Peer Reviewed: Zirconia Stationary Phases for Extreme Separations

HY

full page

Zirconia Stationary Phases for Extreme Separations Composite materials surpass standard silicon and carbon for stability and robustness in complicated reversed-phase column applications. hirty-eight new reversed-phase (RP) columns with various stationary phases were introduced at Pittcon 2001—a stark contrast to the early days of HPLC when, to paraphrase Henry Ford, you could get any kind of column you liked as long as it was an octadecyltrichlorosilane (ODS) modified-silica column (1). Currently, stationary phases for HPLC range from the large family of RPs, including those based on the chemical inertness of zirconia (ZrO2), to highly selective, nearly specific, bioaffinity phases. With an emphasis on RPs, technology is aimed at improving column durability under adverse conditions (especially high pH), column-to-column and batch-to-batch reproducibility, peak tailing toward cationic (basic) analytes, unique selectivities, and speed. Columns are also designed for specific areas, such as LC/MS, and for specific improvements, such as minimal solvent consumption. Recently introduced polar-embedded phases that provide unique selectivity and can be used in highly aqueous media have become particularly popular. Many new phases have emerged from research directed at producing more durable materials, understanding and minimizing silanol–analyte interactions, and addressing the need for more selective separations in all modes of LC (2–5). Another important trend has been the development of specifically nonporous silica and zirconia particles and shorter and wider column geometries for faster analysis times (6). Most new phase development work focuses on silica-based materials; however, silica’s inherent instability at pH > 7–8 and higher dissolution rates at elevated temperatures significantly limit the range of aqueous media conditions. Stabilized silicas, hybrid inorganic–organic particles, and novel support materials—synthetic organic polymers, alumina, and other metal oxides—have been designed to overcome these shortcomings (7–9).

Christopher J. Dunlap Saint Mary's College

Clayton V. McNeff and Dwight Stoll ZirChrom Separations

Peter W. Carr University of Minnesota

N O V E M B E R 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y

599 A

(a) 4.0

Retention factor (k’ )

Butylbenzene 3.0 2.0

Propylbenzene

1.0

Ethylbenzene Toluene Benzene

0

1000

2000

3000

4000

5000

Column volumes (b)

Plate count

12000 8000 4000

0

2000

4000 6000 Column volumes

8000

10000

FIGURE 1. Stability of chromatographic columns. (a) PBD–ZrO2, 150 ⫻ 4.6 mm i.d. column at pH 14. (b) Comparing the same PBD–ZrO2 column (blue) with a C18–SiO2 column (red); lidocaine analyte, pH 7.

Additionally, researchers have stabilized silica by cladding it with metal oxides such as alumina, titania, or zirconia (10). Synthetic organic polymer-based supports, while much more stable than silica under either acidic and alkaline conditions, are much less efficient and less tolerant of solvent changes. Schomburg’s group introduced polybutadiene (PBD)-coated alumina, which spurred interest in non-silica metal oxide-based phases (11). Alumina has a somewhat greater range of usable alkaline pH than silica, but it is rather acid-soluble and is still not stable at pH extremes (8, 12). Because zirconia is nearly insoluble from pH 1 to 14, it led us to develop the polymer-induced colloid aggregation method for preparing monodisperse, high-porosity (0.4–0.5), zirconia microspheres (3 µm diameter) (12, 13). These particles have large (300 Å), well-connected pores suitable for use as substrates for HPLC stationary phases (13). Table 1 lists various zirconia-based stationary phases we have reported and are currently studying. Zirconia’s pH sta-

bility and catalytic inertness are far superior to other available metal oxides; its resistance to dissolution at high temperatures is excellent. The following summarizes some of the recent advances that have been made in zirconia-based HPLC stationary phases.

