Reversed-phase gradient polymer elution chromatography

SEC size-exdusion chromatography RPLC reversed-phase liquid chromatography GPEC gradient polymer elution chromatography ... 

High-performance liquid chromatography (HPLC) techniques are widely used for separation of phenolic compounds. Both reverse- and normal-phase HPLC methods have been used to separate and quantify PAs but have enjoyed only limited success. In reverse-phase HPLC, PAs smaller than trimers are well separated, while higher oligomers and polymers are co-eluted as a broad unresolved peak [8,13,37]. For our reverse-phase analyses, HPLC separation was achieved using a reverse phase. Cl8, 5 (Jtm 4.6 X 250 mm column (J. T. Baker, http //www.mallbaker.com/). Samples were eluted with a water/acetonitrile gradient, 95 5 to 30 70 in 65 min, at a flow rate of 0.8 mL/min. The water was adjusted with acetic acid to a final concentration of 0.1%. All mass spectra were acquired using a Bruker Esquire LC-MS equipped with an electrospray ionization source in the positive mode. 

The investigations mentioned so far aimed at objectives of rather low molar mass. Reversed-phase chromatography of polystyrenes with 17,500 and 50,000 g/mol was performed on C 18 columns with water/tetrahydrofuran gradients or mixtures3). The latter sample was isocratically eluted from 30 nm-pore packing with 85 % THF as a broad band, 87 % THF let the polymer elute in the void volume, whereas 83 % did not produce an observable band at all. 

Many applications have been found for reversed-phase chromatography in HPLC. The composition of the stationary phase is more easily controlled than with the TLC methods, and thus provides more reproducible separations. The use of bonded non-polar phases enables gradient elution to be carried out in a reversed-phase system. This approach has been useful for the analysis of polar compounds and gives improved separations compared with normal-phase HPLC. These methods usually involve separation with systems consisting of Carbowax, C -polymer or similar phases bonded or physically coated on the support. 

DNPH-steroids can be separated by HPLC with several partition systems [31,32] including 1 % /3,/3 -oxydipropionitrile (BOP) on Zipax with eluting solvents containing 0-20% tetrahydrofuran in heptane or 2-methylheptane, or 1% ethylene glycol on Zipax with 3% chloroform in heptane as the mobile phase. Reversed-phase chromatography with 1.0% hydrocarbon polymer (HCP) or 1% cyanoethyl silicone (ANH) on Zipax and methanol-water as the mobile phase can be useful for the separation of several polar steroids. Gradient elution (water to methanol) on octadecylsilane (ODS), Permaphase (chemically bonded on Zipax), also provides a separation of polar DNPH-steroids. The separation of five DNPH-steroids on 1.5% BOP coated on Zipax is shown in Fig.4.13. 

Figure 7. The analysis of one- and two-chain rhGH by reversed-phase HPLC at neutral pH. The chromatography was performed on a Polymer Laboratory PLRP-S column using phosphate-containing mobile phases, and eluted with an acetonitrile linear gradient.

Comprehensive multidimensional liquid chromatography is a relatively new development and has yet to develop a diverse application base. For the time being applications are dominated by the separation of proteins and synthetic polymers. For proteins the first dimension separations are usually based on ion exchange and the second dimension separations on reversed-phase liquid chromatography. Gradient elution was often used for both separation modes with a separation time less than 2 minutes for the second dimension separation and from 30 minutes to several hours for the first dimension separation. Current trends include the use of non-porous particles and perfusive stationary phases for the second dimension separation to reduce the total separation time and wider internal diameter columns in place of packed capillary columns to simplify interface construction and instrument operation and to allow the loading of larger sample sizes. 

Anionic surfactants may be determined by conventional ion chromatography, especially if only one anionic or a simple mixture is present. However, most separations require paired ion chromatography, as described above. One vendor uses the term mobile-phase ion chromatography to describe paired-ion HPLC with a conductivity detector and a system to chemically suppress the baseline conductivity of the mobile phase. A number of variations on this theme have been demonstrated, using either polymer backbone or sihca backbone reversed-phase columns, varying levels of organic solvent in the mobile phase, and either isocratic or gradient elution conditions (3,13-15). 

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