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11.3 High-performance liquid chromatography

11.3.1 Principle

It is evident from equations 11.1 to 11.13 that the resolving power of a chromato-

graphic column is determined by a number of factors that are embedded in equation

11.13. This shows that the resolution increases with:

• the number of theoretical plates (N) in the column and hence plate height (H).
  The value ofNincreases with column length but there are practical limits to the length
  of a column owing to the problem of peak broadening (Section 11.2.4);


• the selectivity of the column,a; and


• the retentivity of the column as determined by the retention factor,k.
  As the number of theoretical plates in the column is related to the surface area of
  the stationary phase, it follows that the smaller the particle size of the stationary
  phase, the greater the value ofN, i.e.Nis inversely proportional to particle size.
  Unfortunately, the smaller the particle size, the greater is the resistance to the flow of
  the mobile phase for a given flow rate. This resistance creates a backpressure in the
  column that is directly proportional to both the flow rate and the column length and
  inversely proportional to the square of the particle size. The back-pressure may be
  sufficient to cause the structure of the matrix to collapse, thereby actually further
  reducing eluent flow and impairing resolution. This problem has been solved by the
  development of small particle size stationary phases, generally in the region of 5 10 mm
  diameter with a narrow range of particle sizes, which can withstand pressures up to
  40 MPa. This development, which is the basis of HPLC that was originally and
  incorrectly referred to ashigh-pressure liquid chromatography, explains why it has
  emerged as the most popular, powerful and versatile form of chromatography. Larger
  particle size phases are available commercially and form the basis oflow-pressure
  liquid chromatographyin which flow of the eluaent through the column is either
  gravity-fed or pumped by a low pressure pump, often aperistaltic pump. It is cheaper
  to run than HPLC but lacks the high resolution that is the characteristic of HPLC. Many
  commercially available HPLC systems are available and most are microprocessor-
  controlled to allow dedicated, continuous chromatographic separations.
  Columns
  The components of a typical HPLC system are shown in Fig. 11.4.Conventional
  columnsused for HPLC are generally made of stainless steel and are manufactured so
  that they can withstand pressures of up to 50 MPa. The columns are generally 3–25 cm
  long and approximately 4.6 mm internal diameter to give typical flow rates of
  1 3cm^3 min–1.Microboreor open tubularcolumns have an internal diameter of
  1–2 mm and are generally 2550 cm long. They can sustain flow rates of 520 mm^3
  min–1. Microbore columns have three important advantages over conventional columns:


• reduced eluent consumption due to the slower flow rates;


• ideal for interfacing with a mass spectrometer due to the reduced flow rate; and


• increased sensitivity due to the higher concentration of analytes that can be used.

446 Chromatographic techniques