What is the ideal stationary-phase support? New stationary phases are best evaluated by comparing them to Unger’s specification of the ideal phase: The particles should have a narrow size distribution and high surface area; the pores must have a diameter appropriate to the size of the analyte and good connectivity to allow for fast analyte mass transfer; and the support material should resist thermal, mechanical, and chemical degradation but have a surface that is both energetically homogeneous yet chemically modifiable (14). Although many synthetic materials have suitable particle sizes and pore structure, the chemical and thermal stabilities of the available stationary phases vary widely. Efficient stationary phases with greater stability than silicabased phases would afford many advantages. Stationary phases that are stable under extremes of pH and other chemical conditions such as phosphate and carbonate buffers (which are particularly hard on silica in solutions at pH > 7) provide a greater range of options for optimizing retention, selection, and peak shape. Less obvious advantages of greater stability are connected to running separations at high temperatures. Until recently, few chromatographers conscientiously optimized temperature in HPLC. Anita and Horvath closely examined the effect of temperature on separation speed and found that increasing the temperature affects mobile-phase viscosity and analyte diffusivity, which affect column dynamics (15). The decrease in eluent viscosity with increasing temperature (estimated at a 5–10-fold decrease for methanol ongoing from 20 to 200 °C) greatly reduces column back pressure, which allows separations at higher flow rates in less time. For example, analytes separated at 30 °C, at reasonable back pressure, and a flow rate of 1.0 mL/min take 11 min; the same separation run at 100 °C is completed in 5000 column volumes of fluid were passed dinate bonds to seven neighboring oxygen atoms. Silica has through the column. Although several groups have greatly improved the staonly four bonds to oxygen atoms, which largely accounts for zirconia’s superior resistance to chemical degradation, espe- bility of silica-based phases by using novel derivatization cially by acid and base. In a 15-day trial under static condi- reagents, the intrinsic stability of the zirconia support cleartions, no dissolved zirconium was detected by inductively cou- ly makes it the better choice when extremes in pH (>11) or pled plasma MS at any pH from 1 to 14 (12). In comparison, temperature (>60 °C) are required (22–24). However, even significant amounts of aluminum were detected after only in the pH and temperature ranges in which silica is considone day when alumina was challenged at pH 3 and 14. Sili- ered stable, some dissolution does occur, especially in phosca was not tested because it dissolves much more readily phate or carbonate buffer mobile phases (24). In practice, than does alumina under basic conditions. bonded-phase silica columns will eventually fail because ei-


601 A

(a)

to 80 °C in neutral solutions, we tested its stability at neutral pH with phosphate buffers at higher than ambient temperature to accelerate column aging and thereby fully compare the two materials in a reasonable pe1 7 riod of time. 4 We saw that the column efficiency of the silica-based column decreased rapidly under these conditions. Zir5 6 conia is modified by prolonged heating in phosphate media, so we did observe minor changes in retention in a slightly more aqueous eluent designed to match retention. Nonetheless, the zirconia-based column main0 2 4 6 10 12 14 8 tained its efficiency and utility (21). Silica columns can Time (min) be greatly stabilized at intermediate and high pH by (b) using high concentrations of organic modifiers in the 6 mobile phase and “sacrificial” precolumns, but they are still not as stable as zirconia columns (25). Figure 2 compares a PBD–ZrO2 column with one of the new generation of stable silica columns specifically designed to withstand high pH. At pH 12, there 7&8 is no apparent change in the elution profile of solutes 3 4 2 9 on the PBD–ZrO2 phase after 5000 column volumes, 5 1 whereas the retention and resolution of the same solutes on the silica-based phase are both severely degraded in less than half the time. Buffers other than 0 2 4 6 8 10 12 14 16 18 phosphate will definitely allow pH much greater than Time (min) 7, but as suggested by the data in Figure 1, current (c) silica-based phases do not have the intrinsic chemical durability of zirconia. 1 The ability to adjust the pH over a wide range can 7 be quite critical to developing a good separation. Use of high and low pH is often helpful in improving band spacing (chromatorgraphic retention factor) and re3 8 4 tention for poorly retained species, and reducing peak 9 6 2 5 tailing, thus making traditionally difficult separations easier to perform. For example, the separation of a set of basic drugs could not be done at pH 7 with either a high-quality silica-based ODS stationary phase or a 0 3 1 2 4 5 6 PBD–ZrO2 column (Figures 3a and 3b). However, Time (min) when the pH was increased to 12 (Figure 3c), the PBD–ZrO2 column separated the compounds in less FIGURE 3. The effect of pH on the resolution of basic drugs. than half the time required for a separation at pH 7. The separation is not possible with this particular sili(a) C18–silica, 150 ⫻ 4.6 mm i.d. column. Mobile phase: 30/70 acetonitrile/20 mM ca-based support material in a phosphate buffer at pH potassium phosphate, pH 7. (b) PBD–ZrO2 150 ⫻ 4.6 mm i.d. Mobile phase: 28/72 acetonitrile/20 mM potassium phosphate, pH 7. (c) Same as (b) but pH 12. Solutes: 12 because the column is unstable under highly alka1, labetalol; 2, atenolol; 3, acebutolol; 4, metoprolol; 5, oxprenolol; 6, lidocaine; line conditions. 7, quinidine; 8, alprenolol; 9, propranolol. Zirconia interacts with ionic analytes quite differently than silica. On silica-based RPs, residual deprother the support or bonded phase will dissolve. Figure 1b tonated silanol groups interact with solutes, especially shows the efficiency of a PBD-coated zirconia column and a amines. Although the average silanol group has a pKa ~7, high-stability silica-based phase as a function of eluent col- ionization of some groups begins at pHs as low as 2, resultumn volumes. Although the manufacturer specifies that the ing in a negatively charged surface over most of the usable hybrid silica column is stable to pH 12 and at temperatures pH range (5). On the other hand, zirconia’s surface is posi2

