Column, short, separation efficiency

Although short columns with standard particle sizes have found many practical applications in pharmaceutical analysis, the compromised separation efficiency prohibits their use in situations... 

Development of short monolithic silica columns with high efficiencies (up to 200.000 plates per meter) and high-speed separations, performance parameters that can be obtained with column packed with sub-2 pm particles, but achieved with monolithic silica columns at very lower system back-pressure. 

Use standard lengths (25 to 30 m). To avoid overloading the column, the capacity can be increased by using a thicker film thickness and a wider diameter. This will not alter the separation efficiency. Occasionally, very short columns (2 m) give better results. 

S.6 Choice of Organic Modifier. Selection of the organic modifier type could be viewed as relatively simple The usual choice is between acetonitrile and methanol (rarely THF). In Chapters 2 and 4 the principal difference in the behavior of methanol and acetonitrile in the column is discussed. In short, methanol shows more predictable influence on the analyte elution, and the logarithm of the retention factor shows linear variation with the concentration of methanol in the mobile phase. Often for the effective separation of complex mixtures of related compounds, this ideal behavior is not a benefit and greater effect of the type and organic concentration on the separation efficiency is required. Acetonitrile as an organic modifier may offer these variations due to the introduction of a dual retention mechanism. The dual retention mechanism was discussed in Chapter 2. 

HPLC columns contain, usually, spherical particle packings, which are carefully sorted to fractions with narrow size distribution to provide high separation efficiency. Totally porous packing materials most frequently used for separations of small molecules in contemporary HPLC have pore sizes of 7-12 nm and specific surface area of 150-400 m /g, but wide-pore particles with pore sizes of 15-100 nm and relatively low specific surface area of 10-150 m /g, or nonporous materials are used for separations of macromolecules. Perfusion materials, designed especially for the separation and isolation of biopolymers, contain very broad pores (400-800 nm) throughout the whole particle, which are interconnected by smaller pores. Column efficiency and flow resistance increase with small particles, and a high pressure has to be used to maintain required flow rate and to keep an acceptable time of analysis. However, the maximum operating pressure is 30-40 MPa, with common instrumentation for HPLC. Hence, short columns should... 

It follows that the lowest dilution or the highest peaks are obtained if a short and, most importantly, thin column with as high a theoretical plate number as possible, is used. The origin of the separation efficiency of the column must come from its fine,... 

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