Application of Liquid Chromatography for Bioanalysis

Normal phase chromatography was developed many years before reversed phase chromatography was investigated. Initially, stationary phases were made of polar materials such as paper, cellulose or silica gel and the mobile phase consisted of non-polar solvents such as hexane or chloroform. Only at a later stage were these phase polarities reversed. Polar solvents such as water and acetonitrile were 

Reversed phase chromatography is the method of choice for the separation of smaller biomolecules such as peptides, amino acids, carbohydrates and steroids, which are soluble in water/acetonitrile mixtures. The separation of proteins can be problematic as organic solvents such as acetonitrile can decrease the protein s solubility and cause denaturisation. 

Buffer systems based on ammonium acetate, phosphate or hydrogen carbonate are usually added at concentrations of about 20 mM to adjust the pH of the mobile phase to values between 2 and 8. Ion pairing reagents can be used at low concentrations, typically 0.1%, to increase the hydrophobicity of charged analytes. They 

In modern chromatography, the separation columns are tightly packed with small particles of about 1-5 pt m in diameter. To achieve ambient flow rates in these columns, high pressures of up to 300-400 bar must be generated. A typical instrumental setup for this high pressure or high performance liquid chromatography HPLC) is shown in Fig. 2.6. 

In recent years, there has been a trend to develop ever smaller liquid chromatography systems. LC systems on micro and even nanoscales have been demonstrated. Shorter and smaller columns with smaller particles offer faster analysis times, decreased solvent consumption and require less sample. The differences between preparative, analytical, micro and nano LC are summarised in Table 2.2. 

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