602 A

3

8&9

A N A LY T I C A L C H E M I S T R Y / N O V E M B E R 1 , 2 0 0 1

tively charged up to pH ~8, where it becomes negatively charged. However, zirconia’s surface charge is greatly influenced by adsorption of the hard Lewis bases found in the carboxylate and phosphate buffers frequently used as eluent additives. These species interact with the coordinatively unsaturated Zr(IV) surface sites, which are strong, hard Lewis acids. In such media, the surface charge depends very strongly on pH, the type of buffer, and its concentration. This difference in surface chemistry means that under conventional conditions, most basic species are strongly tailed on unoptimized silica-based columns. Manufacturers of silica-based columns have made tremendous improvements in the peak shape of basic analytes by using high-purity (metal-free) silica starting materials that eliminate the strongest silanol groups. However, they tend to dissolve more rapidly than do standard silica materials and thus require more frequent replacement. Because zirconia-based phases strongly retain hard Lewis bases, such as carboxylate- and phosphate-containing molecules, a silica column is a decidedly better choice for RP separations of peptides or proteins. However, in the ion- or ligand-exchange mode, Lewis acid–base and ionic interactions provide highly desirable selectivity differences in comparison to silica-based ion-exchange phases. Excellent separations for proteins in ion- and (a) ligand-exchange modes are possible with an appropriate Lewis base-containing eluent (21, 26, 27 ).

peratures >150 °C without significant loss in retention or efficiency upon return to room temperature (29). Additionally, PBD–ZrO2 has been tested with 5000 column volumes of eluent at 195 °C with no changes in the retention characteristics of the column. Because it is now acknowledged that temperature can have a considerable impact on band spacing, separations can often be improved dramatically by simultaneously optimizing temperature and mobile-phase composition (17).

PEI and PBD exhibit excellent resistance to extremes in pH and temperature.

