Page 211 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

P1: ZCK/MBQ P2: GQT Final Pages
Encyclopedia of Physical Science and Technology EN008B-382 June 30, 2001 18:58

678 Liquid Chromatography

TABLE I Quantitative Column Comparison at Optimum tion of acidic compounds such as anti-inflammatory drugs
Velocity a (aspirin, acetoaminophen, and ibuprofen) or basic drugs
Column type N/column N/m N/sec N/psi such as antihistamines. Zirconia columns provide higher
plate counts than polymeric columns and comparable ef-
Conventional 21800 87200 36.8 12.5 ficiencies to silica based columns. Titania has also been
5-µm C-18 silica investigated as a stable HPLC column packing but is less
(250 × 4.6 mm)
well studied.
Microbore 19100 38200 9.05 63.7
10-µm C-18 silica Polymeric packings for HPLC have been developed
(500 × 1.0 mm) largely to overcome the mobile-phase pH constraints of
Small particle 11900 119000 99.2 3.81 silica. In general, for routine use of silica-based pack-
3-µm C-18 silica ings, the pH of the mobile phase must be maintained
(100 × 4.6 mm)
between pH 2 and 7.5. This is because the silica itself
a From McCoy, R. W., and Pauls, R. E. (1982). J. Liq. Chromatogr. can dissolve at alkaline pH, and cleavage of the silox-
5, 1869. Reprinted with permission from Marcel Dekker, Inc. ane bonds holding the functional groups occurs at acid
pH. Another advantage of polymeric packings is that
few residual polar groups, such as nonreacted hydrox-
in Table I. As predicted by the Van Deemter equation, the yls found in silica, are present that can cause peak tail-
number of plates per meter increases as particle diameter ing through hydrogen bonding with certain solutes. Most
decreases. The Van Deemter equation can be expressed as polymer HPLC packings are polystyrene-divinylbenzene
(PS–DVB) resins cross-linked at 10% or greater to en-
HETP = A + Bu + C m u + C s u ,
sure sufficient particle rigidity (Fig. 5). Even so, the col-
where HETP = height equivalent of a theoretical plate, umn pressure limit for most polymers is limited to about
u = mobile phase velocity, A = eddy diffusion term 3000–5000 psi. Most PS–DVB HPLC packings are spher-
dependent on the particle diameter of the packing ical in shape and 5 or 10 µm in size. The pore size and
(d p ), B = longitudinal diffusion term dependent on surface area of PS–DVB particles can also be controlled
the diffusion constant of the analyte in the mobile similar to the range of values given for silica. However,
phase (D m ), C m = the mass transfer term involving the generally polymeric packings are less efficient (20,000–
solute from the bulk mobile phase to the surface of the 50,000 plates/m) than silica packings. Because of the
2
stationary phase and is dependent on d /D m , and C s aromatic (moderately nonpolar) nature of PS–DVB, it is
p
is the mass transfer of the solute into and out of the used primarily for reversed-phase HPLC unless chemi-
stationary phase and is dependent on the square of the cally modified. Functionalization of PS–DVB resins for
film thickness (d f ) of the stationary phase. A good rule of use as ion-exchange packings is the other dominant use of
thumb is N ≈ 3000 L /dp, where L = column length (in these supports. Other polymers such as polyvinylpyridine,
centimeters) and dp = particle diameter (in micrometers). polyacrylamide, polyvinylalcohol, and various floropoly-
The small particle column was lowest in plates generated mers such as derivatized polychlorotrifluoroethylene have
per unit pressure, indicating high-column back pressure also been used as reversed-phase packings. Further de-
could be a problem with some mobile phases. tails of the importance of polymeric supports to ion-
Silica can be used as is for normal phase HPLC in exchange chromatography in particular are provided in
which the mobile phase is nonpolar in nature, such as hex- Section III.
ane/chloroform and the column-packing surface is polar.
For reversed-phase HPLC in which the mobile phase is
F. Column Packing Technique
polar, such as water or methanol, and the column-packing
surface is nonpolar, the silica must be chemically altered. Both the dry-fill and wet-fill packing methods have been
Silanization reactions are carried out to covalently attach used to prepare HPLC columns. The dry-fill approach
long chain hydrocarbon groups to the silica surface. Fur- involves vertical tapping of the column with simultane-
ther details of the importance of silica supports are pro- ous rapping along its side to cause good consolidation
vided in Section III, “Separation Techniques.” of the column bed. This procedure is recommended for
Porous zirconia particles coated with polybutadiene the packing of rigid particles with a diameter greater than
make a reversed-phase HPLC column packing that offers 20 µm, such as pellicular spheres. Small porous particles
both excellent pressure stability and chemical inertness have high surface energies with respect to their mass and
throughout the entire pH range. Because of the complex clump together when attempted to be packed dry. Prepara-
surface chemistry of zirconia, mobile phase additives such tive HPLC columns are often made using this “tap-filled”
as phosphate or fluoride are added to facilitate the separa- method.