Columns Column selection

The aim of any chromatographic separation may be defined as the achievement of an optimal combination of speed of elution, sample size, and resolution of the solutes. Good resolution can only be obtained if there is adequate control over the differential migration rates of a group of solutes as they move down a column (column selectivity) and over the extent of zone dispersion for each of the solutes (column efiiciency). Historically, the various modes of liquid chromatography have been considered as separate and independent phenomena. It is now clear that they all have a common theoretical basis. Column selectivity in HPLC, irrespective of the mode, arises due to differences in the distribution equilibria... 

Three separate factors affect resolution (1) a column selectivity factor that varies with a, (2) a capacity factor that varies with k (taken usually as fej). and (3) an efficiency factor that depends on the theoretical plate number. 

From equation 12.1 it is clear that resolution may be improved either by increasing Afr or by decreasing wa or w-q (Figure 12.9). We can increase Afr by enhancing the interaction of the solutes with the column or by increasing the column s selectivity for one of the solutes. Peak width is a kinetic effect associated with the solute s movement within and between the mobile phase and stationary phase. The effect is governed by several factors that are collectively called column efficiency. Each of these factors is considered in more detail in the following sections. 

The ratio of capacity factors for two solutes showing the column s selectivity for one of the solutes (a). 

Equations 12.21 and 12.22 contain terms corresponding to column efficiency, column selectivity, and capacity factor. These terms can be varied, more or less independently, to obtain the desired resolution and analysis time for a pair of solutes. The first term, which is a function of the number of theoretical plates or the height of a theoretical plate, accounts for the effect of column efficiency. The second term is a function of a and accounts for the influence of column selectivity. Finally, the third term in both equations is a function of b, and accounts for the effect of solute B s capacity factor. Manipulating these parameters to improve resolution is the subject of the remainder of this section. 

Use of column selectivity to improve chromatographic resolution showing (a) the variation in retention time with mobile phase pH, and (b) the resulting change in alpha with mobile phase pH. 

To minimize the mobile phase s contribution to conductivity, an ion-suppressor column is placed between the analytical column and the detector. This column selectively removes mobile-phase electrolyte ions without removing solute ions, for example, in cation ion-exchange chromatography using a dilute solution of HCl as... 

Detectors. The function of the gc detector is to sense the presence of a constituent of the sample at the outlet of the column. Selectivity is the property that allows the detector to discriminate between constituents. Thus a detector selective to a particular compound type responds especially weU to compounds of that type, but not to other chemical species. The response is the signal strength generated by a given quantity of material. Sensitivity is a measure of the abiHty of the detector to register the presence of the component of interest. It is usually given as the quantity of material that can be detected having a response at twice the noise level of the detector. 

Column selection (should the column selection not be obvious or specified, calculations must be carried out for the different types of columns and the final based on economic considerations)... 

The diameter of the column is selected from the volume of sample that is to be processed. As a rule of thumb the maximum productivity is obtained at a sample volume of 2-6% of the bed volume in preparative gel filtration on a 50-/rm chromatographic medium (Hagel et al., 1989). Thus, the required column diameter is calculated from the bed volume needed to cope with the sample volume and the column length needed to give the resolution desired. 

The packing method supplied by the manufacturer of the gel filtration medium may need to be revised according to the column being selected. It is therefore important to have an understanding about the basic principles governing the packing of chromatographic beds. 

TABLE 4.11 Recommended Column Selection Guide for High-Performance Gel-Filtration Chromatography... 

Sample First choice Column selection Alternative Selection criteria... 

FIGURE 6.8 Guidelines for Shodex column selection depending on the molecular weight of polymer. Column Shodex GPC KF-800 series, 8 mm i.d. x 300 mm. Eluent THE. Flow rate 1.0 mUmin. Detector Shodex Rl. Column temp. 40°C. Sample EPIKOTE 828... 

The major parameter for column selection is the intended application. A balance of mobile-phase polarity in comparison with the polarity of the stationary... 

Another important parameter for column selection is the proper choice of sorbent porosity. The pore size of the sorbent determines the fractionation range of the column. The best way of doing this is by looking at the calibration curves of the columns, which are normally documented by the column vendor (cf. Fig. 9.3 for PSS SDV column calibration curves and PSS SDV fractionation ranges) (7). 

An example may show how the different concepts come into effect in a real-life laboratory environment. This example is based on column selections that many laboratories use for ordinary, general-purpose work. 

FIGURE 11.2 Selectivity of several individual pore size columns. This graph shows the resolution capability of Styragel HT 3, HT 4, HT S, and HT 6 columns. The selectivity [Eq. (I)] of these columns vs the molecular weight of polystyrene is plotted. (Courtesy of Waters Corp.)... 

Column selection is greatly simplified and the column is specifically designed for particular application areas. 

The main criterium for column selection is pore size distribution as it is desirable to have maximum pore volume for separation in the molecular weight range of interest. Having determined the upper molecular weight limit required, a column with a suitable exclusion limit should be selected. In the case of individual pore size columns, it is then a question of selecting other columns with complementary calibration curves to comprise a column set covering the re-... 

These three criteria—molar mass interval, eluent, and working temperature—are fixed by the group of samples to be analyzed and considerably restrict the number of suitable columns. The selection has to be done from current lists of the manufucturers. It is useless to collect these data here, as such tables would be antiquated before this book is printed. This chapter deals with the quality of the selected columns. At this stage, columns of the same application profile are compared. The most important properties are (1) the number of... 

COLUMN SELECTION AND RELATED ISSUES FOR ACRYLIC ACID AND ACRYLATE ESTER POLYMERS... 

---