Separation of nucleotides

In relation to separation of nucleotides, Hoffman61 found that adenine nucleotides interacted most strongly with cycloheptaamylose, presumably by inclusion of the base within the cavity of cyclodextrin. When epichlorohydrin-cross-linked cycloheptaamylose gel was used as a stationary phase for nucleic acid chromatography, adenine-containing compounds were retarded most strongly. 

Horvath, C. G., Preiss, B. A., and Lipsky, S. R., Fast liquid chromatography an investigation of operating parameters and the separation of nucleotides on pellicular ion exchangers, Anal Chem., 39, 1422, 1967. 

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. 

Zorbax ODSS (10 pm), Nucleosil ODSS (5 pm) Octadecylsulfonated silica(ODSS) Separation of nucleotides, dinucleotides, transfer-ribonucleic acids MeCN-aqueous phosphate 35 65 v/v, 9.75 mM phosphate, 3.25 mM tetrabutyl ammonium bromide, pH 6.50 MeCN-MeOH-12mM ammonium phosphate30 40 30, v/v/v, pH 6.0 [93]... 

Recently, the principal applicability of CEC to the separation of nucleotides trans-fer-RNAs has been demonstrated using an octadecyl-sulfonated mixed mode silica stationary phase. Although UV absorbance was used for detection of the analytes, it can be expected that such a separation system will be coupled to ESI-MS in the near future. Presently, our laboratory is working on the development of monolithic capillary columns for ion-pair reversed-phase CEC—ESI-MS of nucleic acids. 

Hoffman, N.E. and Liao, J.C. Reversed phase high performance liquid chromatographic separations of nucleotides in the presence of solvophobic ions. Anal. Chem, 1977, 49, 2231-2234. 

Fluorous reverse phase silica gel (FRPSG) has been used in the purification of synthetic DNA fragments.In solid phase DNA synthesis, truncated sequences are often separated from the desired product after deprotection using HPLC or electrophoresis. In order to perform, parallel syntheses and separations of nucleotides the trityl-on purification procedure was developed, in which a lipophilic support material is used to separate the desired and undesired product, followed by deprotection. If the protecting group is labelled with a fluorous group, fiuorous-fiuorous interactions between the FRPSG and the protected nucleotide can be used to aid separation of the aqueous mixture. 

Nucleosides can be analysed by conventional reversed-phase (RP) LC with a buffered mobile phase. The separation of nucleotides is somewhat complicated by the dissociation of the phosphate groups and HINa exchange at these sites. As indicated for the phosphorylated anabolites of nucleoside reverse transcriptase inhibitors (NRTI, Ch. 13.2.4), ion-pair RPLC using 7V,7V-dimethylhexylamine (DMHA) can be applied to both reduce adduct formation and obtain sufficient retention [6-7]. Alternatively, enzymatic dephosphorylation of the nucleotides prior to LC-MS analysis can be performed. 

A major step in the miniaturization of HPLC columns was done early in 1967 by Horvath and co-workers [1,2], when investigating the parameters that influence the separation of nucleotides in a 1-mm-i.d. column. These columns were then named microbore columns. A further step in the miniaturization process was done in 1973, by Ishii and co-workers, by separating polynuclear aromatic hydrocarbons (PAHs) in a... 

Work combining MS and degradation data have also been presented. - Oxidative elimination of the polynucleotide with periodic acid cleaves the 3 -connected nucleotide chain. The terminal base thus liberated is then analyzed, and the process is repeated until the end of the sequence. Another report combined MS with enzymatic digestion data, in particular, the selective cleavage by venom phosphoesterase of the oligonucleotide chain from the 3 end. The separation of nucleotides on a Dowex column and subsequent derivatization leads to the determination of both 5 and 3 terminal nucleotides according to Scheme 1. 

Fig. 3-94. Separation of nucleotide monophosphates. - Separator column IonPac AS4A eluent 0.015 mol/L NaH2P04, pH 3.4 with H3PO4 flow rate 1.5 mL/min detection UV (254 nm) injection volume 50 pL solute concentrations 10 ppm each of CMP, UMP, AMP, and GMP.

T.l.c.— The separation of phosphonic acid derivatives has been studied. Reports on the application of t.l.c. to the analysis and separation of nucleotides and other biologically important phosphates abound. Enzymatic reagents have been used to develop chromatograms of phosphate esters which inhibit cholinesterase. Clean, sharp-edged spots against a dark background are obtained. ... 

