).

(c) PS-DW. PS-DVB supports require hydrophilic surface modification in order to be utilized in modes other than reversed phase in the HPLC analysis of biological molecules. An example of such packing materials are the MonoBead line from Pharmacia Biotech, which employ l@pm PS-DVB with a proprietary mod~cation to give ion-exchange functionalities and a hydrophilic character to the PS-DVB surface. Dionex modifies a PS-DVB support by sulfonation of the surface and subsequent coating of it with monodisperse functionalized latex beads (50-500 nm) (held on the surface by electrostatic forces) to produce various pellicular ionexchange packing materials (065). A hydrophilically modified PS-DVB packing material used for sizeexclusion (PL- GFC, Polymer Laboratories) was tested for the retention of proteins having positive, negative, and neutral charges in a pH 7 mobile phase (037). Ionic retention was minimal for all proteins (no retention with low ionic strength mobile phases), and hydrophobic retention was noted above 0.6 M ammonium sulfate for a-chymotrypsinogen (+) and at higher concentrations for lysozyme (+), ovalbumin (-), and myoglobin (neutral), listed in increasing order of salt concentration needed to cause retention (charge on protein indicated in parentheses) (037). A hydrophilically coated PS-DVB support has been reported which does not show hydrophobic retention of ovalbumin or BSA up to at least 2 M ammonium sulfate in 50 mM sodium phosphate pH 7 mobile phase, indicating that the hydrophobic PS-DVB core can be effectively masked (046).

Protein Recovery. Even with the slight hydrophobic or ionic retention properties, advantages have been demonstrated in protein recoveries for polymer supports. Significantly increased recovery of fibrinogen was noted in comparing the chromatography on a N-hydroxysuccinimide methacrylate packing material (Afti-Prep 10, Bic-Rad; 91% recovery) with chromatography on diol silica (diol prepared from Nucleosil4ooO-10, Macherey-Nagel; 29% recovery) (066). Chromatography of proteins on a C4 silica packing material was generally good under optimized conditions except for hydrophobic proteins, in which the recoveries were unacceptably low, and for basic proteins, which had significant tailing (067). In contrast, chromatography of the same basic and hydrophobic proteins on a native PS-DVB packing material gave significantly reduced tailing and better protein recoveries (067). Protein recoveries of 72-98% have been found for proteins chromatographed on a phenyl-PHAM packing material (068). Improved recoveries were found for various polypeptides using a C4 PVA packing material (Asahipak C4P-50; mean recovery of

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102.9%) in comparison to a Cq silica packing material (Nucleosil 300-5C4; mean recovery 96.4%) (069). In another study, protein and peptide recoveries on native PVA ranged from 86 to 93% and 82 to 106%, respectively (058).

Reproducibility. The reproducibility of polymer packing materials has not been extensively studied. Two studies are sited below, which give different conclusions concerning reproducibility. One paper compared retention and selectivity characteristics of native PS-DVB packing materials from three different manufacturers in the chromatography of erythromycin and related substances and found significant differences (070). Another paper compared chromatographic retention for two different native PS-DVB packing materials and concluded that there were more similarities than differences (071). Clearly, additional studies are warranted.

Summary of Advantages and Disadvantages. The advantages and disadvantages of polymer packing materials in Comparison to silica packing materials are summarized from the discussion above and from the reversed-phase section below. The advantages are as follows: (1) ability to do chromatography at a high pH, (2) ability to chromatograph samples that would ordinarily foul the column (samples that have a complex matrix) owing to the ability of the polymer to withstand periodic cleaning with a strong acid or base, and (3) better recovery of proteins. These advantages result from the higher upper pH limit, the nonactive nature of the polymer surface (i.e., absence of silanols and metal impurities), or both. Unique selectivity characteristics of the polymer packing materials may also be advantageous in separations that cannot be achieved on silica-based supports. The disadvantage of polymer packing materials is higher plate heights at normal HPLC flow rates for rigid low molecular weight analytes. Efficiencies attained for macromolecules, however, should be similar for both polymer and silica packing materials. Other disadvantages include swelling effects when the organic modifier concentration is changed (especially for PS-DVB packing materials) and a limited pressure tolerance for the hydrophilic polymer packing materials.

High-Performance Polymer-Modified Silica (PMS). Over- view. In an attempt to improve silica-based packing materials in terms of masking active sites and increasing the upper limit of pH, several manufacturers have modified the silica surface by covering it with a polymer layer. Most sources refer to these packing materials as polymer-coated silica; this review refers to these packing materials as polymer-modified silica to more accurately reflect the fact that the polymer can be either adsorbed or covalently bound to the silica surface. Reviews of PMS have been published (072-074). Comments in this review will focus on those packing materials in which the manufacturer has disclosed the polymer chemistry involved.

