Columns sample volume

Pre-packed column Sample volume loading per run Sample volume recovery per run Code No. 

Choice of Column, Sample Volume and Flow Rate... 

Sample valve There are two types of sample valves the internal loop valve and the external loop valve. The former are for use with small bore columns (columns having internal diameters of less than 1.5 mm) and the latter are used for larger diameter columns up to semipreparative columns. Sample volumes generally range from 0.5 to 5 pi. 

Fig. 5. Anion-exchange separation of insulin and insulin A- and B-chains, over diethylaminoethyl (DEAF) in a 10.9 x 200 mm column having a volume of 18.7 mL. Sample volume is 0.5 mL and protein concentration ia 16.7 mAf Tris buffer at pH 7.3 is 1 mg/mL for each component ia the presence of EDTA. Eluent (also 16.7 mAf Tris buffer, pH 7.3) flow rate is 1.27 ml,/min, and protein detection is by uv absorbance at 280 nm. The straight line depicts the salt...

Column Si. Size-exclusion chromatography columns are generally the largest column on a process scale. Separation is based strictly on diffusion rates of the molecules inside the gel particles. No proteins or other solutes are adsorbed or otherwise retained owing to adsorption, thus, significant dilution of the sample of volume can occur, particularly for small sample volumes. The volumetric capacity of this type of chromatography is determined by the concentration of the proteins for a given volume of the feed placed on the column. 

Experimental part was provided by device Model Knauer-Compact with UV-detector (b=3 mm) at 250 nm and column Spherisorb-ODS-2 (250x4,6 mm). Sample volume was 1-2 p.1 injected by Reodyne 7725. Concentration range was 0.4-0.5 mg/ml for solutions of studied substances in DMSO. The organic modificator concentration range was 75-85 % w for methanol and 40-60 % w for acetonitrile in eluent (flow rate -1 ml/min). 

The sum expressed by equation (21) lends itself to a digital calculation and can be employed in an appropriate computer program to calculate actual peak profiles. In doing so, however, as (v) is measured in plate volumes and sample volumes are usually given in milliliters, they must be converted to plate volumes to be used with equation (21). To demonstrate the effect of a finite charge and the use of equation (21), the peak profiles resulting from a sample dispersed over the twenty-one consecutive plates of a column are shown in Figure 16. 

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. 

The sum expressed by equation (25) also lends itself to a digital solution and can be employed in an appropriate computer program to calculate actual peak profiles for different volumes of pure mobile phase that have been injected onto an equilibrated column. The values of (Xg) were calculated for a column having 500 theoretical plates and for sample volumes of 20, 50, 100 and 200 plate volumes, respectively. The curves relating solute concentration (Xe) to plate volumes of mobile phase passed through the column are shown in Figure 17. 

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 maximum allowable dispersion will include contributions from all the different dispersion sources. Furthermore, the analyst may frequently be required to place a large volume of sample on the column to accommodate the specific nature of the sample. The peak spreading resulting from the use of the maximum possible sample volume is likely to reach the permissible dispersion limit. It follows that the dispersion that takes place in the connecting tubes, sensor volume and other parts of the detector must be reduced to the absolute minimum and, if possible, kept to less than 10% of that permissible (i.c.,1 % of the column variance) to allow large sample volumes to be used when necessary. 

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))-... 

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. 

If the total extra-column dispersion is shared equally between the sample volume and the detector,... 

Consider the separation depicted in Figure 1. It is assumed that the pair of solutes represent the elution of the solute of interest and its nearest neighbor. Now, when the sample volume becomes extreme, the dispersion that results from column overload, to the first approximation, becomes equivalent to the sample volume itself as the sample volume now contributes to the elution of the solutes. Thus, from Figure 1, the peak separation in milliliters of mobile phase will be equivalent to the volume of sample plus half the sum of the base widths of the respective peaks. 

---