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Reversed phase HPLC is an important technique
for separating large biomolecules, such as proteins
and peptides. Analysts generally employ C18 sta-
tionary phases, because these typically provide the
best separations of related compounds, such as
genetic variants of a protein or complex tryptic
digests. However, limitations often are encoun-
tered when analyzing samples containing complex
mixtures of closely related analytes. Columns con-
taining wide pore silica (e.g., 300Å) are designed
specifically for large molecule analyses, addressing
this need for more resolving power.
Developed on Viva™ wide pore silica, Viva™
HPLC columns have ideal performance character-
istics for separating large molecules and biomole-
cules. Using a reversed phase test mix, we com-
pared column efficiency, peak asymmetry, and
retention for Viva™ C18 columns and four other
C18 wide pore HPLC columns. The Viva™ C18
column ranked highest in retention and selectivity
and produced the best peak symmetry measure-
ments (Table I).
To determine overall separating power, retention,
and peak shape, we evaluated each column with a
protein test mix. The Viva™ C18 column provided
excellent resolution and peak shapes, as Figure 1
shows.
300Å silicas enhance resolution of similar or relat-
ed analytes for several reasons. Large pore materi-
als can provide greater retention because higher
molecular weight analytes can enter more of the
pores and access more surface area. Theoretically,
the more surface to which an analyte has access,
the longer the retention. For analytes with molec-
ular weights greater than 3000, silica materials
with pore diameters in the 250-350Å range yield
the needed retention. Further, the mean pore
diameter within the distribution (e.g., 250Å vs
350Å) can define the selectivity in some separa-
tions, by changing the elution order for certain
analytes.
A 250-350Å mean pore diameter also is important
because silicas with excessive numbers of pores
smaller than 200Å can be more easily fouled by
Excellent Protein Separations fromViva™ HPLC Columns
Best Performance Among Five Tested Wide Pore Columns
By Bruce Albright, HPLC Chemist; Vernon Bartlett, HPLC Manager; Julie Kowalski, Foods, Flavors, and Fragrances Innovations Chemist;
and Becky Wittrig, Ph.D., HPLC Product Marketing Manager
• Best overall performance among five columns evaluated.
• Best resolution and peak symmetry for test proteins.
• C18, C8, C4, and silica columns available; other phases on request.
Table I
Viva™ wide pore C18 columns provide the best overall
performance among five tested columns.
Efficiency
Asymmetry
Retention Time
Column Pressure
Column
(plates/meter)
(biphenyl)
(biphenyl)
(bar)
Viva™ 300 C18
>50,000
1.16
6.30
60
Column A C18
~50,000
1.46
5.77
72
Column B C18
>50,000
1.46
4.96
102
Column C C18
>50,000
1.30
5.89
66
Column D C18
<50,000
1.49
3.79
80
Figure 1
Analysis of a four protein test mix shows the superior
performance of the Viva™ C18 column.
Sample:
Inj.:
20µL
Conc.:
0.08mg/mL each protein
Sample diluent: 0.10% TFA in water / 0.10%
TFA in acetonitrile, 80:20, v/v
Sample temp.: 25°C
Columns:
Dimensions:
150 x 2.1 mm
Particle size:
5µm
Conditions:
Mobile phase:
A: 0.10% TFA in water,
B: 0.10% TFA in acetonitrile,
20% B to 70% B in 30 min.
Flow:
0.20mL/min.
Temp.:
25ºC (or ambient)
Det.:
UV @ 214nm
Viva
™
Wide Pore C18
(cat. # 9514562)
Peak List
MW Ret. Time (min.)
1. ribonuclease A 13,700
11.31
2. cytochrome c
12,327
14.65
3. holo-transferrin 77,000
16.32
4. apomyoglobin
16,951
20.34
Column D
Column C
Column B
Column A
LC_0324
Reversed phase test mix; 150 x 2.1mm C18 phase columns, 5µm particles