Some commercially available PMS packing materials claim pH stability to pH 10 (07,016), with one commercial source claiming stability in the pH range 2-13 (pH-Stable Spherisorb; Phase Separations) (017). It should be noted, however, that polymer modification may not be necessary for high pH stability, as several commercial silica packing materials have a claimed pH stability from 2 to 10, attributed to the use of high-purity silica alone. Some commercially available PMS packing materials are also useful in the chromatography of basic compounds, owing to the shielding effect of the polymer, which covers the active silica surface.

PMS packing materials that have been introduced over the last six Pittsburgh Conferences (up to 1994), in which the polymer chemistry is disclosed, include silica supports modified with

multifunctional silanes (di- or trifunctional chlorc- or alkoxysilanes; reversed phase and others), polysiloxane (reversed phase); polyethylenimine (PEI) (ion exchange), and polybutadiene/maleic acid copolymer (ion exchange). The chromatographic characteristics of these PMS packing materials (except for the ion- exchange polymers) are discussed below.

Multihnctional Silanes. Most manufactures of multifunctional silane PMS do not claim an increase in the upper limit of pH stability or expanded capabilities in the chromatography of basic compounds. One exception to this is a recently introduced Cl8 PMS packing material from YMC, Usphere ODSHSO) (03, which has a claimed pH stability range of 1.1-9.3 (able to withstand more than 500 h at the limits) and an ability to do basic compounds.

A selectivity advantage in resolving similarly structured rigid molecules (shape selectivity) exists for multifunctional silane polymer-Cls PMS packing materials in comparison to monomer- CIS packing materials. This selectivity advantage for polymer- CIS PMS phases in comparison to monomer-Cls phases has been documented for polynuclear aromatic hydrocarbons ( 0 7 8 , carc- tenoids (lutein and zeaxanthin are resolved) (076, 077), and chlorophylls (078). However, there are additional factors that affect shape selectivity (079). For example, ligand chain length is a critical factor, as both polymer- and monomer-& phases have minimal shape selectivity, while both polymer- and monomer-CSo phases have good shape selectivity, equivalent to that of the polymer-C18 phase (075).

Disadvantages of multifunctional silane PMS in comparison to monomer packing materials are lot-to-lot reproducibility prob lems (since the polymer reaction is more difficult to control) and lower efficiencies (080). Variability range and CVs (both defined above) in carbon surface coverage for two commercial sources of CIS PMS (made from multifunctional silanes) were 18.2 (n = 9, CV = 5.4%) (030), and 60.3% (n = 7, CV = 21.3%) (calculated from data given in the paper; the latter data included lots that did not meet separation specifications and thus were rejected for sale) (081). Multifunctional silane PMS has a higher surface coverage of ligand in comparison to monomer phases; however, unreacted silanols are present in both phases.

Polysiloxanes. A characterization of poly(methyloctadecy1si- 1oxane)-PMS packing materials has been published (082). These packing materials could not withstand a 100% aqueous pH 8.4 carbonate buffer mobile phase and, with a few exceptions, were at a disadvantage in comparison to a comparable monomer-Cls silica packing material in terms of stability (082). Reduced plate heights were comparable, but slightly higher, for the poly- (methyloctadecylsi1oxane)-modified silica (polymer thickness of 1.0 nm) compared to a monomer-Clgmodified silica (same silica base used for each, 80% methanol mobile phase, n-butylbenzene solute), being 3.70 (0.13 SD, n = 6) for the monomer-cls packing material and 4.10 (0.23 SD, n = 5) and 3.81 (0.16SD, n = 6) for a polymer-modfied silica and a polymer-modified end-capped silica, respectively (082). In another study, the minimum reduced plate height was found to be 2.6 at a mobile-phase velocity of 0.6 mm/s for a poly(methyloctadecylsi1oxane)-modified Nucleosil 5100 packing material compared to a value of 2.5 at 0.9 mm/s for Nucleosil 5100-ClgE (n-hexylbenzene; 90% methanol mobile phase) (083). However, the slope of the reduced plate height vs linear velocity plot was greater for the polymer-modified packing material at velocities greater than the plot minimum (083). A


comparison of the retention of various compounds on a polymer- modified and a conventional packing material was also done in this study (083). A comparison of chromatography of proteins showed lower efficiencies (though equal recoveries) for a C1 polysiloxane-modified silica packing material compared to a conventional C1 packing material (083). A commercially available polysiloxane-modified silica (Deltabond, Keystone) has a stated pH stability of 2-7 (from company catalog).

