Page 217 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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Encyclopedia of Physical Science and Technology EN008B-382 June 30, 2001 18:58

684 Liquid Chromatography

HPLC. The origin of EOF is the electrical double layer that
is formed at the solid–liquid interface due to the negatively
charged silanols of the capillary wall and the positively
charged cations in solution such as Na . Upon applica-
+
tion of an electric field across the capillary, the positive
ions are attracted to the negative detector end of the cap-
illary and move the bulk flow by viscous drag. The ad-
vantage of EOF over conventional pressure driven flow
is that no column backpressure is generated and the flow
profile is pluglike not laminar in nature. Because of the
pluglike flow profile, eddy diffusion is much lower re-
sulting in the optimum HETP value for CEC being better
by about a factor of two as compared to capillary HPLC
(see Fig. 13). Because of no column backpressure, column
length for CEC does not need to be reduced as the particle
size for the packing particles diminishes. For example, if
a 50-cm capillary HPLC column packed with 5-µm par-
ticles can provide 45,000 plates, the same CEC column
will be expected to give 90,000. However, if the parti-
cle diameter is reduced to 1.5 µm, the capillary HPLC
column can be only 15 cm in length providing 33,000
plates. The CEC column packed with 1.5-µm particles
can still be 50-cm long and now 210,000 plates are avail-
FIGURE 12 On-column focusing large volume injection capillary
able. A representative separation by CEC is shown in
LC-UV separation of retinoids. The injection volume was 100
Fig. 14.
◦
µl, the operating temperature 50 C, and the volumetric flow rate
20 µl/min during sample introduction. The concentration of each
retinoid was 50 ng/ml: (1) all-trans-retinol, (2) 13-cis-retinoic acid,
(3) all-trans-retinoic acid. [From Molander, P., Gunderson, T. E.,
Haas, C., Greibrokk, T., Blomhoff, R., and Lundanes, E. J. (1999).
Chromatogr. A 847, 59–68.]
method must be used employing a noneluting mobile
phase at a higher flow rate and temperature to reduce back-
pressure. Once the sample has been loaded, a valve must
be switched to permit the eluting mobile phase to start
the separation process. Capillary LC because of the low
flow rates is particularly well suited for mass spectrometry
detection. The reduced mobile phase flow rate leads to a
decrease in spray droplet size with an increase in electro-
spray ionization efficiency. Because mass spectrometry is
a concentration sensitive technique, the sensitivity advan-
tage as previously mentioned in comparison to standard
analytical columns is also important.
A relatively new technique called capillary electrochro-
matography (CEC) is a hybrid of capillary electrophore-
sis (CE) and capillary LC. CEC is potentially more ver-
satile than CE or HPLC because separation is based on
both mobility differences (if the compounds are charged)
and reversed-phase retention (effective for neutral organ-
ics and charged compounds with hydrophobic moieties). FIGURE 13 Plots of HETP vs linear velocity for HPLC (top) and
CEC (bottom) for 5-µm particles. Plots are calculated for an an-
Mobile-phase transport through a typical 50–200 µmID −5 2
alyte with k = 5 and a diffusion coefficient of 1.5 × 10 cm /s.

capillary packed with 3-µm particles is achieved by elec- [From Dittman, M. M., and Rozing, G. P. J. (1996). Chromatogr. A
troosmotic flow (EOF) instead of a pressure gradient as in 744, 63–74.]