The expression for calculating the column efficiency (N) can be derived from the plate theory. Column efficiency is measured in theoretical plates (from the plate theory) and is taken as 16 times the square of the ratio of the retention time (the time between the injection point and the peak maximum) to the peak width at the points of inflection.
N = 16 t R2 /w2
The height equivalent to the theoretical plate (HETP) or the variance per unit Length of a column is calculated as the ratio of the column length to the column efficiency (number of plates). If the length of the column is L , then the HETP ( height equivalent to the theoretical plate ) is;
HETP = L / N
Note: The plate model supposes that the chromatographic column contains a large number of separate layers, called theoretical plates . Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates". The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next. It is important to remember that the plates do not really exist ; they are a figment of the imagination that helps us understand the processes at work in the column. They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the better), or by stating the plate height; (the height equivalent to a theoretical plate) (Smaller the HEPT, better the column efficiency).
The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution;
N = 5.55 t R2 /w 1/2 2 (where w1/2 is the peak width at half-height)
Resolution of chromatographic column is the ability to resolve one analyte peak to other. Resolution can be defined as the ratio of the difference in retention time between the two peaks to the mean of their base widths (w av ).
RS= 2(t RA- t RB )/wA + wB = dt R /wav
On the basis of the pressure generated inside the column the liquid column chromatography can be further subdivided as -
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(a) Low pressure liquid chromatography (LPLC) - < 5 Bar.
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(b) Medium pressure liquid chromatography (MPLC) − 6 to 50 Bar.
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(c) High pressure liquid chromatography (HPLC) - > 50 Bar.
In low pressure chromatography, stationary phase are mainly polysaccharides which are mechanically weak. Therefore, even if such particles are produced with small diameters, they would not be sufficiently strong to withstand the high pressure required for high resolution chromatography. Thus, in HPLC silica based particles (5-10 μm) are used. Smaller size of particle (large surface area) reduces size of theoretical chromatographic plat (thus number of plate in a given length is more). Higher number of chromatographic plate results in better resolution .
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