Another type of polysiloxane-modified silica is commercially available (Capcell Pak series, Shiseido), which has properties different from the polysiloxane-modified silica described above. The manufacturer refers to this as a silicone polymer. The silicone polymer-modified silica results from the polymerization of 1,3,5,7 tetramethylcyclotetrasiloxane monomers deposited on the silica surface followed by the introduction of a reversed-phase ligand (084, 085). It has a claimed pH stability of 2-10 (09, 015). This packing material was reported to have a stability of 550 h at pH 9 and 350 h at pH 10 [noting constancy of peak shape and retention time; continuous flow of a mobile phase of methanol/ buffer (70:30 v/v) at 40 "C, using Britton Robinson buffer] (084). This compares to a stability of 20-40 h for conventional reversed- phase silica, using the same mobile-phase conditions (084). The polymer covering was dramatically more effective than end capping in shielding chelating analytes and basic compounds from the effect of silanol and metal impurities (some chelating analytes could not be eluted from the end-capped CIS packing material) (085). The reduced plate height was excellent (2.5 for naphtha- lene in a 70% methanol mobile phase at a flow rate of 1 mL/min) (084). A polymer thickness of 13 A for the c18 silicone polymer is reported (6 A for the Cl8 group alone) (084). HPLC determi- nations using alkaline mobile-phase conditions for this silicone polymer PMS have been reported for porphyrins at pH 7.5 (no alteration in retention time after 1500 injections, at a column temperature of 40 "C) (086) and for the ulcer drug omeprazole and its metabolites at pH 8.5 (at least 200 h with no change in resolution or peak sharpness) (087).

High-Performance Alumina Supports. With the lower efficiencies, and lower pressure tolerances noted for polymer packing materials, development of alternative material supports has been pursued, attempting to maintain the high efficiency and high mechanical strength properties of silica, while extending the pH range and " k i n g the adverse retention properties of silica. Several alumina packing materials are commercially available that have the advantage of an extended pH range (PH 2-13). This type of packing material is either native, and used as a normal- phase packing material, or polymer-coated [most commonly polybutadiene (FBD), polymer layer is not bonded to the support], and used as a reversed-phase packing material. The PBD polymer layer is either unmodified or has alkyl ligands covalently attached to it. Although investigators continue to attempt to bond ligands directly to the alumina surface, no such packing material has been produced to date which has been deemed stable in a variety of acidic and alkaline mobile phases.

Only a few applications of commercially available alumina packing materials have been published. One article gives examples of different separations on various alumina phases, including: peptides and proteins on c18 PBD-alumina, antibiotics (penicillins, cephalosporins, macrolides) and tricyclic antidepres- sants on cyano-PBD-alumina, and opium alkaloids on PBD- alumina (088). Another article gives examples of the separation

of polycyclic aromatic hydrocarbons, steroids, basic drugs, organic bases, and /?-blocking drugs on PBD-alumina (089). Athorough comparison of PBD-alumina with PMS in the chromatography of various proteins showed lower capacities, 50% peak areas (attributed to irreversible adsorption), and greater peak broaden- ing for the PBD-alumina (090). The irreversible adsorption of proteins on PBD -alumina is attributed to the high hydrophobic character of PBD (091). Chromatography of synthetic octapep tides on PBD-alumina, however, was comparable to that of CIS PMS in terms of peak capacities, resolution, and peak areas (090). Thus it appears that PBD-alumina packing materials are best suited for smaller molecular weight molecules.

Chromatography of basic compounds on a c18 polymer- alumina column (proprietary polymer) using a basic mobile phase (pH 10-12) gave poorer (higher) reduced plate heights than most of the base-deactivated silica packing materials (discussed below) using a methanol/phosphate pH 6.25 mobile phase (092). Reduced plate heights (based on half-height peak widths) and 10% peak asymmetries (parentheses) for the c18 polymer-alumina column and the best performing base-deactivated column were 8.3 (1.53) and 4.9 (1.62) for pyridine, and 11.1 (2.4) and 4.7 (1.77) for nicotine, respectively (092). It should be noted that chromatography of basic compounds in water/methanol mobile phases (without being buffered at a basic pH) gave unacceptable peak asymmetries for the alumina-polymer CIS, which indicates a high activity of either the polymer or the underlying alumina surface (092).

High-Performance Porous Graphite. This support is used exclusively as a reversed-phase packing material and is described in the reversed-phase section below.

REVERSED PHASE Described below are advances in reversed-phase packing

materials. High-Performance Polymer Packing Materials. Stability.

The main advantage of polymer-based reversed-phase packing materials is in the chromatography of samples requiring harsh mobilephase conditions, conditions that cannot be used with silica- based packing materials. Alkaline pH was required to optimize electrochemical detection in the determination of cocaine and its metabolites (pH 8.8) (093) and erythromycin (pH 10) (094, necessitating the use of native PS-DVB packing materials. Optimization of on-line fluorescence detection required an alkaline mobile phase (pH 11.5) in the determination of labetalol on Cla PVA (095). Polymer packing materials are advantageous for procedures requiring periodic cleaning. The performance of a native PS-DVB column was maintained for 6 months in the determination of recombinant acidic fibroblast growth factor in cell suspension and lysate samples using a biweekly cleaning procedure employing 60 mL of 50% methanoV0.2 M NaOH (096). Small alkyl silica reversed-phase packing materials (such as CI and C4) are highly susceptible to hydrolysis and are thus limited in their applications. Results for a C4 PVA packing material were mixed when tested for performance in the separation of a four- component sample, maintaining separation and performance after pumping 0.1% trifluoroacetic acid (TFA) (pH 2) for 140 h, but losing resolution (four peaks merged into one peak) after pumping 100 mM sodium phosphate @H 9) for 140 h (097). c18 PVA packing materials, however, have a wider range of pH stability, as no loss of resolution or efficiency was noted after pumping:

480R AnalyticalChemistry, Vol. 67, No. 12, June 15, 7995

(a) a pH 2 mobile phase for 184 h (80% acetonitrile, 0.1% TFA, pH 2); (b) a pH 10 mobile phase for 224 h [lo0 mM sodium borate buffer/acetonitrile (8020), pH 101; and (c) a pH 13 mobile phase for 6 h [0.1 M sodium hydroxide/acetonitrile (8020), pH 131 (041).

