Instant Notes: Analytical Chemistry

t-butanol/benzene RS = 30/20 = 1.5

Note that (i) The plate numbers are slightly higher when half-height peak

widths are used to calculate them due to peak tailing increasing

Wb, hence comparisons of efficiencies are valid only if the same

formula is used throughout.

(ii) The cyclohexane and t-butanol peaks are not fully resolved

(RS=1.2), but the t-butanol and benzene peaks have baseline

resolution (RS=1.5).

There are three approaches to qualitative chromatographic analysis, viz

● Comparison of retention datafor unknown solutes with corresponding data

for standards (known substances) obtained under identical conditions.

For planar chromatography (PCand TLC), retardation factors, Rfvalues,

for standards and unknowns are compared by chromatographing them

simultaneously so as to eliminate variations in laboratory materials and

conditions. For column separations, retention times, tR, or volumes, VR, are

compared by chromatographing standards and unknowns sequentially

under stable conditions with as little time between runs as possible.

● Spiking samples with known solutes.

For column separations where samples are known to contain certain

solutes, a comparison is made between two or more chromatograms run

under identical conditions. The first is of the original sample, and subsequent

ones are obtained after adding a spikeof one of the known solutes. Any peak

in the original chromatogram that is of increased size in a subsequent one

can then be identified as the corresponding spiked solute. However,

unambiguous identifications may not be possible.

● Interfacingthe chromatograph with a spectrometer (Section F).

For column separations, this provides spectral information for each sepa-

rated solute in addition to retention data. Spectra of unknown solutes can be

compared with those in computerized library databases or interpreted

manually, even when pure standards are not available.

Comparisons of retention data alone are not always reliable as many

substances can have identical chromatographic behavior. Two or more

comparisons made under different chromatographic conditions, e.g. different

stationary or mobile phases, reduce the chances of making an incorrect identifi-

cation.

For quantitative chromatographic analysis, in addition to ensuring stable

and reproducible conditions for sample preparation and chromatography,

further specific requirements must also be met viz:

● the analyte (solute) must be identified and completely separated from other

components in the chromatogram;

● standards of known purity must be available;

● a recognized calibration procedure must be used.

For planar chromatography, solute spot areasor densities can be measured

in situ, or the solute spots can be removed, dissolved and measurements made

by another analytical technique such as UV spectrometry(Topic E9).

For column separations, quantitation can be by peak area, peak height or

Qualitative and
quantitative
analysis

2 DtR





(W 1 W 2 )

D2 – Principles of chromatography 129