200

(b)

3

4

100

3

50

1

150

mAU

Zirconia has a melting point of 2750 °C. The postsynthetic treatment of the porous zirconia particles developed in our lab involves sintering at 950 °C for several hours to make the particles mechanically stable. Zirconia is coated by heating elemental carbon to 700–800 °C and cracking hydrocarbons on its surface, which leads to coke formation (28). The high sintering temperature of zirconia leaves the pore structure virtually unchanged and deposits a thin carbon layer. Zirconia-based RP media have been tested in typical RPLC solvent mixtures at tem-

Zirconia, like silica, has a complicated surface chemistry. Zirconia has several types of adsorption sites, some of which are not important chromatographically. However, interactions between zirconia’s surface and carboxylic acids are especially troubling, because this leads to the irreversible absorption of proteins, even when the surface is coated with polymers. These interactions can often be controlled by adding fluoride or phosphate to the eluent, which adsorb to the active Lewis acid sites and thus modulate the interaction with analytes. The adsorption results in a negatively charged surface, which means that the materials then also behave as cation exchangers. Historically, reducing the impact of silica’s unwanted silanol sites on peak shape of amines has been one of the major chal-

40

5

mAU

Temperature stability

Phases based on zirconia

2 8 min

50

4 2

30

5

20

20 min

10 0

0 0

1

2

3

4 5 6 Time (min)

7

8

0

5

10 15 Time (min)

20

FIGURE 4. Separation of basic compounds on zirconia- and silica-based columns. (a) PBD–ZrO2. Mobile phase: 45/55 acetonitrile/20 mM potassium phosphate at pH 12.0. (b) ODS column under same conditions as (a) except 50/50 acetonitrile/20 mM potassium phosphate at pH 7.0. Solutes: 1, nordoxepin; 2, protriptyline; 3, nortriptyline; 4, imipramine; 5, amitriptyline.


603 A

CH3

(a)

chromatographically active stationary phases has been reported, but these materials do not show the level of stability characteristic of the underlying zirconia in terms of resistance to temperature and extremes in pH (11, 30, 31). Physical coating methods (polymer coating and carbon cladding) have proven successful in creating a series of stable stationary-phase materials (8). Another approach is to add hard Lewis bases to the eluent so that they strongly chemisorb onto zirconia’s surface thus blocking adsorption of analytes. Adsorptive modification of the surface effectively blocks the Lewis acid sites while generating useful stationary phases. We used adsorptive modification methods to synthesize a wide range of novel stationary phases (Table 1).

CH3 NO2 CH3 NO2 CH3

CH3 NO2 CH3 CH3 CH3

CH3 NO2

NO2

H3 C

Polymer-coated stationary phases

0

2

4

(b)

6 8 Time (min)

10

CH3

12

14

CH3 CH3

CH3 CH3 NO2

NO2 CH3

NO2

CH3 NO2 CH3

CH3 H3 C

0

4

8 12 16 Time (min)

NO2

20

24

FIGURE 5. Comparison of the selectivity of an ODS–silica column and a carbon-clad zirconia column. (a) ODS column with detection at 254 nm. Mobile phase: 30/70 water/acetonitrile. (b) Carbon-clad zirconia column, 10 ⫻ 0.21 cm i.d., 8 µm particles. Mobil phase: 35/65 water/acetonitrile.

lenges to using it for chromatography. Several methods of blocking these sites while simultaneously introducing chromatographically useful properties have been investigated. Silanization of zirconia using surface hydroxyl sites to attach

604 A


The two polymers used most successfully to coat zirconia are PBD and polyethyleneimine (PEI). Both polymers exhibit excellent resistance to extremes in pH and temperature while imparting useful chromatographic properties to the particles. PBD–ZrO2 is an RP material, whereas PEI–ZrO2 is an anion exchanger. Both phases are well characterized and are used for various separations (26, 32). PBD-coated zirconia. This phase is prepared by impregnating porous zirconia with a solution of PBD and a free radical initiator in hexane, carefully removing the hexane by controlled evaporation, followed by thermal decomposition of the initiator. Polymer-coated phases are quite different from chemically bonded phases because no covalent bonds hold the polymer on the surface. Imagine these materials to be insoluble, highly cross-linked liquids impregnated in the pores of the substrate particle rather than surface coatings. The impregnated polymer causes very little loss in efficiency when properly prepared in a zirconia material with the right pore structure; 120–140,000 plate counts per meter for small solutes on columns packed with 3-µm columns are commonly obtained. The surface of the zirconia is screened and only partly covered in these types of phases. The cross-linked polymer molecule consists of only carbon–carbon and carbon–hydrogen bonds, resulting in considerable residual olefinic unsaturation and making PBD a very stable coating (Figure 1). To be widely adopted, a new RP material should behave similarly to bonded-phase silica. Based on its response to different solutes and changes in mobile-phase composition, PBD–ZrO2 is a true hydrocarbon-like RP material (33). When compared with ODS phases, PBD–ZrO2 exhibits very similar retention, selectivity, and efficiency with nonelectrolyte analytes (29). The major difference is that a slightly weaker (20–25% by volume, organic modifier) eluent should be used to achieve the same retention factors. In contrast, when anionic or cationic analytes are separated