Selectivify. Advantages in separation can result from unique selectivities of polymer-based packing materials, since the polymer differs from silica in the physicochemical properties of the surface. However, it should be noted that the high selectivity of polymer supports for rigid planar and flexible aromatic molecules in comparison to linear alkanes dramatically decreases upon the attachment of c8 or ligands. This is exemplified by the attachment of CIS to TSK G4000PW supports (€'HAM) (048). In this study a values (in 80% methanol) for pyrene/decane (which indicates selectivity for rigid planar over linear alkane molecules) on TSK G4000PW-C1 and G4000PW-Cl8 averaged 12 and 1.0, respectively (0.24 for c18 silica), while the a values (in 80% methanol) for triphenylmethane/decane (which indicates selectivity for flexible aromatic over linear alkane molecules) averaged 6.5 and 0.47 on TSK G~OOOPW-CI and G4000PW-C18, respectively (0.23 for silica) (048). As noted before, PS-DVB shows strong retention of aromatic compounds (035).

Hydrophobicity, measured as retentivity of methylene groups, is given by the following series: silica Cl8 > PS-DVB > CIS PVA > TSK cl8-4Pw in 80% methanol and 70% acetonitrile and silica CIS > TSK Clr4PW > cl8 PVA > PS-DVB in 40% THF (047). The amount of alkyl groups on the TSK PW-CB and -CIS (c8 or

PHAM) is estimated to be 17-33% of that present on c8 silica and c18 silica, leading to less retentivity (035). The CIS polymer packing materials TSK G4000PW-Cl8 and CIS PVA have significantly lower capacity factors for alkanes and alcohols than C18

silica (035, 041). Selectivity differences between PS-DVB and alkyl-bonded silica have been quantitated in terms of solute dipolarity/polarizability and hydrogen-bonding characteristics, with the conclusions that a solute's dipolarity/polarizability is the most important factor contributing to retention on native PS- DVB (higher value, greater retention), while the hydrogen- bonding ability of a solute does not contribute significantly to retention on native PS-DVB (but does contribute significantly to retention on alkyl-bonded silica) (071). The differences in retention on native PS-DVB compared to alkyl-bonded silica are maximized by using methanol as an organic modfier, are reduced (but still significant) when acetonitrile is used, and are minimal when THF is used (071). Use of methanol, however, has the disadvantages of yielding long analysis times and asymmetrical peaks (071). From the above discussion, it can be reasoned that a possible advantage of polymer over silica packing materials is difference in selectivity, as periodically reported in the literature.

Basic Compounds. Advantages in the chromatography of basic compounds are sometimes claimed for polymer reversed-phase packing materials. This has not been rigorously documented, however. Native PS-DVB (098) and CIS PS-DVB (099) performed poorly in the chromatography of basic compounds. Another study reported better separation and peak shapes for antitheilerial agents using a PVA column, in comparison to a base-deactivated column (LiChrosorb €E'-select B, Merck) (0100). However, the best performing base-deactivated column was not chosen for comparison in this study (see discussion below on base- deactivated packing materials).

High-Performance Porous Graphite Packing Materials. Properties. A general review (0101) and a review of biomedical applications (0102) have been published describing porous graphite HPLC packing materials. The designation of porous graphite is preferred to earlier designations of porous graphitic carbon, porous graphitized carbon, PGC, or graphitized carbon (0103). Porous graphite packing materials are made by heating in nitrogen a resin-filled silica gel to 900 "C to form glassy carbon- filled silica, with subsequent dissolution of the silica with hot caustic solution, followed by heating to 2500 "C in argon (which causes graphitization to occur), yielding a material consisting of planar sheets of carbon atoms in hexagonal arrays (0104). Several commercial sources of porous graphite packing materials are available, having respective particle and pore diameters of 7 pm and 250 A (Hypercarb, Shandon Scientific) (01 7) and 6 pm and 500 A ('EK Gel Carbon-500, TosoHaas-Tosoh) (Oll), a pH stability of 1-14, and a pressure stability up to 8000 psi (017).

