HPLC Analysis of Very Polar Compounds in Bioanalysis

Feb 01, 2014
By LCGC Editors
Volume 27, Issue 2

Several high performance liquid chromatography (HPLC) approaches can be used in the analysis of polar small molecules that are classified as pharmaceuticals, metabolites, or biomarkers. Many of these polar compounds can be challenging for traditional reversed-phase separation methods. One approach for analyzing these types of samples is the use of columns having polar retention capabilities such as ion-exchange, hydrophilic interaction liquid chromatography (HILIC), polar-embedded, or polar-endcapped columns. Another approach is aqueous normal phase (ANP) chromatography. This mode has the advantage of having both reversed-phase and normal-phase retention. In a recent web seminar, Joseph Pesek, a professor of Chemistry at San José State University (San José, California, USA), explained the mechanisms, advantages, and disadvantages of these approaches to the analysis of polar compounds. Below, he answers questions raised during the web seminar.

Column Types and Their Properties

If hydrophilic interaction liquid chromatography (HILIC) works now, why did it not work 30 years ago?

Pesek: HILIC was not well understood 30 years ago. However, some of the shortcomings of HILIC were noted at that time and for that reason it was not pursued vigorously.

How is HILIC different from working with plain silica columns?

Pesek: Silica is just one column material used in HILIC. There are many others, each having somewhat different properties. Therefore, as in reversed-phase chromatography, in HILIC it is important to select the right type of column to match the properties of the analyte.

What is the difference between polar-endcapped and HILIC columns?

Pesek: Polar-endcapped columns are similar to traditional reversed-phase columns but instead of using a nonpolar endcapping reagent like a trimethyl group, a more polar group is used to cover some of the remaining silanols. HILIC columns generally are significantly more polar since they are based on bare silica or have bonded polar groups that cover a significant fraction of all of the available bonding sites.

Is silica really a HILIC stationary phase? Several studies suggest that it is very different from bonded phases.

Pesek: It is if you believe that the mechanism for HILIC retention is the presence of an adsorbed water layer on the surface that serves as a medium of analyte partition. It may be different from bonded phases but for most of these, the presence of a layer of water at or near the surface is used to describe retention.

What is your experience with the robustness of most HILIC columns?

Pesek: Most HILIC columns are less robust than traditional reversed-phase columns. This results in part from the fact that the surface can be easily contaminated and is hard to clean. In other cases, the bonded group is not very robust and is susceptible to being cleaved from the surface in mobile phases used for HILIC retention.

Are HILIC or similar columns compatible with ultrahigh-pressure liquid chromatography (UHPLC)?

Pesek: All of the columns discussed in the seminar, including aqueous normal phase (ANP) and HILIC columns, are compatible with UHPLC.

Can I use silica hydride columns for mixed analysis of polar and nonpolar compounds or amphiphilic compounds? If so, is there a possibility that the retention times of polar and nonpolar compounds might be the same (that is, their respective affinities are equal), thus yielding the same retention times?

Pesek: This is relatively unlikely because that would require that both mechanisms be operating at equal efficiency for those particular compounds. In most cases one mechanism would be more predominant than the other and retention would be different. However, even in the unlikely event that both were equal, that would only occur at one mobile-phase composition. Therefore, you could switch to a different isocratic composition or a different gradient and that would shift the relative contributions of the two mechanisms, allowing separation.

Why are there different bonded phases using silica hydride if it is possible to retain both polar and nonpolar analytes on any silica hydride column?

Pesek: The degree of retention in reversed-phase and aqueous normal phase modes on silica hydride columns depends on the degree and type of modification. For columns with no or minimal modification the ANP mode is stronger than the reversed-phase mode. As the degree of modification becomes greater — that is, in columns modified with larger groups or that have a greater degree of surface coverage — the reversed-phase properties increase.

What type of columns can be used to separate both polar and nonpolar compounds?

Pesek: The most versatile columns for this type of separation are silica hydride columns because the amount of retention in both modes can be adjusted by the type of modification and the mobile-phase composition. Polar-endcapped and polar-embedded columns have this same capability because they are also classified as mixed-mode stationary phases. The degree of polar retention depends on the endcapping group or the embedded group.

What type of gradient do you suggest for ANP analysis of hydrophilic and hydrophobic molecules in the same run? I have a large hydrophobic active and a prodrug, as well as five small polar metabolites.

Pesek: The usefulness of ANP separations is that you have two options in selecting the gradient. It would be best to try both a reversed-phase gradient (from 80% to 20% aqueous) and then an ANP gradient (from 20% to 80% aqueous) as an initial screening process. From the preliminary results you can determine which mode is most likely to produce the best separation. Once the direction has been selected, then the gradient can be modified to reach the desired retention and separation of the components in your mixture.

How much efficiency do ANP columns have? Does efficiency have detrimental effects for biological matrices for liquid chromatography–mass spectrometry (LC–MS) analysis?

Pesek: Normal-phase retention generally has lower efficiency than reversed-phased methods. It is highly dependent on the compound being retained. Efficiency doesn't have detrimental effects for biological matrices for LC–MS analysis, but sometimes lower ionization of the compounds is observed in MS detector when compounds are being analyzed in biological matrices. The use of internal standards (usually deuterated compounds) will help to assess how much lower the peaks are.

Are there other bonded phases for ANP chromatography?

Pesek: Yes. As stated in the seminar, polar-embedded, polar-endcapped, and fluorinated phases have some ANP capabilities. Also, graphitized carbon phases have demonstrated some ANP behaviour.

Can you tell us about carbon columns?

Pesek: Carbon columns, or porous graphitized carbon columns, also possess dual retention capabilities. Their structure is porous particles composed of flat sheets of hexagonal carbon. Selectivity in the reversed-phase mode is somewhat different than with traditional C18 columns. The absorptive nature of the carbon surface results in the polar retention observed.

What manufacturers make low-carbon silica hybride columns?

Pesek: The current suppliers of low-carbon silica hydride carbons are Microsolv Technology and VWR in the United States and Hichrom Ltd. in Europe and the UK.

How stable or durable are silica hydride columns?

Pesek: Silica hydride columns are just as durable, and in some cases more durable, than traditional chemically modified stationary phases based on organosilane chemistry. This is due to the direct silicon-carbon at the surface. For columns that are used primarily for polar retention in the ANP mode, these materials are more durable than typical HILIC columns because the hydride surface is not as easily contaminated as ordinary silica and the surface is less susceptible to attack by aggressive mobile phases because of its more hydrophobic nature.

Can you compare silica hydride and ionic liquids? Are there any important advantages of one over the other for retaining polar analytes?

Pesek: It is assumed you are referring to ionic liquid stationary phases. All of these materials contain a charged group that is part of the bonded material. The charged site is usually within the bonded chain, but not always. It is most similar to the polar-embedded phases discussed in the presentation. They have been shown to retain both polar and nonpolar compounds. To date, the range of compounds analyzed with ionic liquid stationary phases is not as broad as with silica hydride phases so the same versatility has not been demonstrated. A good review of ionic liquids in chromatography can be found in the book by Mun and Sim (1).

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