on PBD–ZrO2, the retention order and band spacing usually differ significantly from ODS phases, especially when the eluent is buffered using hard Lewis bases such as phosphate, fluoride, or citrate as previously described. On zirconia phases, these bases adsorb to the surface and impart a high negative charge, thereby affecting the analyte’s interaction with the stationary phase. However, even in the presence of such interactions, good separations for most classes of analytes can be achieved—it is an excellent choice for amines. Figure 4 shows that although both the PBD–ZrO2 and the ODS–SiO2 phases separate a set of basic drugs, the peaks on the silica column are severely tailed. PEI-coated zirconia. Coating zirconia with cross-linked PEI results in a stable and useful anion-exchange phase (33). PEI (a weak anion exchanger) and quaternized PEI (a strong anion exchanger) coated on zirconia have been used for the separation of organic acids and various biomolecules, including nucleotides and proteins (26, 33). Compounds containing Lewis bases can be separated when a competing Lewis base is added to the eluent. The pH and temperature stability of PEI–ZrO2 exceed those of corresponding PEI–SiO2 materials (26). For example, PEI–ZrO2 has been used under alkaline conditions at 100 °C to separate nucleotides, with no indication of column degradation, whereas PEI–SiO2 materials failed after only 30 min. In anion-exchange chromatography, the analyte must be negatively charged to be re-

tained and separated; however, production of an anion from a weak acid can require an excessively high pH. Silica-based phases are more limited because of their instability above pH 7–8, although some of the newer specialized phases can withstand higher pH solutions. PEI–ZrO2 phases can easily handle high pH and successfully separate a broader range of compounds. Both PEI–ZrO2 and quaternized PEI–ZrO2 have similar, if not better, efficiencies than PEI–SiO2 columns and exhibit multimode selectivity (26). The major retention mechanism on zirconia-based phases is ion exchange with significant contributions from hydrophobic interactions on the polymer backbone, cross-linker, and ligand-exchange interactions. Because of this multimode retention mechanism, the selectivity of the PEI–ZrO2 columns is different from most other ion-exchange columns, thereby leading to some unique separations.

Adsorptively generated stationary phases Although the hard Lewis acid sites on zirconia’s surface will adsorb any strong, hard Lewis base in the eluent, which cause chromatographic problems for some types of analytes, we can also use this property to our advantage. Phosphate and fluoride were the first hard Lewis bases used to make novel stationary phases on bare zirconia and are used to block accessible Lewis acid sites on polymer-coated zirconia phases

Table 1. HPLC phases based on porous zirconia. Phase

Synthesis method

Retention mechanism

Typical conditions

Typical analytes

Refs.

Uncoated

—

Anion/cation/ligand exchange, normal phase

Lewis bases in buffered aqueous solution; nonpolar eluents

Benzoic acids, polar analytes

43

Polybutadiene (PBD–ZrO2)

Adsorptively coated, cross-linked

RP

Acetonitrile/water and buffer as needed

Organic amines, basic drugs, nonpolar analytes

12, 29, 34

Carbon (C–ZrO2)

Hydrocarbon vapors cracked RP on zirconia surface

Tetrahydrofuran/water

Positional isomers (e.g., xylenes), diastereomers, nonpolar analytes

28, 30, 40, 41, 42

Phosphate (PO4–ZrO2)