The combined effects of delocalized electron bands and support hydrophobicity give porous graphite unique selectivity characteristics (0102). TFA is used as a mobile-phase modifier for mediating the electronic component to the retention mechanism (which predominates in the chromatography of anions) (0102, 0105,0106), while organic modifiers are used for mediating the hydrophobic component of the retention mechanism. A universal eluotropic series does not exist for porous graphite; E' has a small range and is solute dependent (0101, 0107). Approximate solvent strengths can be established according to the functional groups of the solute (0107). Peak shapes are dependent on the solvent used (0107). Porous graphite is much more hydrophobic than conventional reversed-phase silica, requiring a higher concentration of organic modifier in the mobile phase (90-100% organic content on porous graphite columns equals the retention of only 50% organic content on silica columns) (0104). The selectivity of porous graphite has been studied for systematically modi5ed aromatic compounds (0103,0108-0110). Compounds with greater aromatic content show stronger retention than compounds with greater alkane content, which is the reverse of the order noted for cl8 silica (0103).

Advantages of porous graphite over conventional silica reversed- phase packing materials include stereoselectivity, ability to retain compounds not normally retained by conventional reversed-phase or ionexchange chromatography, increased range of mobile-phase pH, and increased performance with basic solutes. Disadvantages include lower efficiencies and requirements for more extensive sample cleanup. These aspects are discussed below.

Stereoselectivity Porous graphite has been demonstrated to have unique separating ability for geometric isomers, often exceeding the ability of native silica, native alumina, and bonded silica. The mechanism of geometric isomer separation (0103) is attributed to the flat structure of the graphite surface, which consists of hexagonally arranged carbon atoms that have planar geometry. The flat structure of the porous graphite readily discriminates geometric structures that fit onto the surface from those that do not. On the other hand, native silica and alumina are less effective because the surface is geometrically heteroge- neous and because the separation depends on specific dipole and hydrogen-bonding interactions (chemical character that the solute may or may not have). Bonded silica has even less geometric discrimination, because the bonded phase acts more like a liquid, retaining solutes by a partition mechanism. Examples of the


separation of geometrically different solutes on porous graphite are given below.

Advantages in separations of diastereomers of cephalosporin and cephalosporin synthesis byproducts have been reported for porous graphite in comparison to a bonded-silica packing material (0111). Porous graphite was more effective in separating steroids that differ by one additional double bond (prednisolone/hydro- cortisone and prednisone/cortisone) than bonded-silica packing materials (0111). Epimers of estriol, testosterone, and norgestrel were separated on porous graphite; however, P-cyclodextrin was required in the mobile phase (0112). Chromatography of monosaccharides, disaccharides, and cyclomaltaoses (0113), oligosaccharide alditol isomers (01 14, and unsaturated disac- charide isomers from chondroitin sulfate (0115) have been reported. Complementary separating ability has been shown for porous graphite in comparison to other modes of chromatography for oligosaccharide alditol isomers; in some cases porous graphite exclusively achieved the separation, while in other cases other modes were better (0114). Monosaccharides (only weakly retained with a water mobile phase) and disaccharides were separated into anomeric peaks using porous graphite; each anomeric doublet peak could be merged into one peak using millimolar basic mobile phases (0113). In this work, separation of positional isomers of glucosylinositol was achieved with a porous graphite column and not with anion-exchange or reversed- phase columns (0113). Epimers of unsaturated nonsulfated disaccharides of chondroitin sulfate were separated with porous graphite, which could not be attained with bonded silica (0115). It should be noted that a pH 11 mobile phase was required to attain sharp peaks in this latter work (0115).

Other Selectivity Advantages. Another unique selectivity advantage of porous graphite is its ability to simultaneously retain analytes with entirely different chemical properties, retaining both hydrophilic and hydrophobic analytes (ionic and nonionic compounds). One study demonstrated this capability for various compounds, including a simultaneous separation of anionic and cationic technetium compounds, a separation of creatine and creatinine, and a determination of oxalic acid (note that creatine and oxalic acid are difficult to retain on a C18 column) (0105). In another study, oligosaccharides and oligosaccharides linked to one or two amino aids could be retained and separated on a porous graphite column, unlike CIS silica columns, which barely retain these hydrophilic compounds (0116). This work also demonstrated the advantage of porous graphite chromatography in separating chitooligosaccharides, separations that could not be achieved using anion exchange (0116). Confirming results of other studies involving chromatography of oligosaccharides, split anomeric peaks were observed, which merged into one peak in a 10 mM NH40H mobile phase at 70 "C (0116). The sialylated and nonsialylated forms of both oligosaccharides and oligosaccharide alditols are all retained on porous graphite, an advantage over anion exchange, which has minimal retention of the nonsialylated species (011 7). Porous graphite did resolve biantennary from triantennary nonsialylated oligosaccharides; however, it did not resolve monosialylated from nonsialylated oligosaccharides (01 17). Different glycopeptides have also been separated (01 14, 0117).

The scope of porous graphite in its separating ability is not unlimited, as seen from the examples given above. It provides unique separation capabilities in some cases, while lacking the

separating ability of other chromatographic modes in other cases. It is thus useful in conjunction with other chromatographic modes.

