Page 210 - 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

Liquid Chromatography 677

FIGURE 6 Effects of particle size on column performance: (a) 10 µm, (b) 5 µm, and (c) 3 µm. Ultrasphere C-18
columns: (a) 30 cm × 4.6 mm, (b) 15 × 4.6 mm, and (c) 7.5 × 4.6 mm. Mobile phase: 60–40 methanol–water. Flow
rate = 1 ml/min. Temperature = 30 C. Pressure: (a) 810 psi, (b) 1600 psi, and (c) 2250 psi. Peaks: (1) Phenol, (2)
◦
Benzaldehyde, (3) Acetophenone, (4) Nitrobenzene, (5) Methylbenzoate, (6) Anisole, (7) Benzene, and (8) Toluene.
[Reprinted with permission from Beckman/Altex Scientific.]

The pore size of the silica particles must be large enough to be effective as column packings for the reversed-phase
to permit easy entrance and exit of the sample molecules. separation of proteins and are commercially available.
However, since pore size is inversely proportional to the As can be seen in Fig. 6, the advantages of columns
surface area of the packing, the pores should not be exces- packed with smaller particles are faster analysis times,
sively larger than the sample components of interest. For improved solute sensitivity, and decreased solvent con-
relatively small organic or inorganic molecules, a pore size sumption. It can be shown that the peak height maximum
˚
of 60 A is sufficient. For the separation of large molecules (C max ) can be calculated from the following equation,
0.5
˚
such as polymers or proteins, a pore size of at least 250 A C max = (C s V s /V r )(N/2π) , where C s and V s are the con-
is preferred. centration and volume of sample injected respectively, V r
Packing materials for the HPLC separation of is the retention volume, and N = number of theoretical
˚
biomoleculesthathavenotonly500–1500 Aporesbutalso plates. The number of theoretical plates can be calculated
˚
2
a network of 6000–8000 A transecting tunnels have been easily from the chromatographic data as N = 16(t r /w b ) ,
developed by Regnier. These highly porous materials, where t r = peak retention time and w b = width of the peak
when packed into columns, permit mobile phase veloci- at baseline in time units. Because V r is proportional to the
2
ties 2–5 times higher than those for conventional wide pore volume of the column (πr × L, where r = column ra-
silica columns. For the rapid reversed-phase separation of dius and L = column length), C max will increase propor-
peptides and proteins, both small 2-µm porous wide pore tionally to the ratio of the square of the radii as column ra-
˚
(200 A) silica and 2-µm pellicular silica microspheres dius decreases. Going from a column of 4.6 mm to one of
packed in 3-cm columns have been developed. For a five 2.1 mm and assuming all other variables are constant, the
component mixture of proteins, separation times of less improvementinC max canbepredictedtobealmost5times.
than a minute were possible. Nonporous monodisperse An efficiency comparison of different octyldecyl (C-18)
1.5-µm silica beads developed by Unger have been shown modified silica columns using plate count (N) is shown