Chemisorbed by refluxing in phosphoric acid

Cation exchange

Aqueous, buffered phosphate solutions

Proteins

35, 36

PhosphonatedEDTA (PEDTA–ZrO2)

Reactively coated with phosphonated EDTA

Cation exchange

4 mM PEDTA, buffered with a sodium chloride gradient

Proteins

26, 39

Fluoride (F–ZrO2)

Chemisorbed in situ

Mixed mode ion/ligand exchange

100 mM sodium fluoride in buffered Proteins aqueous solution with a sodium sulfate gradient

37, 38

Polyethyleneimine (PEI–ZrO2)


Anion exchange

Sodium acetate buffer

Proteins, nucleotides, organic acids

33

Quaternized polyethyleneimine (QPEI–ZrO2)


Anion exchange/ hydrophobic

Buffered aqueous phosphate solution with sodium chloride

Proteins, inorganic and organic anions

26


605 A

(34, 35). The success of phosphate and fluoride phases led to the examination of other Lewis bases, including a phosphonate analogue of EDTA, which, while still an ion-exchange phase, has rather different selectivity than that of other conventional ion-exchange phases (36). Phosphate. Depending on the vigorousness of the treatment (pH, concentration, temperature, and time), the extent of the modification to zirconia by phosphate solutions can be controlled. A nearly permanent surface modification involves refluxing the particles in dilute phosphoric acid. This results in the conversion of several layers of zirconia to zirconium phosphate. This material is a cation exchanger, which has been used for separating cationic proteins and immunoglobulins from fermentation broths (37 ). The phosphate treatment completely counteracts the effect of the hard Lewis acid sites, allowing for good chromatographic separations of hard Lewis bases. This phase is also quite stable. No change in retention or selectivity is observed up to pH 10. However, above this pH and in the absence of phosphate in the eluent, the surface phosphate groups begin to desorb, changing the properties of the material. This can be partly reversed by flushing the column with a phosphate solution at elevated temperatures. Fluoride. At first glance, the fluoride–zirconia (F–ZrO2) phase appears to be similar to the zirconia–phosphate phase, but F–ZrO2 behaves very differently. First, preparation of F–ZrO2 simply requires flushing a solution of fluoride ion through the column (38). Because the phase possesses unique selectivity compared with other zirconia-based ion-exchange phases, it is excellent for protein separation and can act as a ligand-exchange (or metal affinity) material analogous to calcium hydroxyapatite (39). If retention of proteins decreases, the column is regenerated by flushing the column with sodium hydroxide solution to remove any strongly adsorbed materials and followed by re-equilibrating the column with a fluoride buffer. This phase cannot be used at low pH because of the formation of hydrofluoric acid, which is detrimental to the instrument and the detector’s windows. Phosphonate–EDTA analogues. The usefulness of the phosphate and fluoride phases led us to look for other hard Lewis bases that might provide different selectivities. The treatment of zirconia with a phosphonate analogue of EDTA, ethylenediammine-N,N´-tetramethylphosphonic acid (EDTPA),

resulted in a very useful stationary phase (27). Refluxing the zirconia particles in a solution of EDTPA gives a cation-exchange material (PEDTA–ZrO2) that has unique selectivity. The strong Lewis acid sites on the zirconia surface are effectively blocked, producing a biocompatible stationary phase, which allows reversible desorption of bioactive proteins. Because of the presence of the aliphatic segment in EDTPA, PEDTA–ZrO2 is able to separate proteins and other solutes that phosphate–zirconia cannot. This phase has been successfully used to achieve highly purified monoclonal antibodies from a cell culture supernatant with excellent recovery of biological activity (39).