Basic Compounds. Porous graphite shows improved chromatography of basic compounds in comparison to conventional reversed-phase silica packing materials (without mobile-phase modfication) (0102). These separations usually employ a mobile- phase pH below the pKa of the base, such that the base is positively charged (0102). Use of mobile phases at high pH leads to excessive retention of a basic solute because of increased hydrophobicity (0102). In the same way, use of less hydrophobic ion-pairing reagents such as TFA and triethylamine is preferred for ion-pairing chromatography on porous graphite packing materials (0102). One study showed that the performance of porous graphite was not as good as a basedeactivated silica phase in the chromatography of basic compounds (092). In this study, chromatography of nicotine on a porous graphite packing material gave a reduced plate height of 8.4 and a peak asymmetry at 10% peak height of 2.3 (organidwater mobile phase), compared to values of 4.7 and 1.77, respectively, for the best performing base- deactivated silica packing material (methanol/ 0.05 M phosphate pH 6.25 mobile phase; reduced plate heights were calculated using peak width at half-height). It should be mentioned, however, that the porous graphite packing material did outperform three other base-deactivated packing materials tested in the chromatography of nicotine. However, porous graphite does lack one advantage of base-deactivated packing materials, as base-deactivated packing materials can have the efficiency and peak shape further improved by mobile-phase modification (low pH, ion pairing, amine modifiers).

Disadvantages. Disadvantages of porous graphite include easy contamination of the support due to its strong hydrophobic nature and lower efficiency in comparison to silica-based packing materials. High molecular weight, hydrophobic, and electron-rich compounds are strongly retained on porous graphite, which can contaminate the column. Thus, more extensive sample preparation procedures may be required for porous graphite chromatography (0102).

A wide variation in efficiency performance has been reported for porous graphite packing materials. One study reported efficiencies comparable to silica-based HPLC supports, finding reduced plate heights of less than 3.2 for aromatic amino acids [flow rate 1.0 mWmin; acetonitrile/25 mM potassium dihydrogen phosphate (PH 1.0) (65:35)] and for benzodiazepines [flow rate 1.5 mL/min; acetonitrile/5 mM disodium hydrogen orthophos- phate (PH 10.6) (65:35)] (0118). However, another study reported very low efficiencies, finding reduced plate heights of 14-74 for chlorophenoxyacetic acid congeners [flow rate 0.6 mW min; 7:3 dioxane/water (with up to 50 mM LiC1, sodium acetate, or acetic acid)] (0108). Another work reported reduced plate heights of greater than 7.9 for phenol and benzene (organidwater mobile phase) (092). A manufacturer specifies a reduced plate height for porous graphite packing materials of less than 6.7 (011). No work was published during the review period docu- menting the effect of flow velocity on porous graphite column efficiency.

Reproducibility. Theoretically, porous graphite has an advantage of higher reproducibility, since the surface of interaction is theoretically homogeneous. However there are lot-to-lot differences due to variability in surface defects at the edge of the graphitic sheets, which lead to different retention characteristics


TabFe 2. Summary of Comparison Studies of Base=Deactivated Packing Materials (Reduced Plate Heights and Peak Asymmetry at 10% Peak Height).

Study I* ( 0 9 2 ) pyridine benzylamine nicotine quinine

Inertsil ODS (GL Sciences) (5 pm) 4.9 (1.62) 6.3 (1.73) 4.7 (1.77) 6.8 (1.91) Suplex pKb-100 (Supelco) ( 5 pm) 5.5 (1.64) 21.9 (1.60) 9.0 (2.47) 9.2 (1.72)

29.8 (4.60) LiChrospher RP-8 Select B (E. Merck) (5 pm) 8.2 (2.10) 18.9 (2.04) 23.8 (4.62) Nucleosil CIS AB (Macherey-Nagel) ( 5 pm) 40.7 (3.81) 101 (6.47) 39.2 (4.87) 26.1 (3.64)

Study 2c (0119)

Suplex pKb-100 (Supelco) ( 5 pm) broad peak, not measured SynChropak SCD-100 (Keystone) ( 5 pm) broad peak, not measured Hibar LiChrosorb RP-Select B (E. Merck) ( 5 pm) broad peak, not measured Deltabond Octyl (Keystone) ( 5 pm) 167 (3.93) Chromega -CIS (Fisher) (5pm) broad peak, not measured Zorbax R, (Rockland Technologies) (5 pm) broad peak, not measured Rexchrom ODS (Regis) (5 pm)

18.0 (1.26) 31.6 (3.00) 57.1 (2.13)

Ultracarb ODS-20 (Phenomenx) ( 5 pm) Capcell Pak CIS-SG (Shiseido) ( 5 pm) Chemcosorb 5-ODS-H (Chemco Scientific) ( 5 pm)

Supecosil LC-8-DB (Supelco) ( 5 pm) 230 (6.0) 741 (5.16)

Study 3d (0120)

