All chromatographic techniques are based on an equilibration of solutes between a stationary phase and a mobile phase.

A small volume of sample is placed at the top of the column (filled with stationary phase particles and mobile phase).

Mobile phase sorbent is added and solutes move down the column at different rates determined by their equilibration between the two phases.

We measure the concentration of the eluted molecules as they emerge from the column.

The band spreads out as it moves down the column. The slower eluting molecules exhibit more peak broadening.

1 The efficiency of a column is described by the number of theoretical plates. 2 N = 16(tR/wb)

The narrower the peak, the greater the number of plates. The more theoretical plates, the greater the resolving power. number of theoretical plates. wb = 4s. Retention factor = k = (tR-tM)/tM = t’R/tM.

2 Nsys = 41.7(tR/w0.1) /(A/B + 1.25) This equation is used to calculate the number of plates for a tailing peak.

Asymmetric peak and Foley-Dorsey equation.

2 Peaks are broadened by eddy diffusion, molecular diffusion, and slow transfer rates.

Faster flow decreases molecular diffusion, but increases mass transfer effects.

There will be an optimum flow.

This describes efficiency in GC. van Deemter equation. H = A + B/ū + Cū

A - represents eddy diffusion. It is minimized with small, uniform particles.

B - represents longitudinal (molecular) diffusion (decreases at faster flow).

C - represents interphase mass transfer (decreases at slower flow).

3 Molecular diffusion is small in HPLC (but large in GC). A correction must be made for mass transfer in both the mobile and stationary phase. The Huber and Knox equations do this for calculating H.

van Deempter plots for different particle sizes in HPLC. The smaller particle sizes are more efficient, especially at high flow rates.

Column efficiency is related to particle size. For a well-packed column:

H = (2 to 3) x dp; dp = average particle size.

Plate number as a function of linear velocity for different size particles in a 10-cm HPLC column.

4 Rs= 1/4√N[(a – 1)/a][(k2/(kave + 1)]

Increasing N increases resolution.

Increasing k (retention factor) increases retention time, and broadens peaks.

Increasing the separation factor (a = k2/k1) increases the resolution.

We vary the stationary or mobile phase to increase a or k.

•Sample is injected into a heated port where it is vaporized.

•Analyte in the vapor state distributes between the stationary phase and the carrier gas.

•Gas phase equilibria are rapid, so the number of plates and resolution can be high.

5 Hundreds of compounds can be separated and measured by GC, with very small samples.

Capillary columns have particularly high resolution.

©Gary Christian, , 6th Ed. (Wiley)

These are the most popular columns in GC. They come as:

Wall-coated open-tubular (WCOT) columns – thin liquid film supported on the walls.

Support-coated open-tubular (SCOT) columns – solid micro particles coated with stationary phase attached to walls.

Porous layer open-tubular (PLOT) columns – solid phase particles attached to walls.

6 The narrow capillary columns have high resolution. These columns are now made of fused silica.

Three generations in gas (1979).

Peppermint oil separation on (top) ¼-in x 6-ft packed column;

(center) 0.03-in x 500-ft stainless steel capillary column;

(bottom) 0.25-mm x 50-m glass capillary column.

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