Carbon-clad zirconia phases Elemental carbon can be deposited on zirconia by chemical vapor deposition. The underlying zirconia network supports the carbon layer, making it mechanically stable and giving it a major advantage over other carbon stationary phases. As with all carbon phases, C–ZrO2 has distinctive chromatographic selectivity. Although C–ZrO2 behaves as an RP material, it differs greatly from typical RP materials, such as ODS and PBD–ZrO2, in its separation mechanism. RPs typically separate on the basis of polarity, shape, and size of the solute, while carbon phases separate on the basis of these properties plus ␲–␲ and dipole-induced dipole interactions, which are most evident in the extraordinarily high retention exhibited by multiplanar aromatic ring compounds (40, 41). A strong effect from analyte dipole–phase-induced dipole interactions results in quite different selectivities for singlering compounds. For instance, on conventional RPs, nitrobenzene is retained less than toluene, because it is polar and more water-soluble. However, on any type of carbon phases, nitrobenzene is retained more than toluene. This set of interactions, coupled with the fact that the solid carbon-like surface is not conformationally labile, as are the alkyl chains of bonded phases, enables many interesting separations. For instance, on a typical ODS phase, cis-stilbene and trans-stilbene could not be separated (resolution = 0.15). However, on a carbon phase, these isomers were easily separated (resolution = 6.75) (26). Similarly, the separation of p-xylene and ethylbenzene or diastereomers is quite difficult on conventional RPs but easy on ODS or PBD–ZrO2 phases. The extra ␲–␲ interactions and the geometry of the surface make just enough difference in the energy of interaction to enable

Polar and nonpolar structural isomers can easily be separated on C–ZrO2.

606 A


these separations. Structural isomers, polar as well as nonpolar, such as o-, m-, and p-nitroxylenes (Figure 5a), can also be easily separated on the C–ZrO2 material while such separations are difficult on an ODS–SiO2 phase (Figure 5b) (42).

A look forward Although zirconia-based stationary phases have many advantages over other HPLC stationary phases in terms of thermal and chemical stability and flexibility with regard to pH, buffers, and high temperatures without sacrificing efficiency, they are far from problem-free. The strong interactions between surface sites and certain solutes such as proteins still need to be addressed. New phases with novel selectivities have been developed on the basis of interactions provided by these sites. The development of new phases based on the current set of materials and further investigation into the long-term reproducibility of zirconia-based materials are under way. The extraordinary thermal and chemical stability of these materials opens the way to many new ideas in chromatography, including the use of 100% water eluents in RPLC, which enables the use of flame ionization detectors, new techniques such as T3C, and the ability to manipulate selectivity and peak shape by deprotonating any amine.

(10) (11) (12) (13) (14)

(15) (16) (17) (18) (19) (20) (21) (22)

Much of this work was supported by grants from the National Institutes of Health and the National Science Foundation.

Dunlap is an assistant professor at Saint Mary’s College, and is interested in chromatographic stationary phases and environmental separations. McNeff is vice-president for research at ZirChrom and is interested in application of ultra-stable stationary phases. Stoll is a senior research associate at ZirChrom and focuses on bioanalytical separations. Carr is a professor at the University of Minnesota–Twin Cities. His research focuses on the development of ultra-stable HPLC phases, theory of chromatography, and bioanalytical chemistry. Address correspondence to Dunlap at Department of Chemistry and Physics, Saint Mary’s College, Notre Dame, IN 46656 or [email protected].

(23) (24) (25) (26) (27) (28) (29) (30) (31) (32)

References (1) (2) (3) (4) (5) (6) (7) (8) (9)

Majors, R. E. LC–GC 2001, 19, 272. Kirkland, J. J.; Adams, J. B.; van Straten, M. A.; Claessens, H. A. Anal. Chem. 1998, 70, 4344. Wirth, M. J.; Fatunmbi, H. O. Anal. Chem. 1993, 65, 822. Nawrocki, J. J. Chromatogr., A 1997, 779, 29. McCalley, D. V. LC–GC Europe 1999, 10, 638. Barder, T. J.; Wohlman, P. J.; Thrall, C.; DuBois, P. D. LC–GC 1997, 15, 918. Benson, J. R.; Woo, D. J. J. Chromatogr. Sci. 1984, 22, 386. Haky, J. E.; A. Raghami; Dunn, B. M. J. Chromatogr. 1991, 541, 303. Trüdinger, U.; Müller, G.; Unger, K. K. J. Chromatogr. 1990,

