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Reversed phase HPLC is an important technique
for separating largebiomolecules, such as proteins
and peptides. Analysts generally employ C18 sta-
tionaryphases, because these typicallyprovide the
best separations of related compounds, such as
genetic variants of a protein or complex tryptic
digests. However, limitations often are encoun-
teredwhen analyzing samples containing complex
mixtures of closely related analytes.Columns con-
taining wide pore silica (e.g., 300Å) are designed
specifically for largemolecule analyses, addressing
this need formore resolvingpower.
Developed on Viva™ wide pore silica, Viva™
HPLC columns have ideal performance character-
istics for separating largemolecules 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 rankedhighest in retention and selectivity
and produced the best peak symmetry measure-
ments (Table I).
To determine overall separating power, retention,
andpeak shape, we evaluated each columnwith a
protein testmix.TheViva™C18 columnprovided
excellent resolution and peak shapes, as Figure 1
shows.
300Å silicas enhance resolutionof similar or relat-
ed analytes for several reasons. Large poremateri-
als can provide greater retention because higher
molecular weight analytes can enter more of the
pores and accessmore surface area. Theoretically,
the more surface to which an analyte has access,
the longer the retention. For analytes withmolec-
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.
A250-350Åmeanporediameter also is important
because silicas with excessive numbers of pores
smaller than 200Å can be more easily fouled by
ExcellentProteinSeparations fromViva™HPLCColumns
Best PerformanceAmongFiveTestedWidePoreColumns
By Bruce Albright, HPLC Chemist; Vernon Bartlett, HPLCManager; Julie Kowalski, Foods, Flavors, and Fragrances Innovations Chemist;
and BeckyWittrig, Ph.D., HPLC Product MarketingManager
• Best overall performanceamong five columns evaluated.
• Best resolutionandpeak symmetry for test proteins.
• C18, C8, C4, and silica columns available; other phaseson request.
Table I
Viva™wideporeC18 columnsprovide thebest overall
performanceamong 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
ColumnD C18
<50,000
1.49
3.79
80
Figure1
Analysisof a four protein testmix shows the superior
performanceof theViva™C18 column.
Sample:
Inj.:
20µL
Conc.:
0.08mg/mL each protein
Sample diluent: 0.10%TFA inwater / 0.10%
TFA in acetonitrile, 80:20, v/v
Sample temp.: 25°C
Columns:
Dimensions:
150 x 2.1mm
Particle size:
5µm
Conditions:
Mobile phase:
A: 0.10%TFA inwater,
B: 0.10%TFA in acetonitrile,
20%B to 70%B in 30min.
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
ColumnD
Column C
Column B
Column A
LC_0324
Reversed phase test mix; 150 x 2.1mm C18 phase columns, 5µm particles
