Nucleic acids, HPLC analysis

Sawai, H., Analysis and purification of synthetic large oligodeoxyribonucleotides by HPLC on RPC-5 like resin, Nucleic Acids Res., 17, 113, 1986. 

HPLC is frequently employed in the analysis of amino acids, peptides, proteins, nucleic acids, and nucleotides. HPLC is also often used to analyze for drugs in biological samples (see Workplace Scene 16.2). Due to the complex nature of the molecules to be analyzed, these techniques tend to be more complex than HPLC applications in other areas of analytical chemistry. For example, separation of nucleotides or amino acids is more difficult than testing for caffeine in beverages, even though the same instrument and same general methods would be employed. A variety of columns and mobile phases are regularly employed. 

Most HPLC instruments monitor sample elution via ultraviolet (UV) light absorption, so the technique is most useful for molecules that absorb UV. Pure amino acids generally do not absorb UV therefore, they normally must be chemically derivatized (structurally altered) before HPLC analysis is possible. The need to derivatize increases the complexity of the methods. Examples of derivatizing agents include o-phthaldehyde, dansyl chloride, and phenylisothiocyanate. Peptides, proteins, amino acids cleaved from polypeptide chains, nucleotides, and nucleic acid fragments all absorb UV, so derivatization is not required for these molecules. 

As far as quantitative chemical derivatization GC analysis is concerned, it is necessary to mention especially the work of Gehrke and his collaborators, who specified the fundamental concepts of quantitative GC analysis combined with the chemical derivatization of sample compounds and applied them to the accurate determination of the twenty natural protein amino acids and other non-protein amino acids as their N-TFA-n-butyl esters [5 ], the urinary excretion level of methylated nucleic acid bases as their TMS derivatives [6], TMS nucleosides [7] and other investigations. Further examples include a computer program for processing the quantitative GC data obtained for seventeen triglyceride fatty acids after their transesterification by 2 NKOH in n-butanol [8], a study of the kinetics of the transesterification reactions of dimethyl terephthalate with ethylene glycol [9] and the GC-MS determination of chlorophenols in spent bleach liquors after isolation of the chlorophenols by a multi-step extraction, purification of the final extract by HPLC and derivatization with diazoethane [10]. 

Improvements in column technology, detector sensitivity and the development of new detection systems, have made possible the routine separation of picomole quantities of nucleic acid components in complex physiological matrices. The very sensitivity of most LC systems, however, which is invaluable in the analysis of biological samples, is often the limiting factor because of inadequate or ambiguous identification methods. Although tremendous advances have been made in the on-line combination of HPLC with spectroscopic techniques [e.g., mass spectrometry, Fourier transform infrared (FT/IR), nuclear magnetic resonance], their application has not become routine in most biochemical and biomedical laboratories. 

HPLC in Nucleic Acid Research Methods and Applications, edited by Phyllis R. Brown 29. Pyrolysis and GC in Polymer Analysis, edited by S. A. Liebman andE. J. Levy 30. Modem Chromatographic Analysis of the Vitamins, edited by Andre P. De Leenheer, Willy E. 

Ion-exchange chromatography (lEC) is one of the oldest HPLC modes. Today it is used for separations of small inorganic ions or of ionic biopolymers such as oligonucleotides, nucleic acids, peptides and proteins rather than in the analysis of ionic small-molecule organics, for which RPC or IPC usually offer higher efficiency and better control of selectivity and resolution. 

Newer technologies that replace assays traditionally performed by electrophoresis are emerging, some of which are attractive alternatives for the clinical laboratory as they achieve analysis of nucleic acids with less hands-on time and with far greater throughput because of automation. These include pyrosequencing, mass spectrometry, and HPLC. 

Quantitative analysis of the oxidative DNA lesion, 2,2-diamino-4-[(-2-deoxy-P-D-erythro-pentofuranosyl) amino]-5 (2 H)-oxazolone (oxazolone) in vitro and in vivo by isotope dilutioncapillary HPLC-ESI-MS/MS. Nucleic Acids Res., 34, 5449-5460. 

LC-separation of low molecular-weight constituents of nucleic acids and intact nucleic acids was reviewed by Zadrazil [358,359], Brown [360] described an enzyme peak shift method verifying peak identities of nucleotides, Singhal [361] reviewed separation and analysis of nucleic acids and their constituents by ion-exclusion and ion exchange column chromatography, and Brown [31,362] summarized the latest developments and state-of-art in HPLC of nucleic acid constituents. Plunkett [363] dealt with the use of HPLC in research of purine nucleoside analogs. 

A series of reviews has been published on the analysis of oligonucleotides using CE [183-185]. A comprehensive review on separation of mono, oligo- and polymeric nucleic acids by this method has also been published [186], In contrast to RP-HPLC and ion-exchange chromatography, CE - which has been established as the third technique of choice -is a purely analytical method [187]. Ion-exchange HPLC fails particularly with SODNs [185]. RP-HPLC does not at present allow baseline separation of SODNs, which differ by only one base pair [188]. Thus, control of preparative purifications by RP-HPLC has become an important application of CGE. Table 11 illustrates the resolution efficiency of CGE when compared with other methods. 

The HPLC analysis of nucleic acid components can be divided into sections according to the chemical nature of the components. For... 

Gelfi, C., et al.. Analysis of antisense oligonucleotides by capillary electrophoresis, gel-slab electrophoresis, and HPLC a comptaison. Antisense Nucleic Acid Drug Dev, 6, 47, 1996. 

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