Choice of column

Although sample size will most greatly influence the choice of column, the chemical and physical differences between TSK-GEL SW columns and TSKGEL PW columns will also affect choice. Eor example ... 

The choice of columns used for 2DLC is based upon experience with the sample and resolution required. The HPLC column descriptors of selectivity, resolution, peak capacity, sample capacity, degree of sample recovery, and speed of separation have been discussed previously (Unger et al., 2000). Columns with higher peak capacity and sample capacity (IEC, HIC, NPLC, and RPLC) are preferred in the first dimension, and higher speed columns (SEC and RPLC) are better in the second dimension. This is discussed in detail in Chapters 2 and 6. 

Thus, the column diameters chosen for the two dimensions are determined by the amount of sample available and will dictate the flow rate ranges available to use. In split-flow systems, where only a portion of the first-dimension effluent is injected into the second dimension, the choice of column size is unlimited and the two methods can be developed independently. In comprehensive systems where the entire sample from the first dimension is injected into the second dimension, the flow rates are generally lower in the first dimension to accommodate the lower injection volumes into the second dimension. For example, for a 1-mm ID column in the first dimension with a flow rate of 50 (tL/min and a sampling rate of 1 min, 50 pL could be injected onto the second dimension. A 50-(lL injection onto a4.6-mm ID column flowing at 1 mL/min should be accommodated fairly well based upon its composition. In Chapter 6, the first dimension column diameters are estimated based upon the injection volume and sampling rate into the second dimension. 

Adequate resolution of the components of a mixture in the shortest possible time is nearly always a principal goal. Establishing the optimum conditions by trial and error is inefficient and relies heavily on the expertise of the analyst. The development of computer-controlled HPLC systems has enabled systematic automated optimization techniques, based on statistical experimental design and mathematical resolution functions, to be exploited. The basic choices of column (stationary phase) and detector are made first followed by an investigation of the mobile phase composition and possibly other parameters. This can be done manually but computer-controlled optimization has the advantage of releasing the analyst for other... 

The choice of column should be made after careful consideration of mode of chromatography, column-to-column variability, and a number of other considerations [3-5]. A short discussion on columns and column packings is given below. The column packings may be classified according to the following features [2] ... 

C. The Choice of Column Diameter Sensitivity, Detector Compatibility, Bandspreading... 

This choice of columns and rows is completely arbitrary and switching the columns for the rows will yield equivalent results, but for the sake of consistency, this analysis will use the row column convention stated above. This data structure has been illustrated diagrammatically in Figure 6. In Figure 6, there are two hypothetical excipients excipient A has an absorption peak at 2100nm and excipient B has an absorption peak at 1400 nm. In this hypothetical example, absorbance measurements were taken at 100 nm intervals from 1000 to 2500 nm thus, there are 16 variables corresponding to the absorption data at the 16 wavelengths and there are two samples one for each excipient. 

Understanding the van Deemter equation is important if one is to obtain the best possible performance of chromatographic columns, as indicated by a low value of h. A more thorough discussion of the equation has been given by Schupp (1). Each of three major terms are affected by either the choice of column materials or the manner in which the column is prepared or operated. 

The choice of column size will depend on the sample size As a rule, the sample size should not exceed 5% of the total column volume. 10-30 mg of protein/cm2... 

Depending on the choice of column, flow rates and temperature program are important parameters for the qualitative analysis of citrus oils. It is also important to have the same temperature program for quantifying compounds. Replication of injections for generating a standard curve is vital. Injections should be done on the same day by the same technician. A standard curve with an R2 value >0.9 is sufficient. 

Many reports (78-84) investigated the differences in packed and capillary supercritical fluid chromatography. Unfortunately, the rift between packed and capillary column users of SFC impeded the development of the science. This rift is a likely cause of the current low interest in SFC. Ideally, the unique features of the mobile phase is the area of scientific exploration that should be exploited. Choice of column size or type should be dependent upon the analytical problem to be solved. 

Samples are injected onto the turbulent-flow column similar to single-column methods. The analytes of interest are retained in the turbulent-flow column while the large macromolecules are eluted to waste. Once the analytes are separated from the matrix, the samples are then eluted into the analytical column. The characteristics of the analytical column determine the peak shape and separation seen at the MS detector. Flow rates which are compatible with the mass spectrometer can then be used and the chromatograms are based on conventional HPLC parameters. The key to dual-column methods is that the retentive properties of the analytical column must be sufficiently stronger than that of the turbulent-flow column the dual-column approach is performed in such a manner so that the mobile-phase composition needed to elute the analyte from the turbulent-flow column does not elute the analyte from the analytical column. The sample is then focused at the head of the analytical column until the mobile-phase conditions are changed to elute the analyte. The choice of columns is critical to the success of dual-column methods. Table 10.2 lists some of the applications of dual-column methods found in the literature. 

Notably it was the weepage limit that ultimately determined the choice of column diameter. It was necessary to provide gas velocities high enough to prevent significant weepage of liquid through the plate holes. Appendix G contains a comparison of the results for various column diameters, and also includes the weepage calculations. 

By varying the geometries in the program, an optimum design can be achieved. The choice of column diameter was a critical decision in the optimization procedure. In order to evaluate the effect of changing the column diameter, various column diameters were used and the number of trays required was determined. The results of these test runs are shown in Table G.l... 

Merely, the choice of column dimension demonstrates the awareness of cost reduction for organic solvents as well as analytical run time and thus of sample throughput and economic efficiency. Only 2 % of published reports make use of a column with an inner diameter (I.D.) as small as 1 mm [12] corresponding to a flow of 50 ul/min. Columns with an I.D. of 2.0-2.1 mm are used in 48 % of reports determining a flow between 200 and 500 ul/min and half of all applications is performed with a column I.D. between 3.0 and 4.6 mm and a flow ranging from 500 to 1,000 ml/min (Tables 5-8). 

---