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they will be apart and the greater the separation. Any change in stationary phase, however, will change the retention of all solutes proportionally and thus the separation will only increase, if the peak widths remain unchanged. Increasing the amount of stationary phase will usually increase the thickness of the stationary phase film, which, as is shown in Dispersion in Chromatography Columns will increase peak dispersion. It follows that there will be a specific stationary phase loading that provides the best compromise between separation and band dispersion (6) and thus provides the maximum resolution. The loading can be quite critical for open tubular columns in GC. Thus, the stationary phase loading cannot be increased indefinitely to separate the peaks as, eventually, the peaks will start spreading to a greater extent than they are being separated. Increasing the stationary phase load on a GC column (packed or open tube) will allow the sample

The Control of Chromatographically Available Stationary Phase (Vs) The volume of stationary phase that is made available to the solutes can be controlled in a number of ways. Firstly, the stationary phase loading on the column can be varied to adjust the retention as required. A specific stationary phase loading may be selected, to either improve the resolution, or to reduce the analysis time, or in some instances, to increase the sample load. Sometimes, the stationary phase loading is reduced so the column is more amenable to specific compounds (e.g. to prevent proteins from being denatured). Secondly, the stationary phase can contain molecules of a special shape that can only make

The core houses the inner frit, through which the eluent percolates and exits at the base of the column to a detector and hence to a fraction collector. The outer frit constitutes the column inlet, and consequently the sample has initially an extremely large area of stationary phase with which to interact. This renders the loading capacity of the radial flow column also very high. It is interesting to note, that as the solute progress radially through the stationary phase bed towards the center, the effective cross-sectional area of the column will become smaller. Consequently, the plate volume of the column will decrease (see Plate Theory and Extensions ) as the solute moves to the center which will result in the solute being concentrated. However, as the solute bands progressively decrease in

cutting is a common technique in preparative separations and, as shown by the example earlier in the chapter, can eliminate significant peak dispersion which can result in either low product purity or reduced yield. 4. Under certain circumstances the column dimensions can be increases to improve both load and product yield. There are two ways of increasing the size of the preparative column, by increasing its length and by increasing its diameter. Increasing the diameter increases the loading capacity and maintains the same separation time. It does not, however, increase the resolution. Increasing the column length increases the separation time and the resolution but does not increase the loading capacity, if the maximum efficiency is needed. If the sample consists of a simple pair of substances (e.g., the separation of a pair of enantiomers) then increasing the column length will allow multiple samples to be separated in the column at one time and, thus, will increase

volume and the maximum sample mass. It is also seen that increasing the column length will also increase the column efficiency (unless it is accompanied by an corresponding increase in the particle diameter). However, increasing the column efficiency will have the opposite effect, as seen by equation (1), it will reduce the maximum sample load. Consequently, if the necessary efficiency to achieve the required separation has been obtained, then if the column is lengthened to increase the loading capacity for optimum performance, either the flow rate will need to be increased to reduce the efficiency and thus maintain the maximum loading, or the particle size will need to be increased to reduce the efficiency to its required value. However, an increased flow rate will also reduce separation time and thus increase sample throughput. Conversely, the alternative use of larger particles will increase column permeability and thus the column can be operated at a lower pressure and be

degrade the column performance. This type of sample valve might also be used with short columns, perhaps 4.6 mm in diameter and 3 cm long packed with support particles only 3 m in diameter or, with small microbore columns (e.g., 10 to 50 cm long and 1 mm I.D.) . For columns providing peaks of larger volume, the external loop valve with six ports can be used. In this valve, three slots are cut in the rotor in a manner that will allow any two adjacent pair of ports to be connected. In the loading position (shown on the left of the lower diagram), the mobile phase supply is connected directly to port (4) and the column to port (5). This arrangement, causes the mobile phase to flow directly through the column. At the same time, the ends of the sample loop are also connected to ports (3) and (6). The sample loop is charged by a syringe through port (1), and by way of the rotor slot to the sample loop which is connected to port (6). The third slot in the rotor connects the sample