Handbook of Size Exclusion Chromatography and Related ...

operate at aflow rate that can easily be maintained with the available HPLC
instrumentation. Recent studies have demonstrated that capillary SEC columns
can be packed with equivalent or higher efficiencythan SEC columns of standard
dimensions. An example is shown in Fig. 2, in which the efficiency of 28- and
50-mm ID columns were evaluated using bovine serum albumin (BSA), chicken
ovalbumin, and bovine a-chymotrypsinogen as test solutes at linear velocities
(based on atotally excluded solute) varying from 0.01 to 0.9 mm/s (49). The
microcolumnswerepackedwith4.5-mm,150 A ˚ ,ZorbaxGF-250XLparticlesthat
were treated with azirconium salt and derivatized with adiol functionality.The
diffusion coefficients ( 10 7 cm 2 /s) for these proteins, ranging in molecular
weight from 69,000 to 43,000 and 26,000, were experimentally determined to be
5.65,6.68,and8.23,respectively.Notethattheoptimumreducedplateheightwas
as low as 2for BSA and as high as 4for a-chymotrypsinogen. In all cases, the
reduced velocity at hmin was approximately 5. As measured by the half-height
method, the efficiency of a30 cm 50 mm ID column compared favorably with
that of astandard 25 cm 9.4 mm ID column filled with the same packing
material, and the performance of the capillary column was much better when
calculated by statistical moments or based on the Dorsey–Foley equation (50).
BecauseofthelargerIDwhenoperatingastandarddiameterSECcolumnat
aflowrateof1mL/min,thelinearvelocityis2.5timeslowerthanwhenthesame
flowrateisusedona4.6-mmIDcolumn.Thus,anSECcolumnisoperatedcloser
to the velocity at which the column performs at optimal efficiency.As discussed,
however, at least a10-fold drop in flow rate is required for the column to perform
near its optimum for most proteins. This effect is illustrated in Fig. 3, in which a
protein test mixture is separated at various flow rates on a25 cm 4.1 mm ID
column packed with 10-mm, 250 A ˚ ,amide-bonded silica (51). Clearly,resolution
improves with decreasing flow rate: the optimum efficiency had not yet been
reached at aflow rate of 65 mL/min or alinear velocity at 0.13 mm/s.
AccordingtoEq.(2),reducedvelocityisinverselyproportionaltothesolute
diffusion coefficient. Under the same conditions, solutes of varying molecular
weightshowoptimalcolumnperformanceatdifferentflowrates.Thisisillustrated
in Fig. 4. The relationship between the logarithm of molecular weight (MW) and
the otimal flow rate is plotted for 50 peptides and glycine (MW 50–10,000)
analyzed under denaturing mobile-phase conditions (52). As shown, the optimal
flow rate is inversely and linearly related to log MW. Over the narrow molecular
weight range, the optimum flow rate decreases roughly 2-fold for a 10-fold
increase in molecular weight.
2.5 Porosity
Except for nonporous particles, all packing materials contain a variation of pore
sizes around a mean value. This pore size distribution determines the range of
© 2004 by Marcel Dekker, Inc.