Anion-exchange HPLC

The separation of nucleic acids by using strong anion-exchange stationary phases has become very popular (Gait et al., 1982 McLaughlin and Romanuik, 1982). The separations depend on the charge of 

Mobile phases containing phosphate buffers have often been used in the preparative purification of oUgodeoxynucleotides. However, the necessity to subsequently desalt the product limits the use of such mobile phases furthermore, problems associated with intramolecular and intermolecular interactions (hydrogen bonding) can occur, although these can largely be overcome by including formamide in the eluent (Newton et al., 1983). An alternative mobile phase is aqueous triethylammonium acetate which can be removed by lyophilisation and in which these molecular interactions are limited (Fig. 11.1.11). 

A comparison of anion-exchange, reversed phase and reversed phase ion-pair modes for the separation of small oligonucleotides concluded that anion-exchange provides the best resolution although column life-time may be shorter (Haupt and Pingoud, 1983). 

---

Figure 11.4 Analysis of in vitro synthesized RNAs. 32P-Radiolabeled RNAs (48 nucleotides) capped with m7Gp3G (A and C) or m27,3 °Gp3G (B and D) were digested with either RNase T2 (A and C) or RNase T2 plus tobacco acid pyrophosphatase (TAP) (B and D) followed by anion-exchange HPLC on a Partisil 10SAX/25 column as described in the text. Fractions of 1 ml were collected, and the Cerenkov radiation was determined. The elution times of the following standard compounds, detected by ultraviolet (UV) absorption, are indicated with arrows 3,-CMP (Cp), S UMP (Up), 37-AMP (Ap), 3 -GMP (Gp), 3, 5 -m7GDP (pm7Gp), 3, 5 -GDP (pGp), 5 -GDP (p2G), 5 -GTP (p3G), and guanosine-SCtetraphosphate (P4G).

A similar set of conditions was utilized by Reif et al.76 to analyze folic acid, using leucovorin as an internal standard. Other authors77 have used similar conditions to study extensively the identity of possible leucovorin contaminants. In addition to these partition /ionization surpression methods, anion exchange hplc has been used to separate CF from other naturally occurring folates,78 and some correlation has been made between the number of glutamyl residues and retention time. 

J. A. Day, M. Montes-Baydn, A. P. Vonderheide, and J. A. Caruso, A Study of Method Robustness for Arsenic Speciation in Drinking Water Samples by Anion Exchange HPLC-ICP-MS, Anal. Bioanal. Chem. 2002,373, 664. 

Other methods used to characterize and identify DOP involve bioassays with Chlorella to study the biological availability and biouptake of the HMW SRP fraction (4, 6). These bioassays indicate that the algal growth responds similarly to HMW SRP and to PO. A preference for PO 3- was detected, and not all of the reactive HMW fraction was used. Enzymatic assays used by Herbes et al. (13) tentatively identified inositol hexaphosphate as part of the DOP. Using an anion-exchange HPLC system with a phosphorus-specific post-column reactor, Minear and co-workers (15,16) possibly have detected inositol hexaphosphate, DNA, and nucleotide fragments in lake waters. 

CF RP-anion-exchange HPLC with UV, electrochemical, and fluorescence detection. ... 

Figure 8.9. Anion-exchange HPLC analysis of a plasmid sample. (a) Qiagen-purified plasmid (standard), (b-d) Analysis of streams within the process (b) after lysis (c) after clarification/ concentration (d) after ion exchange. (Reprinted with permission from Ref. 7.)...

Proteolytic secretion variants of recombinant human growth hormone were isolated by anion-exchange HPLC but compared analytically to the reference standard using reversed-phase (C4) HPLC with an ammonium bicarbonate/acetonitrile mobile phase rather than the more commonly used TFA/acetonitrile mobile phase [297]. 

Changes in bioengineered expression systems can alter the number and composition of carbohydrate residues attached to recombinant or monoclonal antibody products [301]. The use of high pH anion-exchange HPLC and pulsed amperometric detection (Dionex Corp) has enabled the analytical profiling or mapping of the characteristic biantennary carbohydrate structures following their enzymatic release from both murine and humanized monoclonal antibodies [302]. 

S. McSheehy, J. Szpunar, Speciation of arsenic compounds in edible algae by bidi-mensional size-exclusion anion-exchange HPLC with dual ICP-Ms and electrospray MS/MS detection, J. Anal. Atom. Spectrom., 15 (2000), 79- 87. 

Ausserer, W. A., and Biros, M. L. (1995). High-resolution analysis and purification of synthetic oligonucleotides with strong anion-exchange HPLC. BioTechniques 19, 136-139. 

Monitoring phosphorylation reactions by anion exchange HPLC... 

Day, J.A., Montes-Bayon, M., Vonderheide, A.P., Caruso, J.A. A study of method robustness for arsenic speciation in drinking water samples by anion exchange HPLC-ICP-MS. Anal. Bioanal. Chem. 373, 664-668 (2002)... 

Quantification of Carbohydrates by Anion-Exchange HPLC and Amperometric Detection... 

---