Maximum sample volume

Maximum Sample Volume that Can Be Placed on a Chromatographic Column... 

Having established that a finite volume of sample causes peak dispersion and that it is highly desirable to limit that dispersion to a level that does not impair the performance of the column, the maximum sample volume that can be tolerated can be evaluated by employing the principle of the summation of variances. Let a volume (Vi) be injected onto a column. This sample volume (Vi) will be dispersed on the front of the column in the form of a rectangular distribution. The eluted peak will have an overall variance that consists of that produced by the column and other parts of the mobile phase conduit system plus that due to the dispersion from the finite sample volume. For convenience, the dispersion contributed by parts of the mobile phase system, other than the column (except for that from the finite sample volume), will be considered negligible. In most well-designed chromatographic systems, this will be true, particularly for well-packed GC and LC columns. However, for open tubular columns in GC, and possibly microbore columns in LC, where peak volumes can be extremely small, this may not necessarily be true, and other extra-column dispersion sources may need to be taken into account. It is now possible to apply the principle of the summation of variances to the effect of sample volume. 

Equation (22) allows the maximum sample volume that can be used without seriously denigrating the performance of the column to be calculated from the retention volume of the solute and the column efficiency. In any separation, there will be one pair of solutes that are eluted closest together (which, as will be seen in Part 3 of this book, is defined as the critical pair) and it is the retention volume of the first of these that is usually employed in equation (22) to calculate the maximum acceptable sample volume. 

Now, the maximum sample volume (Vi) that can be placed on the column that would restrict the increase to less than 5% has been shown to be. 

The effect of sample volume on peak width has been considered and treated theoretically in Chapter 6 however, it is of interest to determine the maximum sample volume that can be tolerated with modern columns packed with small particles. The maximum sample volume is defined by the following equation,... 

Equation (5) was used to calculate the maximum sample volume for a series of columns having different lengths and internal diameters and packed with particles 3 p... 

Figure 1. Curves Relating Maximum Sample Volume to Column Diameter for Columns of Different Length Packed with Particles 3 p in...

It is seen that columns having diameters less than 2 mm will only tolerate a maximum sample volume of a fraction of a microliter. Although larger volume valves can be used to inject sample volumes of this size, the dispersion from the valve is still likely... 

The use of 5 pm particles permits the use of much longer columns due to the increased permeability. This, in turn, permits the use of much larger sample volumes. In fact, for a column 20 cm long, eluting a solute at a (k ) of 5, the maximum sample volume that can be used without increasing the peak variance by more than 10% will... 

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... 

There remains the need to obtain expressions for the optimum column radius (r(opt)), the optimum flow rate (Q(opt)), the maximum solvent consumption (S(sol)) and the maximum sample volume (v(sam))-... 

It is seen from equation (46) that, as would be expected, the maximum sample volume decreases as the separation ratio decreases, i.e., with difficulty of the separation. 

Maximum Sample Volume and Maximum Extra-Column Dispersion... 

In a packed column the HETP depends on the particle diameter and is not related to the column radius. As a result, an expression for the optimum particle diameter is independently derived, and then the column radius determined from the extracolumn dispersion. This is not true for the open tubular column, as the HETP is determined by the column radius. It follows that a converse procedure must be employed. Firstly the optimum column radius is determined and then the maximum extra-column dispersion that the column can tolerate calculated. Thus, with open tubular columns, the chromatographic system, in particular the detector dispersion and the maximum sample volume, is dictated by the column design which, in turn, is governed by the nature of the separation. 

The expression for the maximum sample volume can be found in Chapter 12 equation (45) and is as follows,... 

Employing the data from Table 1 the values already calculated for (ropt) and (Lmin) the maximum sample volume was calculated for a range of values for (a) and the results are shown as a graph in Figure 10. 

Figure 10. Graph of Log of Maximum Sample Volume against the Separation Ratio of the Critical Pair...

It is seen that the maximum sample volume ranges from a fraction of a microliter when (a=1.01) to several ml when (a) = 1.2. The small sample volume results from... 

The expression defines the maximum sample volume is given in chapter 13, equation (21) and is reproduced as follows. 

Employing equation (25), the effect of the separation ratio of the critical pair on the maximum sample volume was calculated using equation (25) and the results are shown in Figure 17. 

It is seen that, in all cases, the maximum sample volume is extremely small ranging from about 100 pi for a very difficult, lengthy separation to less than a tenth of a... 

Employing equation (1), curves relating maximum sample volume to the capacity ratio of the first eluted peak for different separation ratios were calculated and constructed and the results are shown in Figure 2. 

Figure 2. Curves Relating Maximum Sample Volume to the Capacity Ratio of the First Peak for Different Separation Ratios...

The capacity ratios, efficiencies and separation ratios are given in Table 1, together with the maximum sample volume as calculated from equation (1). Comparing the maximum sample volume used for benzene given in Table 1, with the actual sample volumes for benzene shown in Figure 3, it would appear that equation (1) can be used with confidence for calculating (Vl). In a similar manner, using the same... 

TABLE 7.1 Recommended Maximum Sample Volumes for Different SEC Column Dimensions... 

Column dimension Bed height (cm) Maximum sample volume (mi)... 

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