Columns selection

Selecting a column for an HPLC separation is a matter of asking yourself a series of questions (Fig. 5.4). You first must determine how much material you wish to separate in a single injection (preparative vx. semipreparative vs. analytical). The next question involves the separation mode to be employed (size exclusion vx. ion exchange vx partition). Finally, there is the question of solubility controlling solvent and column selection in all modes. 

Are the primary differences in polarity Partition columns are available that vary in polarity from nonpolar (octyldecyl), through intermediate polarity (octyl and cyanopropyl), to polar (silica). Some columns have similar polarities, but differ in their specificity. Qg and the phenyl column have similar polarities, but Ci8 separates on carbon chain length, while phenyl separates fatty acids on both carbon number and number of double bonds. Phenyl columns also resolve aromatic compounds from aliphatic compounds of similar carbon number. In another example of similar polarities, C8 is a carbon number separator while cyanopropyl selects for functional groups. 

Assuming we have selected the proper mode of chromatography, will the mixture dissolve in the mobile phase Ion-exchange columns must be run in polar-charged solvents. Size separation columns are not, in theory, affected by solvent polarity, and size columns for use in both polar and nonpolar solvents are available. In partition chromatography, we have nonpolar columns that can be run in polar or aqueous solvents, and polar columns that are only run in anhydrous, nonpolar solvents. Intermediate columns such as cyanopropyl or diol can be run in either polar or nonpolar solvents, although often with differing specificity. An amino column (actually a propylamino) acts in methylene chloride/hexane like a less polar silica column but in acetonitrile/water 

Solvents Hexane Benzene CH2CL2 CHCL3 THF AN MeOH H20 

When I make a diagram of column polarities versus solvent polarities, I tend to think of the columns as being a continuous series of increasing polarity from Cis to silica C18, phenyl, C8, cyano, C3, diol, amino, and silica (Fig. 5.5). Under that, I have their solvents in opposite order of polarity from hexane under Ci8 to water under silica hexane, benzene, methylene chloride, chloroform, THF, acetonitrile, i-PrOH, MeOH, and water. The cyano column and THF are about equivalent polarity. In setting up a separation system, we cross over nonpolar columns require polar mobile phase and vice versa to achieve a polarity difference. 

The stationary phases available for HPLC are as numerous as those available for GC. As mentioned previously, however, adsorption, partition, ion exchange, and size exclusion are all liquid chromatography methods. We can therefore classify the stationary phases according to which of these four types of chromatography they represent. Additionally, partition HPLC, which is the most common, is further classified as normal phase HPLC or reverse phase HPLC. Both of these are bonded phase chromatography, which was described in Chapter 11. Let us begin with these. 

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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. 

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)... 

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. 

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-... 

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

While samples such as these have obviously been the focus for much GC X GC work in the past, the technology still remains to be demonstrated for many other sample types. It is likely that in the near future, as many more applications are studied, a general theory-or at least a guide to column selection for GC X GC applications-will reveal a logical approach to selection of phases that embodies the principles of orthogonality of separation. 

Figure 15.8 Multidimensional GC-MS separation of urinary acids after derivatization with methyl chloroformate (a) pre-column cliromatogram after splitless injection (h) Main-column selected ion monitoring cliromatogram (mass 84) of pyroglutamic acid methyl ester. Adapted from Journal of Chromatography, B 714, M. Heil et ai, Enantioselective multidimensional gas chromatography-mass spectrometry in the analysis of urinary organic acids , pp. 119-126, copyright 1998, with permission from Elsevier Science.

Column selection remains the most important factor in successful enantiomeric separations. The CSPs most likely to be effective in SFC are those that have been employed under normal phase conditions in LC. In fact, the tremendous body of knowledge that has been accumulated for LC can also guide column selection in SFC [66]. The likelihood of success with a particular CSP can generally be gauged after one or two injections [67]. If no evidence of separation is observed, another CSP should be investigated. 

As an example, if this were the final column selection, then the column trays = 8-condenser-reboiler = 6 theoret-... 

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