Extra Column Dispersion - Rapid Separations Employing Gradient Elution > Page 52
The mixing T consists of a drilled out Swagelok T union which contains two 1/16 O.D., 0.10 in. I. D. connecting tubes that leads from either pump to the center of the T. The ends of the tube are cut at an angle leaving a triangular pace at the center. The tube from the sample valve enters the stem of the T and is also 1/16 in. O.D. and 0.010 in. I. D., and is tapered to a flat on two sides so that it fits into the angle of the two tubs from the pumps at the center of the T. The mixing volume so formed is about 20 ml.
From the mixing T the solvent passes to a suitable sample valve (usually with an internal volume loop) and then to the column. The layout of a suitable apparatus for gradient elution that can be employed with low volume small bore columns, including the low dead volume T mixer, is shown in figure 26. It should be emphasized that modern chromatographs may, or may not, be designed for use with very small capacity, fast, high resolution columns and therefore, the specifications of the instrument must be closely examined if it is intended for such use.
Rapid Separations Employing Gradient Elution
Rapid LC separations are relatively easy to accomplish with isocratic development assuming low dispersion instrumentation with a fast response is available. However, if a sample mixture contains components that extend over a wide polarity or molecular weight range then gradient elution development will be necessary and fast gradients are almost impossible to form with conventional LC solvent programmers. As a consequence, for high speed gradient separations, a unique procedure must be used in conjunction with specially designed apparatus.
The solution to the problem of fast gradient generation is to employ a preformed gradient a concept that was first introduced by Snyder and Saunders (13) as long ago as 1969. A diagram of a gradient preformer is shown in figure 27. In the particular apparatus that was used to provide the fast analyses that are described below the required gradient was formed in a column 25 cm long, 4.6 mm I.D. packed with glass beads 40 mm in diameter.