peaks on bonded phase silicas because of adsorptive interactions with residual
silanols and metallic impurities in the silica. The problem is reduced by end-
capping, a process of blocking the sites with trimethylsilyl ((CH 3 ) 3 -Si-) groups
(Fig. 4(b)),and by using highly purified silica (<1 ppm metals).
Size exclusion and ion-exchange stationary phases are either silica or
polymer based. Sulphonic acid or quaternary ammonium groups provide cation
and anion-exchange capabilities respectively, but slow rates of exchange leading
to poor efficiencies and low sample capacities have limited their use, except for
ion chromatography(Topic D7).
Chiralstationary phases have been developed for the separation of mixtures
of enantiomers but are expensive and have a very limited working life.
The availability of a wide range of bonded phases together with polymeric
materials, has resulted in the development of a number of modesof HPLC
(Topic D7). The more important stationary phases and their characteristics are
summarized in Table 3.160 Section D – Separation techniques
Table 2. A comparison of conventional and microbore HPLC columns
Conventional columns Microbore columns
Tubing Tubing
Stainless steel Stainless steel
Lengths 3, 10, 15, 20 and 25 cm Lengths 25 and 50 cm
Coupled lengths 1 m or more
(^1) ⁄ 4 ≤outside diameter (^1) ⁄ 4 ≤outside diameter
Internal diameter 4.6 mm Internal diameter 1 or 2 mm
Stationary phase (packing) Stationary phase (packing)
Porous, microparticulate silica, chemically-modified Porous, microparticulate silica, chemically-modified silica
silica (bonded phases) or styrene/divinyl benzene (bonded phases) or styrene/divinyl benzene co-polymers
co-polymers
Mean particle diameters 3, 5 or 10 mm with a narrow Mean particle diameters 3, 5 or 10mm with a narrow
range of particle sizes range of particle sizes
Operating pressures Operating pressures
500–3000 psi (35–215 bar) 1000–5000 psi (70–350 bar)
Typical mobile phases Typical mobile phases
Hydrocarbons +chlorinated solvents or alcohols for Hydrocarbons +chlorinated solvents or alcohols for
normal-phase; methanol or acetonitrile +water or normal-phase; methanol or acetonitrile +water or
aqueous buffers for reversed-phase aqueous buffers for reversed-phase
Flow rate 1–3 cm^3 min-^1 Flow rate 10–100ml min-^1
Modified instrumentation
Solvent delivery system capable of accurate flow control
down to 10ml min-^1 or less
Small volume sample injection valves
Small volume detector cells
Performance Performance
Efficiency increases with diminishing particle size, Very efficient and sensitive, but slow
but column life for 3 mm particles is shorter
Separations on 3 cm fast columnsin less than Solvent consumption only a quarter that of conventional
1 minute columns