Liquid column chromatography, isolation

The main toxic pore forming component of P. marmoratus secretion, named pardaxin, was isolated by liquid column chromatography (5). Originally two toxic (5) polypeptides, Pardaxin I and II, were isolated. However, their primary sequences have been found to be identical (6) therefore, the two components most probably represent different aggregates of one polypeptide. This finding is in contrast to the secretion of P. pavonicuSj which contains three toxic polypeptides (8). Pardaxin is a single chain, acidic, amphipathic, hydrophobic polypeptide, composed of 33 amino acids and with a mass around 3500 daltons (5,6). The primary sequence is (6) NHj-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Ile-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-Gly-Gly-Gln-Glu-COOH. 

The step 1 product was diluted with 1500 ml toluene to a final concentration of 0.013 mol/L. The mixture was then treated with 1,3-dimethyl-3-phospholene oxide and heated to 90°C while the cyclization reaction was monitored by Fouier transform infrared (FTIR). When the characteristic—NCO peak was eliminated in the macrocyclic carbodiimide (2134 cm-1), the reaction was stopped, concentrated, dried, and the crude product isolated. The crude product was purified by liquid column chromatography on silica gel using either ethyl acetate or ethyl acetate/ -hexane, 9 1, respectively, and the product isolated. 

A certain amount of qualitative information can be obtained by means of so-called multidimensional chromatography (1). Tlris is a combination of different chromatographic techniques in which fractions from a primary separation step are transferred online to a secondary separation step. Multidimensional gas chromatography (GC), for example, involves coupling of GC columns of different selectivities so that the primary column isolates the fraction of interest, and the secondary column takes care of the final separation of that fraction. Using multidimensional liquid chromatography (LC), determination of androgen hormone residues in cattle liver has been possible (2). 

For metabolite isolation, 1.5 liters of pooled urine were applied to a XAD-2 resin column first. The ethyl acetate extract obtained containing 85 % of the radioactivity was applied upon evaporation to semipreparative HPLC on a Zorbax RX C18 column (9.4 x 250 mm, 5 pm) using gradient elution. Fractions obtained were further separated by isocratic elution on the semipreparative column. The metabolite fractions obtained were finally purified by preparative thin-layer chromatography. Liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS analysis was applied to the isolated metabolite fractions for structure elucidation. 

Prior to structural elucidation and possible eventual synthesis, the isolation of component phenolic lipids in a pure state is essential. The cold methods of thin layer chromatography (TLC), column chromatography (CC), flash chromatography, high performance liquid chromatography (HPLC) and hot methods (GC), often after derivatisation, are well established. Argentation versions of these separatory methods are less common but are desirable for the rapid separation of unsaturated constituents. 

Well established techniques 170, 171) for the isolation and separation of depsides from mixtures of lichen acids include fractional crystallization, preparative layer chromatography and column chromatography. Vacuum liquid chromatography 124) provided a convenient and efficient modification of the latter technique and was used in separating a complex mixture of tridepsides from the crude extracts of Parmelia damaziana. 

This section is concerned with the isolation of organic acids as a group prior to further chromatographic separation and determination, primarily gas chromatographic. Separation and analysis by liquid-column chromatography are described in Section 4.2. 

Figures 2 through 9 are infrared spectra of fractions collected from partition columns, gas chromatography, thin-layer chromatography, or a combination of these separation techniques. Figure 10 is the infrared spectrum of a compound isolated by gas chromatography after hydrolysis of a pyrethrum concentrate. In this case the compound is a long-chain ester. All the infrared spectra were made with a Perkin-Elmer Model 221 instrument. The following operating parameters were used. A liquid demountable cell with a 0.01-mm path length was employed.

The need for a more definitive identification of HPLC eluates than that provided by retention times alone has been discussed previously, as have the incompatibilities between the operating characteristics of liquid chromatography and mass spectrometry. The combination of the two techniques was originally achieved by the physical isolation of fractions as they eluted from an HPLC column, followed by the removal of the mobile phase, usually by evaporation, and transfer of the analyte(s) into the mass spectrometer by using an appropriate probe. 

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

One of the most complex separation schemes utilizes flash liquid chromatography and PLC to obtain petropophyrins both from geochemical samples or those synthesized and used subsequently as standards [110]. Ocampo and Repeta [111] described the scheme of petroporphyrins isolation in which at the first step the sediment extract is fractionated into ten fractions on silica gel using dichlo-romethane (fractions 1 to 4), a mixture of dichloromethane-acetone with increasing acetone concentrations (for fractions 5 to 9), and, at last, dichlo-romethane methanol (4 1) (fraction 10). Next, the fifth fraction was separated on silica PLC plates using dichloromethane-acetone (97.5 2.5 v v v) as a developer. Two purple bands (with Rj 0.53 and 0.50) were recovered from silica and purified further on a silica gel column with dichloromethane-acetone (97.5 2.5, v v v) as an eluent. The emiched fraction was then separated by PLC with the same solvent mixture, and the purple bands containing two bacteriopheophytin allomers were recovered with acetone. 

Specifically for triazines in water, multi-residue methods incorporating SPE and LC/MS/MS will soon be available that are capable of measuring numerous parent compounds and all their relevant degradates (including the hydroxytriazines) in one analysis. Continued increases in liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/API-MS/MS) sensitivity will lead to methods requiring no aqueous sample preparation at all, and portions of water samples will be injected directly into the LC column. The use of SPE and GC or LC coupled with MS and MS/MS systems will also be applied routinely to the analysis of more complex sample matrices such as soil and crop and animal tissues. However, the analyte(s) must first be removed from the sample matrix, and additional research is needed to develop more efficient extraction procedures. Increased selectivity during extraction also simplifies the sample purification requirements prior to injection. Certainly, miniaturization of all aspects of the analysis (sample extraction, purification, and instrumentation) will continue, and some of this may involve SEE, subcritical and microwave extraction, sonication, others or even combinations of these techniques for the initial isolation of the analyte(s) from the bulk of the sample matrix. 

---