Ultrabase CS (Shandon-SFCC) ( 5 pm) 14.3 (2.67) Zorbax R,-Cg (Rockland Technologies) (5 pm) 62.5 (4.23) Ultrabase CIS (Shandon-SFCC) ( 5 pm) 73.5 (4.82) Nucleosil 100-5 C18AB (Macherey-Nagel) (5 pm)

14.7 (2.79) 20.4 (2.54)

LiChroCART Superspher 100 RP-18e (Merck) (4 pm)e LiChroCART Superspher 60 RP-8e (Merck) (4 pm)' 250 (6.37)

Values in parentheses. Conditions: mobile phase methanoU0.05 M phosphate pH 6.25 (55:45, v/v); 20 "C; flow rate 1 " i n ; theoretical plates determined by peak widths at half-height. Conditions: mobile phase acetonitrile/25 mM potassium phosphate pH 6.2; ambient temperature; flow rate 1 or 2 mIJmin; theoretical plates determined by Foley-Dorsey statistical moments method; solute is codeine.

Conditions: mobile phase methanoUO.15 M NaHzP04 (3565, v/v); apparent pH 5.2; 40 "C; flow rate 1 mL/min; theoretical plates determined by Foley-Dorsey statistical moments method; solute is 3,4 dihydro-6-hydroxy-NjVjV,2,5,7,8-heptamethyl-2H- 1-benzopyran-2-ethanaminium 4-methylbenzene sulfonate. e Conventional reversed-phase column, not base-deactivated.

of different lots (0101). As of yet, reproducibility has not been thoroughly characterized for porous graphite packing materials.

High-Performance Base-Deactivated Packing Materials. Theoretically, base-deactivated reversed-phase silica packing materials do not require mobile-phase modification for the chromatography of basic compounds. These packing materials have been developed by various manufacturers. Most manufacturers do not reveal the exact process by which these packing materials are produced. In general, however, most of these packing materials employ high-purity silica, which have a low trace-metal content. From this starting point, the various manufacturers may utilize different strategies to further deactivate the silica surface, including one or more of the following procedures: doing excessive end capping, attaching a dense surface coverage of alkyl ligand, covering the silica surface with a polymer layer, employing proprietary treatment($ to modify the reactivity and/or distribution of the silanols on the surface, attaching novel ligands, electrostatically shielding the silica surface, employing other reaction chemistries, or doing other silica pretreatment procedures. As with conventional silica packing materials, most of these packing materials are limited to a pH range of 2-8 (although some manufacturers claim an upper pH limit of 10 for some of the polymer-modified silica packing materials).

Table 2 (092, 0119, 0120) summarizes the data of three studies which compared the performance of base-deactivated packing materials from different manufacturers. As seen in Table 2, there is a significant difference in the performance of base- deactivated packing materials from different manufacturers. It should be pointed out that study 1 calculated column efficiency from peak width at half-height data, which gives falsely low values for reduced plate heights, while studies 2 and 3 calculated peak efficiency using an empirical equation based on a modified statistical moments approach, which gives a more accurate estimate of reduced plate heights. Data in study 3 of Table 2 also contain a comparison of the performance of base-deactivated

columns with that of conventional reversed-phase columns in the chromatography of a basic compound.

What is clear from Table 2 is that some base-deactivated packing materials are only marginally better in performance than conventional reversed-phase silica packing materials. This was also documented in another study comparing the performance of base-deactivated packing materials (099). From these comparison studies, two packing materials standout as the best performers, Inertsil ODS (GL Sciences) and Suplex pKb-100 (Supelco). An extensive study chromatographing 32 basic drugs on a Suplex- pKb-100 base-deactivated column showed a range of reduced plate heights from 4.5 to 16.7 for all compounds (calculated with modified statistical moments analysis) and asymmetries (10% peak height) of 5 2 for all but 6 compounds (099). Increased concentration of buffer in the mobile phase to 0.15 M was found to improve plate number and peak symmetry for a base- deactivated packing material (0120).

Use of base-deactivated packing materials have been reported for the following clinically relevant analytes: anthracyclines and metabolites (098); diltiazem and metabolites (0121, 0122); diltiazem and metabolites and celiprolol (0123); tamoxifen and metabolites (0124); tamoxifen, toremifene, and metabolites (0125); dapsone, monoacetyldapsone, and pyrimethamine (0126'); S-adenosyl-L-methionine and metabolites (0127); nicotinamide and metabolites (0128); nucleosides and its derivatives (0129); and various basic drugs, PTH amino acids, B vitamins, and pyrimidines (0130).

High-Stability High-Performance Packing Materials. An issue with conventional reversed-phase silica packing materials is stability. The pH range for operation of conventional silica- based packing materials is 2-8. As noted above, an increased usable pH range has been documented for polymer (HEMA pH 2-12; PS-DVB 1-13), PBD-alumina (PH 2-13), and porous graphite (PH 1-14) packing materials. Efforts have also been undertaken to increase the stability of silica-based reversed-phase

AnaiyticalChemistiy, Vol. 67, No. 12, June 15, 1995 483R

packing materials. As noted above, siliconemodified silica has a reported stability in the pH range 2-10, while a proprietary polymer-modified silica has a reported stability in the pH range 2-13. Studies have been done that document increased stability of silica-based packing materials with increased ligand surface coverage (0131), increased alkyl chain length (0132, 0133), increased bulkiness of the reversed-phase ligand (0131, 0132, 0134, 0135) use of multifunctional silane chemistry (0131, 0136), and special preparation of the silica base (0131, 0137).

