Overload column

Contemporary development of chromatography theory has tended to concentrate on dispersion in electro-chromatography and the treatment of column overload in preparative columns. Under overload conditions, the adsorption isotherm of the solute with respect to the stationary phase can be grossly nonlinear. One of the prime contributors in this research has been Guiochon and his co-workers, [27-30]. The form of the isotherm must be experimentally determined and, from the equilibrium data, and by the use of appropriate computer programs, it has been shown possible to calculate the theoretical profile of an overloaded peak. 

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. 

The technique of column overloading is only feasible if more than adequate resolution is possible between the solute of interest and its nearest neighbor. Many samples require a column to be constructed that will only just separate the solutes of interest and under these circumstances the loading capacity must be increased without overloading the column. It has been shown in earlier chapters that the maximum sample (mass or volume) that can be placed on a column is proportional to the plate volume of the column and the square root of its efficiency. Thus, the maximum sample mass (M) will be given by... 

Errors in the molecular weight data from HPSEC are usually due to improperly prepared samples, column dispersity, or flow rate variations. The sample to be analyzed should be completely dissolved in the mobile phase and filtered prior to injection onto the column. A plugged column inlet frit will invalidate results. In addition, do not load the column with excess sample. Column overloading affects the accuracy of data by broadening peaks, reducing resolution, and increasing elution volume. For best results, the concentration of the injected sample should be as low as possible while still providing adequate... 

In all modes of chromatography, high sample loads distort peak shapes and cause an overall decrease in efficiency due to column overload. Sample loads may be increased by using organic solvents to enhance the solubility of the sample or by using higher column temperatures to lower the viscosity of... 

Column dimensions mainly determine the quantity of sample to be separated. However, because the SEC process is driven by size separation and is diffusion controlled, special care has to be taken to keep optimized separation conditions, especially when going to smaller internal diameter columns. Overloading and excessive linear flow rates can be observed quite often in these typese of columns. For this reason, standard 8-mm i.d. columns are commonly used, as they are rugged and have a good tolerance toward separation conditions. 

Sample loading must be reduced in accordance with the column inside diameter. Polymers exhibit high solution viscosity, and in order to avoid band broadening due to viscous streaming the sample concentration must be reduced for narrow-bore columns. Overloading effects become noticeable at much lower concentrations using 4.6-mm columns compared to 7.5-mm columns because of the effective sample concentration in a smaller volume column. 

Once the linear gas velocity of the carrier gas has been set, the splitter vent flow should be adjusted between 15 and 100 ml/min. Use lower values to improve detection and higher values to improve GC resolution and decrease column overload. 

However, the major contribution to peak asymmetry is usually a result of column overload and the two effects that can occur are depicted in figure 9. 

Peak Asymmetry Resulting from Column Overload... 

The easiest way for an analyst to obtain small quantities of a component of a mixture is to overload an analytical column. In order to exercise this technique, the solute of interest must be well separated from its closest neighbor. The column can then be overloaded with sample until the peak dispersion resulting from the overload, causes the two peaks to touch at their base. There are two types of column overload, volume overload and mass overload. In practice, it is often advantageous to employ a combination of both methods and a simple procedure for doing this will be given overleaf. 

Unfortunately, this procedure can only be successful if the critical pair can be well resolved and column overload is a practical solution to the problem. Often, due to the complex nature of practical mixtures, values for these conditions are not realized and the optimum column... 

The parameters that influence the injection plug width will be discussed in a further section of this chapter. In practice, it is necessary to keep the width of the injection plug smaller than 1% of the total length of the capillary, in order to avoid column overloading [27]. 

The system has been used for a wide range of appHcations, the secret of its success being the attention to detail and a series of interlocking safety alarms. Several key parameters are continuously monitored and the built-in safety alarms will automatically shut the system down if there is a fault. As can be seen from Fig. 4.10, the balance the user has to make with these systems is between column overload and throughput. As the size of the injection increases, so the quahty of the chromatogram decreases and the purity of the fraction suffers. A compromise has to be reached in terms of the purity desired and the throughput that has to be maintained. 

The analysis of Equation 10.10 indicated that the loading factor and the plate number are the essential parameters—besides the type of the isotherm—to control the band profile arising from column overload. The Shirazi number connects those two terms and can be regarded as a universal quantity that controls the band profile. 

---