Instant Notes: Analytical Chemistry

modes that enable mixtures of either anionic or cationic solutes to be separated.

Size-exclusion (SEC) and chiral chromatography (CC) are two additional

modes used for separating mixtures of high relative molecular mass solutes and

enantiomers, respectively.

Earlier forms of liquid chromatography, used for relatively large scale separa-

tions and known as classical column chromatography, are based on large glass

columns through which the mobile phase flows by gravity compared to the

pressurized systems used in HPLC.

The rate of migration of a solute through a stationary phase is determined by its

distribution ratio, D, which in turn is determined by its relative affinity for the

two phases. In the context of chromatography, Dis defined as the ratio of the

total solute concentration, CS, in the stationary phase to that in the mobile phase,

CM, i.e.

D (1)

Thus, large values of Dlead to slow solute migration, and small values of D

lead to rapid solute migration. Solutes are elutedin order of increasing distribu-

tion ratio. The larger the differences between the distribution ratios of the

solutes in a mixture, the more easily and quickly they can be separated. Because

the interaction of solutes with the stationary phase slows down their rate of

migration relative to the velocity of the mobile phase, the process is described as

retardationor retention.

● Column separations (GC and LC). For the separation of mixtures on

columns, solutes are characterized by the length of time they take to pass

through, i.e. their retention time, tR, or by a retention factor, k, that is directly

proportional to D. The retention time and the retention factor are related by

the expression

tR= tM(1 + k) (2)

where tM(sometimes written as t 0 and known as the dead time) is the time

taken by a nonretainedsolute to pass through the column. A nonretained

solute migrates at the same velocity as the mobile phase, having a distribu-

tion ratio and retention factor of zero; hence tR=tM. Solutes whose Dand k

values are greater than zero are proportionately retarded, having retention

times longer than tM, e.g.

if k= 1, tR= 2 tM

if k= 2, tR= 3 tMetc.

Chromatographic conditions are generally adjusted so that kvalues are

less than about 20, otherwise retention times become unacceptably long.

Practical values of kare easily calculated using a re-arranged equation (2)

k= (3)

For size-exclusion chromatography (Topic D7), the retention volume, VR,

is used to characterize solutes. This is the volume of mobile phase required to

elute the solute from the column. Retention times are directly proportional to

retention volumes at a constant flow rate, so equation (2) can be re-written as

(tR-tM)




tM

CS

CM

Solute migration
and retention

D2 – Principles of chromatography 121