Page 55 - Subyek Encyclopedia - Encyclopedia of Separation Science

50 I / CHROMATOGRAPHY/ Derivatization

particle diameter is decreased. Consequently, most Supercritical Fluid Chromatography
separations in LC are performed with a total of about
In SFC, mobile-phase modiRcation of the stationary
5000}20000 theoretical plates that is largely inde-
phase and its dependence on Suid density, together
pendent of the particle size. However, since the reten-
with the variation of Suid density along the length of
tion time at a constant (optimum) mobile-phase velo- the column, result in additional sources of zone
city is proportional to the column length, this arbit- broadening that cannot be treated in an exact way.
rary Rxed number of plates is made available in Packed columns used in SFC are identical in type to
a shorter time for shorter columns packed with small- those used in LC. When separations can be achieved
er diameter particles. Thus the principal virtue of with a modest number of theoretical plates (up to
using particles of a small diameter is that they permit about 80 000), then packed columns provide much
a reduction in the separation time for those separ- faster separations, perhaps up to an order of magni-
ations that do not require a large number of theoret- tude, than open-tubular columns, which are generally
ical plates. preferred when very large numbers of theoretical
Conventional column diameters in analytical LC at plates are required.
3}5 mm are comparatively large so as to minimize
zone broadening from extracolumn effects in
earlier instrument designs and have become the de Systems with Electroosmotic Flow
facto standard dimensions, even though instrument Plug Sow in CEC results in a smaller contribution to
capabilities have improved over time. Smaller the plate height from Sow anisotropy and transaxial
diameter columns have been explored to reduce diffusion compared with pressure-driven column
mobile-phase consumption (which is proportional liquid chromatography, while contributions to the
to the square of the column radius) and to enhance plate height that are Sow-proRle-independent are the
mass detection through reduction in peak volumes, same. The absence of a pressure drop in electroos-
but offer no improvement in the intrinsic column motically driven systems provides the necessary con-
efRciency, except perhaps for columns with a ditions to achieve a larger total number of theoretical
low column diameter-to-particle size ratio. Capillary plates in CEC in a reasonable time through the use of
columns of 0.1 to 0.5 mm internal diameter smaller particles and longer columns (see Table 2 and
packed with 3}10 m particles can be used in Figure 11). Under normal operating conditions CEC
relatively long lengths for the separation of complex columns have the potential to provide column plate
mixtures, where a large number of theoretical numbers 5}10 times higher than LC columns. Ulti-
plates is required. Such columns probably minimize mately the performance in CEC is limited by Joule
the contribution form Sow anisotropy while at the heating, which causes additional zone broadening
same time providing a better mechanism for the dissi- and restricts applications of CEC to the use of micro-
pation of heat caused by the viscous drag of the columns, since columns with a small internal dia-
mobile phase moving through the packed bed. The meter ((100 m) are required for efRcient heat
operation of these columns is still pressure-limited dissipation. The dominant cause of zone
and separation times an order of magnitude greater broadening in MEKC is axial diffusion, with signi-
than for GC have to be accepted as the price for high Rcant contributions from slow sorption}desorption
efRciency. kinetics between the analyte and micelles and elec-
The enhancement of intraparticular mass transport trophoretic dispersion arising from the polydispersity
is particularly important for the rapid separation of
biopolymers, whose diffusion coefRcients are
perhaps 100-fold smaller than those of low molecular
Table 2 Achievable theoretical plate numbers in HPLC and
weight compounds in typical mobile phases used in CEC
LC. Also, the high surface area porous packings used
for small molecules may be too retentive for bio- Particle size HPLC CEC
polymers with a signiRcant capacity for multisite in- ( m)
teractions. For these compounds short columns Length Plates/ Length Plates/
column
(cm)
(cm)
column
packed with 1.5 and 2 m pellicular or porous par-
ticles are used for fast separations. Longer columns 5 5 55 000 50 115 000
containing perfusive particles of a large size with 3 25 45 000 50 170 000
large diameter through-pores to promote convective 1.5 10 30 000 50 250 000
transport can also be used for fast separations. Per- Column pressure drop"400 atm for HPLC and the field strength
fusive particles are also used for the preparative-scale (30 kV in CEC for operation at the minimum point in the van
separation of biopolymers. Deemter plot.