There are two theories to explain chromatography

Plate theory - older; developed by Martin & Synge in 1941

Rate theory - currently in useProposed by van Deemter in 1956Accounts for the dynamics of the separation

View column as divided into a number (N) of adjacent imaginary segments called theoretical plates Within each theoretical plate analyte(s) completely equilibrate between stationary phaseand mobile phase

Chromatographic principleThe molecules of the mixture interact with the molecules of the Mobile and Stationary PhaseRetardation of rate of movement of moleculesEach molecule interacts differently with MP and SPDifferent distribution coefficients and different net rates of migration

Greater separation occurs with: greater number of theoretical plates (N) as plate height (H or HETP) becomes smaller L = N H or H = L / N where L is length of column, N is number of plates, and H is height of plates or height equivalent to theoretical plate (HETP)

The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution by various methods likehalf-height methodUSP method

N = 5.55 tR2/ w1/22 = 16 tR2/ w2 where: tR is retention time w1/2 is width at h0.5 w is width measured at baseline

N is a ratio of tR and of Wb which is 4

Where: N = Number of theoretical platesVe = elution volume or retention time (mL, sec, or cm)h = peak heightw1/2 = width of the peak at half peak height (mL, sec, or cm)

Nmax = 0.4 * L/dp where: Nmax - maximum column efficiency L - column length dp - particle size So, the smaller the particle size the higher the efficiency!

Band spreading - the width of bands increases as their retention time (tR) or retention volume (VR) increases

A band exhibiting a width of 4 mL and a retention volume of 49 mL, is eluted from a column. What width is expected for a band with a retention volume of 127 mL eluting from the same analyte mixture on the same column?

ANS: 10.4 mL

The smaller HETP, the narrower the eluted peak

It is not unusual for a chromatography column to have millions of theoretical plates Columns often behave as if they have different numbers of plates for different solutes present in same mixture

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