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Minimum Analysis Time 10. lOmin
Column Efficiency 3871
Optimum Flow-Rate 961 ml/min
Maximum Sample Volume 100 ml Solvent Consumption per Analysis 9695 ml
Column Wall Thickness 0 20 cm
Column Weight 852g
(It should be noted that the column weight does not include flanges)
The program can be modified slightly, to calculate the minimum analysis time required to isolate 5 gram of material, at a given inlet pressure, from a series of mixtures of differing separation difficulty. The results obtained are shown as curves relating separation time to separation ratio in figure 5.
Graph of Analysis Time against Separation Ratio for a Load of 5g
Figure 5 shows that here is a minimum in the analysis time at a separation ratio of about 1,06, Now, from the theory of analytical column design, it would be expected that, if the column was fully optimized, the analysis time would decrease continuously as the separation ratio of the critical pair increased. The reason that a minimum exists in figure 5 arises from the limitations place on the column by the minimum aspect ratio of unity and
the minimum column length of 5 cm. Thus, as the load of 5 gram requires a column of specific diameter to avoid undesirable peak dispersion, the column can not be reduced to the optimum length without rendering the column aspect ratio less than unity. As a result the column length must be greater than the optimum causing an extension of the analysis time. This situation is exacerbated as the separation ratio increases.
The program can also be modified to show how the optimum pressure required to effect the preparative separation of 5g of material in the minimum time, changes with the separation ratio, of the critical pair. The results are shown in figure 6 as curves relating the logarithm, of the inlet pressure to the separation ratio.
Figure 6 Graph of Log. Inlet Pressure against Separation Ratio for a Load of 5g
? Column 5
1.1 1.2 Separation Ratio
Figure 6 shows the effect of the three limits imposed on the column design, the need to maintain an aspect ratio greater than unity, restricts the column length to a minimum of 5 cm and confines the analysis time to between one minute and one hour. Without the restriction, the column with the minimum analysis time would be operated at the maximum pressure available. However, as a result of the limitation on column length, the optimum pressure must be reduced to allow the optimum particle diameter to be ircreased. This, in turn, allows the column length to be increased to
maintain a column aspect of unity while achieving the minimum practical analysis time and the required column loading. !t is seen that the interactions between the column properties, and their effect on analysis time, are inherently complex and, unfortunately, become more so, as further external limitations are applied to the column design. It should also be noted that the more simple the separation (i. e. the smaller (a)) the lower the optimum operating pressure).
Column Overload as an Alternative to Column Design
Prior to the development of a rational procedure for the design of preparative columns, large scale separations were carried out by employing the technique of column overload. Column overload, although not providing optimum separations, was partly successful, but could only function well where overload was possible, i.e. where the separation ratio of the critical pair was large e.g. greater than I 5 and preferably 2, 3 or even 4. Unfortunately, with the majority of LC separation problems met in practice, this is not possible, due to the complexity of the mixture. Even with very careful mobile phase selection, the necessary large values for (a) are not often attainable. Nevertheless, the technique of column load can be useful under some circumstances and thus, the basic principles of column over load will be briefly discussed. The theory of column overload has been considered by a number of workers in the field (7-13) and more recently in a paper by Knox and Piper (14) and in a number of papers by Guiochon and his co-workers (15-18).
For any given chromatographic system, there is a limiting charge that can be placed on a column before the resolution is impaired. Loss of resolution from column overload can arise from two causes, either excessive sample feed volume or excessive sample mass The theory of moderate sample volume overload has already been considered in the applications of the Plate Theory. The theory of excessive sample volume overload will now be discussed.
Sample Volume Overload
Consider the situation depicted In figure 7 To determine the band dispersion that results from a significant, but moderate, sample volume overload the principle of the summation of variances can be applied. However, when the sample volume becomes excessive, the band dispersion that results from the overload becomes equivalent, to a first approximation, to the sample volume itself. In figure 6, two solutes are depicted that are eluted from a column