Polar-Modified Stationary Phases: An Ideal Choice for the Analysis of Nucleotides

Ritu Arora, Eugene Chang, Anita DerMartirosian, and Linda Lloyd, Varian, Inc.

Out of the several options available for retaining polar compounds like nucleotides, this application note focuses on the use of polar-modified C18 bonded phases for their analysis.

There are several options available for the analysis of hydrophilic compounds like nucleotides. Due to the presence of phosphate groups in their structure, they can be separated by anion exchange chromatography or more routinely, analyzed by reversed phase chromatography. In the latter mode, they could be retained on conventional alkyl bonded phases with the aid of ion-pairing agents or mixed-mode phases like polar-modified chemistries and fluorinated phenyl phases.

Results and Discussions

This paper discusses the advantages and disadvantages of the various choices available to the end-user trying to select the best solution for this class of compounds. Table I lists the strengths and weaknesses of those choices for the analysis of nucleotides.

Table I: Strengths and weaknesses of various bonded phases for nucleotide analysis

1. Reversed Phase

a. Polaris C18-A

Mixed-mode polar-modified stationary phases like 200 Å silica-based Polaris C18-A, in general, can help retain polar analytes under highly aqueous conditions. The polar functionality allows increased resistance to hydrophobic collapse under these conditions. Figure 1 shows the separation of ten deoxyribonucleotides on this column. The gradient starts with a 95% aqueous eluent, indicating a high aqueous environment, which is compatible with the design of the chemistry, such that minimal reduction in retention times can be expected over a period of time. Despite the presence of the polar group, ion-pairing through the use of tetrabutyl ammonium hydroxide was used to create a more hydrophobic complex of each analyte, which would retain on the column.

Figure 1

b. Pursuit PFP

Pursuit PFP is another mixed-mode 200 Å reversed phase suitable for polar compound retention under pure aqueous / high aqueous eluents without being susceptible to phase collapse. By incorporating dipole–dipole as well as pi–pi and hydrogen bonding mechanisms, this pentaflourophenyl phase delivers optimal separation of polar analytes. Figure 2 is an example of seven nucleotides/nucleosides run on this column using 100% aqueous MS-compatible conditions.

Figure 2

Since the PFP ligand involved in this bonded phase is less dense when compared to the corresponding C18 phase (both 200 Å products), resolution of complex mixtures is not as great as that seen on Polaris C18-A.

Figure 3

c. Pursuit XRs Ultra 2.8 C18

Nucleotides can also be analyzed through conventional alkyl bonded chemistries using ion-pairing agents. Figure 3 illustrates a high speed separation of a mix of adenine nucleotides within 3 min on a new line of "Fast LC" columns. Table II provides details of MS transitions used for each analyte. While these are suitable for high speed, high-resolution separations, pure aqueous or high aqueous eluents cannot be used on these columns without the possibility of a phase collapse. Thus, traditional C18 and C8 bonded phases are not very popular for the analysis of polar analytes.

Table II: MS-MS details of adenine nucleotides

2. Anion exchange

By using a hydrophilic, strong anion-exchange resin-based packing material like PL-SAX 8 μm 1000 Å, a mix of 12 standard mono-, di- and triphosphate nucleotides can be resolved in under 20 min using a salt and pH gradient (Figure 4).

Figure 4

Due to the high concentration of salts used in ion-exchange methods, instrument lifetime may be affected as a result of salt precipitation. Besides, involatile salts may not be compatible with certain detectors, like ELSD or MS.

Conclusions

Polar-modified bonded phases such as Polaris C18-A serve as ideal candidates for their analysis due to their improved water wettability allowing usage of high aqueous eluents, increased resistance to phase collapse, and improved resolution of fairly complex mixtures.

Varian, Inc.

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