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Independently Tested at The University of Aberdeen, UK An Independent Comparison of Leading C18 Column Brands Introduction
In recent years significant improvements have
been made in the quality of bonded silica particles used in HPLC. An
increased understanding of the chemical nature of the silica surface and
the manufacture of purer silicas have led to a tightening of physical
specifications. Additional improvements have also been made in bonding
and column packing techniques. Consequently, there has recently been a
dramatic increase in the number of improved C18 reversed phase columns
available to the chromatographer.
Problem
With so many different C18 column brands to choose
from, finding the right column for a particular separation can be a very
time consuming and expensive exercise. Comparison of manufacturers
quoted results cannot always be relied upon to accurately predict column
performance under searching test conditions.
Solution
In order to differentiate between columns and
substantiate manufacturers claims, a number of well recognised tests
have been performed on a selection of leading HPLC column brands by an
independent test laboratory. The results reported are for previously
unused columns of identical dimensions, purchased directly from either
the manufacturer or their approved distributor and tested under highly
controlled conditions.
Test Protocol
Three tests have been selected which accurately
represent column performance under searching conditions. Many
"validated" columns are evaluated using such tests, but comparison
between columns is not normally possible due to the slightly different
evaluation conditions employed. Columns have been ranked by efficiency
on all three tests, measured as N 0.1. As shown by Figure 1, this
calculation ensures both column efficiency and peak tailing are
considered -factors which affect the final resolution obtained. An
overall ranking has then been determined by averaging the ranking of
each column on each test.
Independent Test Conditions
| Neutral Molecules: |
Column Dimensions:
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15cm x 4.6mm id
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Sample:
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1)uracil 2)dimethyl phthalate 3)toluene 4)biphenyl 5)phenanthrene
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Mobile Phase:
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80:20 MeOH/H2O
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Flow Rate:
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1.0 ml/min
| |
|
Temperature:
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22 °C
| | Basic Molecules:
|
Column
|
15cm x 4.6mm id
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|
Dimensions:
|
| |
|
Sample:
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1)norephedrine 2)nortriptyline 3)toluene 4)imipramine
5)amitriptyline
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|
Mobile Phase:
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80:20 MeOH/25mM KH 2 PO 4 (pH 6.0)
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Flow Rate:
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1.0 ml/min
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|
Temperature:
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22 °C
| | Acidic Molecules:
|
Column
|
| |
|
Dimensions:
|
| |
|
Sample:
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1)uracil 2)4-hydroxybenzoic acid 3)acetylsalicylic acid 4)benzoic
acid 5)2-hydroxybenzoic acid 6)ethyl paraben
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|
Mobile Phase:
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35:65 MeCN/0.1%TFA in H 2 O
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Flow Rate:
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1.0 ml/min
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|
Temperature:
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22 °C
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Ranking - Neutral Molecules 
Summary
Column efficiencies were seen to differ greatly. The
use of high efficiency columns is recommended to reduce analysis time.
Column length can be reduced without loss of resolution.
Ranking - Basic Molecules 
Summary
C18 bonded columns show significant differences in
chromatography for basic compounds. These variations are generally
caused by undesirable secondary silanol interactions with the silica
surface. Increasing silanol activity results in a deterioration of
chromatographic performance. Highly inert "base deactivated" C18 columns
with very low levels of silanol activity all exhibit similar
selectivity, with differences limited to efficiency and peak shape.
Ranking - Acidic Molecules 
Summary
Testing with acidic molecules shows similar trends to
those seen with basic molecules. Smaller particle size, highly inert
"base deactivated" phases are again seen to offer improved separations.
Final Ranking
The results from all three tests have been
analysed and compared. As an aid to column comparison, a final ranking
has been determined by averaging the test rankings for each column (see
Table 1). This final ranking of column performance is illustrated by
Figure 2.
|
.
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Test Ranking Position
|
.
|
|---|
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Column Type
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Neutral
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Basic
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Acidic
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Average
|
|---|
|
.
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Symmetry 3.5 C18
|
2
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2
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2
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2
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.
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Prodigy 5 ODS-3
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8
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4
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6
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6
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.
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XTerra MS 3.5 C18
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11
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7
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3
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7
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.
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HyPurity 5 C18
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10
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5
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9
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8
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.
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Hypersil 5 ODS
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5
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11=
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10
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8.67
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.
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XTerra MS 5 C18
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12
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8
|
11
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10.33
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Figure 2 - Final Ranking 
Conclusion
Significant differences in efficiency, peak shape
and selectivity are seen with C18 bonded columns when evaluating acidic
and basic compounds. These variations are caused by undesirable
secondary silanol interactions. The selection of a high efficiency base
deactivated phase with very low silanol activity will prove highly
beneficial for the majority of analyses.
ACE® C18 materials were the highest performing 3 µm and 5 µm phases
respectively. Superior column efficiency and peak shape are combined to
provide excellent separations with acidic, basic and neutral molecules.
Trademarks and registered trademarks appearing in this catalogue are
registered with the following companies;ACE -Advanced Chromatography
Technologies;Genesis -Jones Chromatography;Hypersil and HyPurity
-ThemoQuest Corporation;Prodigy -Phenomenex Inc;Spherisorb, Symmetry and
XTerra -Waters Corporation;Zorbax -Agilent Technologies. Comparative
separations may not be representative of all applications.
Further Information
A more comprehensive guide comparing over
100 commonly used C18 phases is now available. Subject areas discussed
include:
-
phase specifications
-
efficiency and selectivity comparisons
-
causes of peak tailing with acidic and basic molecules
-
column reproducibility
-
optimising selectivity
Please contact your distributor now to request your free copy.
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