Reverse phase chromatography gradient elution

Reverse phase chromatography is finding increasing use in modern LC. For example, steroids (42) and fat soluble vitamins (43) are appropriately separated by this mode. Reverse phase with a chemically bonded stationary phase is popular because mobile phase conditions can be quickly found which produce reasonable retention. (In reverse phase LC the mobile phase is typically a water-organic solvent mixture.) Rapid solvent changeover also allows easy operation in gradient elution. Many examples of reverse phase separations can be found in the literature of the various instrument companies. 

An example of a separation carried out in reversed-phase and in HILIC is shown in Figure 16. The analytes are rather polar, morphine and morphine 3- -glucuronide. On the reversed-phase column, the glu-curonide elutes first, since it is more polar than the parent compound. On the HILIC column, it elutes last. In addition, a gradient is used in the HILIC separation to elute both compounds in the same time frame. This demonstrates clearly the difference in retention between HILIC and reversed-phase chromatography. 

FIGURE 5.4 Effect of the gradient dwell volume, V7>. the elution volume, Vj, in reversed-phase chromatography. Solute neburon, retention equation (Equation 5.7) with parameters a=A, m = 4. Linear gradients 2.125% methanol/min (a) from 57.5% to 100% methanol in water in 20min ( i = 50) (b) from 75% to 100% methanol in water in 11.75 min (k = 10). Vg uncorrected calculated from Equation 5.8, Vg + Vg, Vg, added to Vg uncorrected, Vg corrected calculated from Equation 5.21. (A) A conventional analytical C18 column, hold-up volume y ,= ImL flowrate l.OmL/min. (B) A microbore analytical C18 column, hold-up volume y = 0.1mL flow rate 0.1 mL/min. 

Elution in reversed-phase chromatography is often carried out using a gradient, produced from water and some water-miscible organic solvent. The solute components are thus distributed between the stationary and mobile phases mainly on the basis of their polarities. In reversed-phase chromatography hydrophilic compounds elute before hydrophobic ones. 

Fig. 24. Reversed-phase chromatography of oligomers. Gradient elution of phenyl-oligo(ethylene glycol) homologs on a RP 8 column (250 x 4.6 mm d0 = 6 nm dP = 7 pm). Gradient water/acetonitrile program as indicated 25 °C flow rate 2 ml/min UV detection at 254 nm. (From Ref. S7> with permission)...

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. 

Offline precolumn derivatization is the most common alternative in this respect it involves separating the esters obtained from the organic acids by reversed-phase chromatography, which amply surpasses solvophobic chromatography (i.e., the use of undissociated acids as such) and allows gradient elution techniques to be applied, thanks to the wider lipophilicity range covered by the derivatized compounds. 

Reverse-phase chromatography was used by Lawrence and Cbarbonneau (16) for the separation of cyclamate from saccharin, aspartame, acesulfame-K, alitame, sucralose, and dulcin. The system consisted of a Supelcosil LC-18 column and gradient elution of 0-100% mobile phase B [20 mM KH2P04 (pH 3.5) acetonitrile, 8 2 v/v] in mobile phase A [20 mM KH2P04 (pH 5.0) acetonitrile, 97 3 v/v]. 

For flavones in citrus peel oils, separations were accomplished with isocratic mobile phases of 38% and 40% acetonitrile in H20 (1). The extracts of peel and cold-pressed peel oils were diluted in ethanol and analyzed by reversed-phase on various C18 columns with good results. For the dilute citrus oils, gradient elution was preferred, to prevent the accumulation of terpenes on the column. With normal-phase chromatography, the elution order is reversed terpenes elute with the solvent front and are not a problem. 

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. 

Isocratic elution is the most popular method for removing solutes from a column, but gradient elution is useful, especially in reversed-phase chromatography, for the removal of solutes with widely varying k values. 

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