Columns length and

Lmin) is the minimum column length, and (e) is the fraction of the column occupied by the mobile phase. 

The analytical capability of a SEC column is sometimes judged by the peak capacity, which is the number of unique species that can be resolved on any given SEC column. This number will increase with decreased particle size, increased column length, and increased pore volume. Because small particlesized medium generally has a lower pore volume and a shorter column length, peak capacities of ca. 13 for fully resolved peaks can be expected for high-resolution modern media as well as traditional media, (see Eig. 2.5). It was found that SEC columns differ widely in pore volume, which affects the effective peak capacity (Hagel, 1992). 

Generally, optimizing the selectivity by choosing a gel medium of suitable pore size and pore size distribution is the single most important parameter. Examples of the effect of pore size on the separation of a protein mixture are given in Fig. 2.15. The gain in selectivity may then be traded for speed and/ or sample load. However, if the selectivity is limited, other parameters such as eluent velocity, column length, and sample load need to be optimized to yield the separation required. 

Many operating variables, such as sample volume, flow rate, column length, and temperature, must be considered when performing any separation. The relative importance of these variables for Toyopearl HW-55F resin columns has been specifically evaluated. For example. Fig. 4.47 shows the relationship between column efficiency, or height equivalent of a theoretical plate (HETP),... 

As we continue lowering the pressure, GC is the final limiting case when the mobile phase has zero solvent strength over the entire column length and where temperature is the only effective control parameter. Gas chromatography is shown in Figure 7.3. 

Column length and diameter these are set according to the minimum number of plates required and the maximum acceptable pressure-drop, and calculated by taking into account the constraints given by the Van Deemter and Darcy s laws. 

The time taken for an analyte to elute from a chromatographic column with a particular mobile phase is termed its retention time, fan- Since this will vary with column length and mobile phase flow rate, it is more useful to use the capacity factor, k. This relates the retention time of an analyte to the time taken by an unretained compound, i.e. one which passes through the column without interacting with the stationary phase, to elute from the column under identical conditions (to). This is represented mathematically by the following equation ... 

Often, the retention time is used but, as discussed above in Section 2.3, this absolute parameter changes with column length and flow rate and this precludes the use of reference data obtained in other laboratories. To make use of these reference data, the capacity factor (k ), which removes such variability, must be employed. 

Experimentally the resolution in GPC 1 was increased by the long column lengths and low flow rate but degraded somewhat by the high sample loadings. The high flow rate in GPC 2 was only necessary because in these exploratory studies many different runs had to be performed and necessitated short analysis times. 

The peak volume is directly proportional to the square of the column diameter and the column length and decreases with Increasing column efficiency (decreases for smaller particle packings). The concentration at the peak maximum, C—, is given by... 

Koyama, J., Nomura, J., Shiojima, Y., Ohtsu, Y., and Horii, I., Effect of column length and elution mechanism on the separation of proteins by reversed-phase high-performance liquid chromatography, /. Chromatogr., 625, 217, 1992. 

Given the construction of the Poppe plot, the number of plates, the column length, the peak capacity, and the particle diameter are determined in the Schoenmakers et al. (2006) scheme all for the first-dimension column. These are then used to determine the second-dimension parameters that include the particle diameter, the number of plates, column length, and peak capacity. Other variables are utilized and optimized from this scheme. 

Several of these points are met by applying the optimization steps discussed above, e.g., using smaller particles, shortening column lengths, and reducing solvent viscosity to reduce backpressure. When we consider a virtual column—a packed bed in a purely theoretical sense— we commonly accept that reducing particle size proportional to column length results in columns with at least the same theoretical efficiency. This is true, but only in the theoretical world. 

The relationship between column length and particle size is L = NH = Nhdp = 10,000 x 2 x 1.5 x lO cm = 3 cm. Assuming the column has a reduced plate number of 2 at its optimum flow velocity, v = vopt= 1, then a 3 cm column could produce 10,000 plates when packed with 1.5 /.mi particles. 

As mentioned in Section 11.8.4, the parameters that are most important for a qualitative analysis using most GC detectors are retention time, tR adjusted retention time, t R and selectivity, a. Their definitions were graphically presented in Figures 11.16 and 11.17. Under a given set of conditions (the nature of the stationary phase, the column temperature, the carrier flow rate, the column length and diameter, and the instrument dead volume), the retention time is a particular value for each component. It changes... 

---