Solvent gradient column method

A reverse-phase chromatography matrix is a hydrophobic surface, such as silica. The protein mixture is loaded in a relatively hydrophilic solvent, so that proteins with hydrophobic patches on their surfaces will bind to the column. The column is then eluted with a solution of increasing hydrophobicity and the proteins are eluted in order of their hydrophobicity as they are displaced by the solvent gradient. This method also separates protein mixtures into many different groups. 

The rise in popularity of HPLC is due in large part to its high-performance nature and the advantages offered over the older, noninstrumental open-column method described in Chapter 11. Separation and quantitation procedures that require hours and sometimes days with the open-column method can be completed in a matter of minutes, or even seconds, with HPLC. Modern column technology and gradient solvent elution systems, which will be described, have contributed significantly to this advantage in that extremely complex samples can be resolved with ease in a very short time. 

This method requires the least sophisticated equipment and relies heavily on the unique characteristics of the column to separate the carotenoids (Craft et al., 1992 Epler et al., 1992). It incorporates the use of a polymeric Cl 8 column, which has been shown to offer unique selectivity for structurally similar compounds such as geometric isomers. The addition of a second detector or use of a diode-array detector permits the simultaneous analysis of tocopherols, but not retinol. If the method is modified to incorporate a solvent gradient, retinol can be measured also (MacCrehan and Schonberger, 1987). 

Moret et al. (67,68) studied all the parameters that influence amine recovery under conditions where a liquid-liquid purification step with an organic solvent follows the acid extraction, prior to derivatization with DBS and RP-HPLC analysis. The optimized methods of sample preparation for different foods, including cheese, meat, and fish, are given. The same research group (69) optimized the extraction conditions for Phe, Put, Cad, His, Tyr, Spe, and Spd. Food samples were first mixed with TCA and centrifuged and then basified and extracted with BuOH/CHCl3 (1 1). The BAs were then derivatized with DNS and separated on a Spherisorb 3S TG column with an ACN-H20 gradient. The method was applied to samples of tuna, salmon, and salami. 

Gradient systems let you control flow rate and solvent/buffer changes to improve chromatographic separations. They can be used to sharpen separations and to speed column re-equilibration. A four-solvent gradient system is useful for methods development when equipped with methanol, acetonitrile, ammonium acetate buffer, and formic acid solution. But, many quality control laboratories prefer to use inexpensive isocratic systems that run a constant-composition premixed mobile phase for rapid separations. 

In order to familiarize himself with macromolecules, he spent a postdoctoral year (1950-1951) with Professor A. Tiselius in Sweden. Together with Aim and Hagdahl they developed the method of gradient elution analysis. On his return to England, he showed how to apply temperature gradients to columns which, with solvent gradients, permit high polymer fractionation. These are called Baker-Williams columns. 

Isolation methods using solvent gradient in Sephadex LH-20 column. Further purification by semipreparative HPLC... 

Perhaps the worst problem of gradient elution separations is the need to reequilibrate the column with the initial solvent before a second sample can be run. An often-quoted rule of thumb is that up to 20 column volumes of the initial solvent may be necessary for this reequilibration process. The best test of reequilibration is the elution time of a weakly retained solute. These solutes will be greatly affected by an incompletely equilibrated stationary phase, and the retention time will vary. Cole and Dorsey have described a simple and convenient method for the reduction of column reequilibration time following gradient elution reversed-phase chromatography (119). Their method utilizes the addition of a constant 3% 1-propanol to the mobile phase throughout the solvent gradient to provide consistent solvation of the stationary phase. They noted reductions in reequilibration times of up to 78% ... 

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