Columns for Reversed-Phase LC Separations in Highly ...

1 COLUMN WATCH. Columns for Reversed-Phase LC Separations in Highly Aqueous Mobile phases Ronald E. Majors, Agilent Technologies, Wilmington, Delaware, USA, and Matthew Przybyciel, ES Industries, West Berlin, New Jersey, USA. October's Column Watch discussed the phenomenon of phase collapse and ways to solve the problem and successfully separate polar analytes using mobile-phase systems with a very high percentage of water. In this month's column, Majors and Przybyciel describe Columns that can deal with this difficult situation and suggest some commercial Columns for separating polar compounds in Highly aqueous environments.

2 Reversed-Phase chromatography is by far compounds can frequently interact with of silanol groups, and more-reproducible the most widely used mode in high unreacted silanols on the silica gel because and denser bonding of alkyl chains. Most performance liquid chromatography it is virtually impossible, at least with silica-based Columns today are built on (HPLC).1 It allows chromatographers to monomeric bonding, to remove or cover all Type B base materials and the most manipulate the mobile phase by changing of the silanols because of steric reasons, popular phases of those are based on C8.

3 Organic solvent type, solvent composition especially with long alkyl chain phases such and C18 alkylsilane chemistries. When and pH; by adding modifiers such as as octadecylsilane (C18). This interaction is separating basic compounds, the base- surfactants, chiral reagents, competing most problematic when the packing is used deactivated phases are also popular and bases and ion-pair reagents; or by at intermediate pH values of pH 4 8 at many of them are available on the adjusting experimental conditions such as which silanols and many basic compounds flow-rate and temperature. Originally, are partially ionized.

4 Researchers believed that they would need Twenty years ago, the low-purity silica Conventional Reversed-Phase only a few stationary phases to achieve gel used for most HPLC Columns caused Chromatography in Highly virtually any separation they encountered. high surface acidity and a tailing problem Aqueous Mobile phases Indeed with a C18 column and the ability with basic compounds. The first modified Even with these improved alkylsilane to adjust the myriad parameters, stationary phases were the so-called base- surfaces, certain experimental conditions chromatographers have achieved many deactivated bonded silicas.

5 These base- can cause separation or stationary-phase successful Separations . Still, sometimes deactivated phases were not deactivated problems. For example, both very high pH. analysts can neither find the necessary with base, but the term implied that the (higher than pH 10) and low pH (lower selectivity nor obtain a rugged and column had been treated to provide than pH 2) conditions can present reproducible separation easily, no matter minimal interaction and tailing with problems with stationary-phase and how many parameters they adjust. strongly basic compounds. Among the column For very polar analytes, treatments were acid washing of the silica other problems can arise.

6 For solubility Early Modified Surfaces in before bonding, single- or multiple- reasons, many polar compounds prefer a Reversed-Phase Chromatography reactant endcapping, covering or Highly aqueous mobile phase and can be New and modified Reversed-Phase encapsulating the surface with polymeric retained only with a minimal concentration chromatography stationary phases have phase and other unspecified proprietary of organic modifier, sometimes less than 5%. been introduced throughout the years to methods. Later, researchers developed In October's Column Watch, we provide more separation power.

7 In Type B silicas, which had less acidic demonstrated and discussed the Reversed-Phase chromatography, basic surfaces, a more homogeneous distribution phenomenon of phase collapse and 2 LC GC Europe December 2002. Column Watch mentioned approaches to solve the Most silica-based Columns today are built on Type B base problem and successfully separate polar materials and the most popular phases of those are based analytes using mobile-phase systems with a very high percentage of water, even as on C8 and C18 alkylsilane chemistries. great as 100% In this month's column, we will present ways to deal with can be useful but can sometimes be and the predominant separation this difficult situation.

8 In addition, we will detrimental. Their utility comes when the mechanisms are adsorption (not suggest typical commercial Columns that alkyl function alone provides insufficient partitioning) onto the alkyl phase and the chromatographers can use to separate separation selectivity and the presence of polar interactions of the analyte with polar compounds in Highly aqueous silanols can cause polar interactions with silanols. The phase-bonding densities are environments. polar functionality on analytes. The lower for chains longer than C4 and they Several aspects of the design of resulting mixed mechanisms can yield have a greater degree of conformational stationary phases can help retain polar improved Separations , but the packings freedom.

9 Partitioning (not adsorption) into analytes under Highly aqueous conditions, might be difficult to reproduce batch-to- the alkyl chains can occur if the chains are including batch unless the ligand-to-silanol ratio is Highly solvated and extend away from the non-endcapped, short-chain alkyl phases exactly the same. surface. When the amount of organic hydrophilic, polar-endcapped, and polar- Surface silanol groups also interact with solvent is decreased to less than 5 10%. enhanced stationary phases water (hydrogen bonding), so the available with some of these long-chain packings, polar-embedded alkyl phases alkyl surface is determined by the high the phase solvation decreases and the long-chain alkyl phases density of the bonded phase in some phases tend to collapse and cause the wide-pore-diameter phases .

10 Instances, especially for short-chain alkyl problems discussed in last month's phases (smaller than C4). In those Non-endcapped, Short-Chain situations, little free volume exists between On the detrimental side, these unreacted Alkyl phases bonded chains and little shielding of the silanols can ionize at intermediate-to-high When unreacted surface silanol groups are silanols occurs. As Kasakevich and pH values (pH 5 and greater) and can present on an alkyl bonded silica gel, they co-workers5 pointed out, these short-chain undergo ion-exchange interactions that impart a degree of polarity that sometimes phases present little risk for phase collapse, cause severe tailing for basic compounds Table 1: Examples of Reversed-Phase chromatography Columns for use in Highly aqueous mobile phases .