(33) (34) (35) (36) (37) (38) (39) (40) (41)

535, 111. Stout, R. W.; DeStefano, J. J. J. Chromatogr. 1985, 326, 63. Bien-Vogelsang, U.; Deege, A.; Figge, H.; Kohler, J.; Schomburg, G. Chromatographia 1984, 19, 170. Rigney, M. P.; Funkenbush E. F.; Carr, P. W. J. Chromatogr. 1990, 499, 291. Sun, L.; Annen, M. J.; Lorenzano-Porras, F.; Carr, P. W.; McCormick, A. V. J. Colloid Interface Sci. 1994, 163, 464. Unger, K. K.; Trudinger, U. In High Performance Liquid Chromatography; Brown, P. R., Hartwick, R. A., Eds.; Wiley & Sons: New York, 1989; p 145. Anita, F. D.; Horvath, C. J. Chromatogr. 1988, 435, 1. Yen, B.; Zhao, J.; Brown, J. S.; Carr, P. W. Anal. Chem. 2000, 72, 302. Snyder, L. R. J. Chromatogr., B 1997, 689, 105. Smith, R. M.; Burgess, R. J. J. Chromatogr., A 1997, 785, 49. Mao, Y.; Carr, P. W. Anal. Chem. 2000, 72, 4413. Baes, C. F.; Mesmer, R. E. The Hydrolysis of Cations; Wiley & Sons: New York, 1976. Nawrocki, J.; Rigney, M. P.; McCormick, A.; Carr, P. W. J. Chromatogr., A 1993, 657, 229. Kirkland, J. J.; Henderson, J. W.; DeStefano, J. J.; van Straten, M. A.; Claessens, H. A. J. Chromatogr., A 1997, 762, 97. Kirkland, J. J.; van Straten, M. A.; Claessens, H. A. J. Chromatogr., A 1998, 797, 111. Claessens, H. A.; van Straten, M. A.; Kirkland, J. J. J. Chromatogr., A 1996, 728, 259. Law, B.; Chan, P. F. J. Chromatogr., A 1989, 467, 267. McNeff, C.; Carr, P. W. Anal. Chem. 1995, 67, 2350. Clausen, A. M.; Carr, P. W. Anal. Chem. 1998, 70, 378. Weber, T. P.; Jackson, P. T.; Carr, P. W. Anal. Chem. 1995, 67, 3042. Li, J.; Hu, Y.; Carr, P. W. Anal. Chem. 1997, 69, 3884. Yu, J.; El Rassi, Z. J. Chromatogr., A 1993, 631, 91. Rigney, M.P. Ph.D. Thesis, University of Minnesota, Minneapolis, 1989. McNeff, C.; Zhao, Q.; Carr, P. W. J. Chromatogr., A 1994, 684, 201. Li, J.; Carr, P. W. Anal. Chim. Acta 1996, 334, 239. Sun, L.; Carr, P. W. Anal. Chem. 1995, 67, 2517. Schafer, W. A.; Carr, P. W.; Funkenbusch, E. F.; Parson, K. A. J. Chromatogr., A 1991, 587, 137–147. Clausen, A. M.; Subramanian, A.; Carr, P. W. J. Chromatogr., A 1999, 831, 63. Schafer, W. A.; Carr, P. W. J. Chromatogr., A 1991, 587, 149. Blackwell, J. A.; Carr, P. W. J. Chromatogr., A 1991, 549, 43. Blackwell, J. A.; Carr, P. W. J. Chromatogr., A 1991, 549, 59. Jackson, P. T.; Carr, P. W. CHEMTECH 1998, 28, 29. Jackson, P. T.; Schure, M. R.; Weber, T. P.; Carr, P. W. Anal. Chem. 1997, 69, 416.


607 A

Peer Reviewed: Zirconia Stationary Phases for Extreme Separations

Recommend Documents