Size separation efficiency optimization

Using SEC, most accomplishments of the commonly used RP-LC technique can be employed, such as various detector options, high sample loading capacity, variability in stationary phases and up-scaling option. Often in SEC, non-size-exclusion effects such as electrostatic and hydrophobic interactions between the analyte and stationary phase may be observed. The separation efficiency can be improved by optimizing the mobile phase, flow rate, column length, and sample volume. Practical guidelines for SEC method development have been described [42]. 

Similar to the findings in DNA analysis the separation efficiency decreases with increasing field strength. Optimal conditions are around 200 V cm-1. It has also been reported [11] that the mobility decreases with increasing ionic strength. Because the size of the PSS depends also on the buffer counterions, an influence of the counter-ions on mobility has been observed [11]. 

Resolution in SEC is dependent on the slope of the calibration plot J log M/dv and efficiency, and these two parameters should be manipulated to optimize resolution (20). Calibration slope can be decreased by the addition of more columns in series, and the effect on resolution is illustrated in Figure 7. Efficiency is dependent on particle size, and smaller particle size, higher efficiency columns are generally preferred. The effect of particle size on the separation of polystyrene oligomers is shown in Figure 8. Column sets should comprise packing materials of the same particle size because the full potential efficiency of... 

The plate number A/ is a compound-specific measure (it, therefore, applies to each individual peak) for the separation efficiency of a column under clearly defined mobile phase and temperature conditions. It will change over the column lifetime and can also be influenced by the HPLC instrument. The plate number increases proportional to the column length L, provided all other conditions remain constant (except for the colunm pressure). It also increases at constant column length when the stationary phase particle size, particle architecture, or bonding chemistry is optimized in way that accounts for less band dispersion. The respective columns exhibit increased separation efficiency per unit column length. Once the plate number of a column or method is increased, one can separate more analytes or separate analytes with better resolution under otherwise constant conditions. The formula to calculate the plate number from the peak parameters retention time and peak width at base Wi, or peak width at half height Wj, is as follows ... 

In addition to these larger solid core particles, smaller sub-2 pm solid core particles with particle sizes of 1.7 and 1.3 pm are commercially available. However, their theoretical potential with respect to separation efficiency and speed of analysis, which certainly exists, cannot be used in practice. This is because no HPLC systems are available with the necessary small volumes (of both the capillaries and detector cells) and which also have the data acquisition rate required to detect and record the separations achieved on these columns without loss. Today s modern UHPLC systems are already quite well optimized, but their performance is by far not good enough for these applications the manufacturers face further challenges here. As with all materials, it is highly recommended to check the batch-to-batch reproducibility especially of solid core phases. 

The separation characteristics of a considerable variety of other TLC supports were also tested using different dye mixtures (magnesia, polyamide, silylated silica, octadecyl-bonded silica, carboxymethyl cellulose, zeolite, etc.) however, these supports have not been frequently applied in practical TLC of this class of compounds. Optimization procedures such as the prisma and the simplex methods have also found application in the TLC analysis of synthetic dyes. It was established that six red synthetic dyes (C.I. 15580 C.I. 15585 C.I. 15630 C.I. 15800 C.I. 15880 C.I. 15865) can be fully separated on silica high-performance TLC (HPTLC) layers in a three-solvent system calculated by the optimization models. The theoretical plate number and the consequent separation capacity of traditional TLC can be considerably enhanced by using supports of lower particle size (about 5 fim) and a narrower particle size distribution. The application of these HPTLC layers for the analysis of basic and cationic synthetic dyes has also been reviewed. The advantages of overpressured (or forced flow) TLC include improved separation efficiency, lower detection limit, and lower solvent consumption, and they have also been exploited in the analysis of synthetic dyes. 

Charged macromolecules, such as proteins or polymers, are often separated elec-trophoretically. The rate of migration through an electric field increases with net charge and field strength. Molecular size of analytes and viscosity of separation media both have inverse relationships with rate of migration. These variables must all be taken into account in order to optimize the conditions for an efficient electrophoretic separation. 

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