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Excellent Protein Separations from a Wide Pore HPLC Silica with a Narrow Pore Diameter Distribution
by Bruce Albright, Vernon Bartlett, Julie Kowalski, Rebecca Wittrig, Ph.D., Restek Corp.
Reversed phase HPLC is an important technique for separating intermediate to large biomolecules, but limitations are encountered when analyzing complex mixtures or closely related analytes, such as tryptic digests or genetic variants of a single protein. Column packings based on wide pore silicas (e.g., 300 Å) have been designed to address this need for more resolving power because, theoretically, higher molecular weight analytes can enter the pores and access more surface area, promoting greater retention.
Silicas with 250 to 350 Å pore diameters offer the retention needed for analytes with molecular weights greater than 3000. Silicas with excessive numbers of pores smaller than 200 Å can be fouled more easily by larger molecular weight debris, and silicas with a high percentage of pores larger than 500 Å can be impractically fragile for conventional HPLC applications. Also, the mean pore diameter within the distribution (e.g., 250 Å vs. 350 Å) can redefine the selectivity in some separations, by reversing the elution order for certain analytes. A narrow distribution around the mean pore diameter better ensures that selectivity is maintained, and aids in separating closely related analytes that differ only slightly in hydrodynamic size (molecule size in solution).
Among the commercially available materials we have tested, Viva™ wide pore silica provides the greatest available surface area represented by 250 to 350 Å pores, with a highly desirable pore volume and pore diameter distribution (Restek Advantage, 2005 v1, pp 1-2, available on request). Among tested C18 columns, the Viva™ column ranked highest in retention and peak symmetry measurements (Table I). In evaluations to determine overall separating power, retention, and peak shape, the Viva™ column provided excellent resolution and symmetric peaks (Figure 1).
Measurably superior physical characteristics and strong test performances show Viva™ HPLC columns are an excellent choice for analyzing large molecules or biomolecules. Currently, C18, C8, C4, and silica columns are offered; other phases are available on request.
Table I. Viva™ column performs best among tested wide pore columns.
Efficiency
Asymmetry
Retention Time
Column Pressure
Column
(plates/meter)
(biphenyl)
(biphenyl)
(bar)
Viva™ C18
52,146
1.16
6.30
60
Column A
49,720
1.46
5.77
72
Column B
51,813
1.46
4.96
102
Column C
57,326
1.30
5.89
66
Column D
41,666
1.49
3.79
80
Columns: 150 × 2.1 mm, 5 μm particles; sample: 10 μL reversed phase test mix — uracil (0.02 mg/mL), benzene (3.00 mg/mL), naphthalene (0.50 mg/mL), biphenyl (0.06 mg/mL); mobile phase: water:methanol, 25:75 @ 0.2 mL/min.; detection: UV, 254 nm.
Figure 1. Test proteins show the superior performance of the Viva™ C18 column.
Columns: 150 × 2.1 mm, 5 μm particles; sample: 20 μL protein test mix — 1: ribonuclease A (MW 13,700), 2: cytochrome c (MW 12,327), 3: holo-transferrin (MW 77,000), 4: apomyoglobin (MW 16,951); mobile phase: A: 0.1% TFA in water, pH 2.0, B: 0.1% TFA in acetonitrile, 20% B to 70% B in 30 min. @ 0.2 mL/min.; detection: UV, 214 nm. LC_0324
Restek Corp.
110 Benner Circle
Bellefonte, PA 16823
Restek Corporation
110 Benner Circle
Bellefonte, PA, 16823
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