Shortchain (i.e., C1, C4, and CN) and phenyl reversed-phase silicas are particularly susceptible to hydrolysis, particularly in acidic mobile phases. Monomer organosilanes used in the preparation of conventional silica reversed-phase packing materials, contain the reversed-phase functionality (i.e., C18, Cg, C4, CN, etc.) and two methyl groups bonded to the silicon atom. A stable silica reversed-phase packing material has been developed, which substitutes bulky groups (such as isopropyl or isobutyl) for the two methyl groups (0132, 0134). The bulky groups provide steric protection from hydrolysis of the Si-0-Si bond, the bond that anchors the alkylsilane to the silica surface. This line of packing materials is commercially available as Zorbax SB (Rock- land Technologies) having CN, phenyl, Cg, and C18 ligands (07, 011, 013). These sterically protected packing materials are discussed below.

Retention (capacity factor for 1-phenylheptane) on a sterically protected (cyanopropy1)silica was maintained, while that on a conventional (cyanopropy1)silica packing material was reduced to 4096, after pumping 4500 column volumes [equivalent to 14 8-h days of operation (031)l of acetonitrile/water (50:50 v/v), both being 0.1% TFA at 50 "C, at 1.0 mWmin (0132). Retention (capacity factor of toluene) was unchanged on a sterically protected CIS silica up to at least 27 000 column volumes, while it was reduced to 68% (27 000 column volumes), to 60% (17 000 column volumes), and to 10% (4500 column volumes) for the conventional clg silica columns Zorbax Rx-clg, Nucleosil CIS, and YMC Basic c18, respectively, using an acidic mobile phase [methanoVwater (50:50 v/v) 1.0% TFA, pH <l, 90 "C, 1.0 mL/ min mobile phase] (0135). The sterically protected Clg silica can be used at an upper pH limit of 9.0, having a column lifetime of 13 000 column volumes using a methanol/O.Ol M sodium phosphate (40:60 v/v), pH 9.0, 22 "C, 1.0 mL/min mobile phase, after which peak asymmetry and plate height increased for N,N- dimethylaniline (0135).

PACKING MATERIALS FOR HlGH=SPEED ANALYSIS OF MACROMOLECULES

An important advance in packing material technology is the development of packing materials that chromatograph macromolecules at high efficiencies and high flow rates. Three approaches have been used to achieve this: use of nonporous packing materials, use of very large pore diameter macroporous packing materials, and use of perfusion packing materials. Demonstrating the speed of analysis, mixtures of five proteins were separated within 15-20 s using nonporous (0138) and perfusion (0139) packing materials and a mixture of six proteins was separated in 60 s using a native PS-DVB packing material with 4000 A diameter pores (0140).

Articles have been published characterizing silica (0141- 0147) and polymer (0148-0151) nonporous packing materials. Separations of proteins (0152-0156), proteins and peptides

(0157, 0158), peptide mapping (0159), hemoglobins (OXO), glycated hemoglobin AK (0161), and oligonucleotides and DNA fragments (0162-0167) have been reported.

Perfusion packing materials were first reported in 1990 (0168, 0169) and are commercially available (POROS series, PerSeptive Biosystems). These packing materials are used for both prepara- tive and analytical work. Articles reporting analytical applications include the determination of neuropeptides (0170) and peptide mapping (01 71). Theoretical and quantitative aspects of perfusion chromatography (01 72, 01 73) and of perfusion and nonporous packed capillaries (01 74) have been presented.

Various packing materials, including nonporous and perfusion, have been compared in the anion-exchange chromatography of DNA restriction fragments (01 75).

ACKNOWLEDGMENT The author gratefully acknowledges the help of Yunyu Zhou,

Yansheng Liu, Cindy Wang, and Eumelia Tipa in the prepartion of this review.

David .J..An&rson received .his B.S.. in chemjstry om Bucknell University in 1977and hzs Ph.D. in analyttcal chemistlyj&m Iowa State University in 1986. From 1986 to 1988 he was a Postdoctoral Fellow in Clinical Chemistry at the Mayo Clinic. He is current1 an Associate Professor and Director of Clinical Chemishy in the Aepartment of Chemist at Cleveland State Uvivenity. His research interests are the use ,of HyLC,methodology in clinacal anal szs, s ecfically, the determination offibrtnogen-related species as m a r d m ohemostasis and throm- bosis, the determination of glycosylated variants of proteins as markers for cancer, and the determination of isoenzymes in cardiovascular disease and inherited disorders.

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