Figure 12.8 Separation of nucleotides and related compounds by anion exchange [reproduced with permission of Vieweg Publishing from D. Perrett, Chromatographia, 16, 211 (1982)]. Conditions stationary phase, APS-Hypersil, 5pm mobile phase. A-0.04M KH2PO4 (pH 2.9), B-0.5 M KH2PO4 + O.8M KCI (pH 2.9), linear gradient from A to B in 13 min UV detector, 254 nm.

Figure 6.14. Separation of nucleotides. Conditions Hamilton PRP-XlOO column 150 x 4.1 mm A 25 mM citric acid pH 5.4 B 173 25 mM citric acid acetoni-trile, linear gradient 0 to 50% B from 0 to 10 min (Courtesy Hamilton Co)...

Both inorganic and organic anions may be determined by anion chromatography with direct UV detection. Figure 6.14 shows a separation of nucleotides with 25 mM citric acid buffered at pH 5.4 as the eluent. A gradient elution with increasing amounts of acetonitrile was used to speed up elution of the later peaks. 

Macroporous silica gel with poly(9-vinyladenine) either coated or immobilized on the surface has been reported to give practically useful h.p.l.c. columns for the separation of nucleotides, displaying base-selective recognition ability.333... 

This is a CE analog of conventional zone gel electrophoresis for the separation of macromolecules based on size. The capillary is filled with a porous polymer gel, and molecular sieving occurs as the molecules move through the gel, that is, separation is based on both electrophoretic mobility and molecular size. Very high resolution is achieved. The trend is to fill the capillary with a liquid gel matrix (pumpable gel solutions, such as deriyatized celluloses dissolved in the run buffer). This allows replacement of the gel in the capillary to eliminate contamination problems from the sample matrix that occurs with fixed gels.. This technique is widely used for separation of nucleotides in deoxyribonucleic acid (DNA) sequencing (Chapter 25). 

FiyJSl Gel electrophoresis separation of nucleotides. Each vertical lane represents a different sample. In each lane, bases are eluted in order of size, the smaller ones traveling the fastest. Each band has one of four base colors, which identifies the end base on the oligonucleotide corresponding to that band. (Courtesy of University of Washington Genome Center.) (See your CD, auxiliary data, for a colored picture of the lanes, showing the four different nucleotide colors.)... 

Folley, L.S., Power, S.D. and Poyton, R.O. (1983) Separation of nucleotides by ion-pair, reversed-phase high-performance liquid chromatography use of Mg(ll) and triethylamine as competing hetaerons in the separation of adenine and guanine derivatives, jouma/ of Chromatography 28i, 1 99-207. 

A comparison between TLC and PC for the separation of nucleotides is given in Figure 21.12. A one-dimensional development using identical conditions shows the superiority of cellulose-layer TLC over PC for separating various mixtures (a different mixture for each vertical column of spots). Note that, in the same development distance, the isomeric T- and 3 -nucleotides (spots 1 to 4) were only partially resolved by PC but were fully resolved by TLC, whereas spots 7 to 9 were completely resolved only by TLC. The reduced degree of spot diffusion in TLC can be readily observed. Standardization of procedure is of considerable importance. For details on TLC standardization, the reader is referred to the book by Stahl [3]. 

Figure 21.12. Comparative TLC and PC separation of nucleotides. A Cellulose thin-layer chromatogram—development distance, 10 cm in 91 min. B Paper chromatogram run under identical conditions—development distance, 10 cm in 134 min paper Schleicher and Schull 20436. The solvent used for both was saturated ammonium sulfatejl M sodium acetate isopropanol (80 18 2). Each vertical column of spots corresponds to separate mixtures separated. Samples (1) 3 -AMP (2) 2 -AMP (3) 3 -GMP (4) I -GMP (5) 2 - and3 -GMP (6) 2 - and3 -UMP (7) 5 -AMP (8) 5 -ADP (9) 5 -ATP. (,A = adenosine, G = guanine, C = cytidine, M = mono-, D = di-, T = tri-, P = phosphate.) From K. Randerath, Biochem. Biophys. Res. Comm., 6, 452 (1961-62), by permission of Academic Press.

For preparative ion-exchange separations of nucleotides a volatile eluent is desirable and the use of ammonium carbonate and ammonium acetate have been described (Linz, 1983). 

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