Modern HPLC columns

It appears from table 7.3b that modern HPLC columns impose very stringent demands on the detection (and recording) system. Typical time constants of current LC detectors are in the range of 0.3 to 0.5 s [710], which is not even sufFicient to allow the use of a 20 cm, 5 pm column (column III in table 7.3b). 

Improvement of the resolution of poorly resolved analytes then could be pursued in two different ways either by increasing the efficiency or by improving the selectivity. The resolution value equal to 1.5 is usually regarded as sufficient for the baseline separation of closely eluted peaks and if we consider that typical average efficiency of modern HPLC column is equal to 10,000 theoretical plates, then we can calculate the selectivity necessary for this separation to get a resolution of 1.5. It will be also useful to compare what would be required in terms of efficiency and selectivity to improve the resolution from 1 to 1.5. Corresponding calculations are shown in the Table 1-1. 

A modern HPLC column is either stainless steel or plastic tube filled with packing material (adsorbent) and arranged with end-fittings designed to provide sealed connection with the eluent inlet and outlet lines and to retain packing material inside while allowing liquid to pass through. 

It is also important to understand, that retention in RP-LC is never solely based on a pure hydrophobic or dispersion interaction, but always influenced by secondary retention mechanisms. These secondary mechanisms can be both of wanted or of an unwanted nature. A good understanding of these relationships is extremely helpful for selectivity optimization and the fundamentals are discussed in more detail in Chapter 4 on modern HPLC columns. 

I 4 Comparison and Selection ( Modern HPLC Columns Comments on Table 4.1... 

Chapter 4 is about the comparison and choice of modern HPLC columns Stefan Lamotte, Stavros Kromidas, and Frank Steiner give an overview of different columns and come forward with proposals for pragmatic tests for columns as well as column portfolios, depending on the separation problem. 

A number of modern HPLC columns have been used for DNA and RNA separations. One example is a silica-based C18 material available from Varian Corporation (Walnut Creek, CA). There have been recent reports on monolith polymeric materials providing excellent separations [30-32]. The remarkable performance of the DNA and RNA separation columns is based on a number of properties including the porosity of the packing material, its polarity, the absence of metal contamination, and the small size and narrow size distribution of the packing material. 

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