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diameter. There are other problems that need addressing, once packed, the practical lifetime of a column is also uncertain. The changes in performance of a preparative HPLC column that occurs with time depends upon the stability of the packed bed. Frequently, the bed settles after operation for even a short time and the top of the column needs to be repacked. Sometimes channels are formed in the bed, in which case the entire column has to be repacked. The rate of settling again depends upon thediameter of the column. This bed instability arises because there is a significant change in wall support as the column diameter increases. In analytical columns the walls are relatively close to the center of the column and 'bridges' of packing particles can be formed across the bed, as shown in Figure 16. These bridges allow the longitudinal forces acting on the packing within the column to be dissipated to the walls. When a column is packed, it is never in its optimal configuration and there

;               It is seen from equation (43) that, if an LC column is operated at its optimum linear velocity, the maximum efficiency obtainable for well retained peaks will be directly proportional to the inlet pressure  available (P) and the square of the particle diameter of the packing. Thus, the larger the particle diameter, the greater efficiency attainable at a given pressure. This is because, as the particle diameter is increased the column permeability is also increased allowing a longer column to be used. The permeability increases as the square of the particle diameter but the variance per unit length only increases linearly with the particle diameter. Thus, doubling the particle diameter will allow a column four times the length to be used but the number of plates per unit length will be halved. Consequently, the column efficiency will be increased by a factor of two. It is also seen that the

. This limits the pressure in contemporary chromatographs to an absolute maximum of about 10,000 p.s.i. and more often (due to the pressure limitations of the sample valve) to about 6000 p.s.i. Thus, if the pressure is limited, then to utilize longer columns the particle diameter must be increased to reduce the flow impedance and allow the longer column to be operated at the optimum mobile phase velocity. The use of larger particles to reduce flow impedance and thus permit the use longer column is possible because, at the optimum velocity, the inlet pressure decreases as the square of the particle diameter but the efficiency is only reduced approximately linearly with the particle diameter (thids is true for packed columns only). Thus, doubling the particle diameter allows the column length to be increased by a factor of four and as the plate height will be increased by a factor 2 the net result will be to double the number of theoretical plates

by increasing the dimensions of the column both in GC and in HPLC. However, this approach has distinct limitations. If the column radius is increased, unless special packing techniques are employed, the packing procedure becomes inefficient and the packing itself unstable. In addition to maintain the optimum mobile phase velocity, the flow rate will need to be substantially increased and the consumption of mobile phase will eventually become economically impractical. Conversely, if the column length is increased, then the impedance to flow will become greater leading to high column pressures. If large column radii are employed, then the mechanical strength of the column system will limit the maximum permissible pressure. Consequently, lengthening the column will eventually require the particle diameter to be increased to provide adequate permeability. Increased particle diameter will, in turn, reduce the column efficiency, which may impair the resolution of the compounds of

The column was operated close to its optimum velocity and, even with a 2 m column the analysis time extended over 40 hr. The number of peaks disclosed by the chromatogram is about 150. The separation was developed isocratically by a 75% v/v acetonitrile/water mixture. It is seen that there is now a clear resemblance to a GC separation carried out on a capillary column. Unfortunately the analysis times are far from comparable. It is seen that extra-column dispersion can arise in the sample valve, unions, frits, connecting tubing, and the sensor cell of the detector. The maximum sample volume, i.e., that volume that contributes less than 10% to the column variance, is determined by the type of column, dimensions of the column and the chromatographic characteristics of the solute. In practice, the majority of the permitted extra-column dispersion should be allotted to the sample volume, as a large sample volume may be necessary to handle a

with lower diffusivities (higher molecular weights). this is an inherent problem with small bore capillary columns which is extremely difficult to obviate. In an attempt to overcome this problem, larger diameter open tubular columns have been employed that would permit on-column injection. The columns have an I.D. of about 0.056 in., which is slightly greater than the diameter of a specific hypodermic needle. The injection system is shown in figure 7.     Figure 7. Device for On-Column Injection in Large Bore Capillary Columns   Unhappily, this type of injector also is far from ideal, not so much from poor accuracy and precision but from its effect on column resolution. On injection, the sample breaks up into discrete segments, due to bubble formation in the first part of the column. As the solvent evaporates the sample is deposited at two or more locations along the column. When development commences, each local concentration of sample acts as a unique injection