Transcript An Efficient Approach to Column Selection in HPLC Method

A Chromatographic Comparison of
Silica-C18 HPLC Columns
Charles H. Jersild
Alltech Associates, Inc.
2051 Waukegan Road • Deerfield, IL 60015
Phone: 1-800-ALLTECH • Web Site: www.alltechWEB.com
PP044
Introduction
Silica-based C18 columns are the most commonly used columns for
HPLC. The number of available silica-based C18 columns has risen
greatly in the past few years and now stands at more than 150. The
proliferation of C18 columns has made it difficult to choose the right
column for an application or as an appropriate backup column.
Typically chromatographers choose HPLC columns by comparing the
packing media specifications supplied by the manufacturer (i.e. surface
area, particle size and shape, carbon load, endcapping, pore diameter,
pore volume, bonding density, bonding type, etc.). This type of
comparison, although sometimes useful, can not be used to accurately
predict a column’s performance. Column selectivity and peak shape are
largely influenced by the underlying silica, and to a lesser extent, the
bonding procedure used rather than the packing material’s physical
characteristics. Therefore, the best way to compare columns is by their
chromatographic performance.
In 1995, Steffeck and co-workers1 developed a chromatographic test
and graphed the results in order to simplify comparisons of silica-based
C18 and C8 HPLC columns based on their retention, symmetry, and
selectivity for polar and non-polar compounds. The test mix used in
that work contained acidic, basic, and neutral probes. In this study, we
used a similar approach using a different test mix, and we ran the tests
at two different pH values (pH 2.5 and pH 7). Additionally, we used a
test mixture containing polyaromatic hydrocarbons to assess the
columns’ shape selectivity as described by Sander and Wise2.
To compare column performance for a wide variety of compounds under
both acidic (pH 2.5) and neutral (pH 7.0) conditions we used the test
mixture shown in Figure 1. It includes polar and non-polar probes that
are acidic, basic, or neutral. Each probe has a specific purpose in the
test mix. Uracil is used as a void-volume marker. 3-Butylpyridine is a
basic amine that tests the silanol activity toward bases. Tailing of this
peak is an indicator of interaction with acidic silanols.
Phenol is a weak acid used in conjunction with 3-butylpyridine to test
the activity of the underlying silica. Their elution order is an indicator
of the degree of silanol exposure. Columns with little silanol exposure
retain phenol longer than 3-butylpyridine. A reversal in elution order
indicates that there is a significant population of active silanols.
4-Phenylbutyric acid is a carboxylic acid that tests silanol activity
towards acids. This peak tails when silanol activity towards acids is
significant. 4-Phenylbutylamine is a strong base that tests silanol
activity towards amines. N,N-diethyl-m-toluamide (DEET) is another
polar probe. The selectivity of this weakly basic molecule with a neutral
probe such as propylbenzene is good indicator for the degree of
endcapping on a bonded phase. Non-endcapped phases give lower
alpha values for this pair than do endcapped phases. Quinizarin is a
chelator. It is used to check for residual metals in the packing. When
residual metals are present, quinizarin will either tail or irreversibly
adsorb onto the column packing. Propylbenzene and butylbenzene are
neutral probes that test the capacity and hydrophobic selectivity of the
bonded phase.
To assess column shape selectivity we used an empirical test described
by Sander and Wise2. The test uses Standard Reference Material® 869a
(SRM 869a) shown in Figure 2, which contains three polyaromatic
hydrocarbons (PAH). We added acetone as a void volume marker. The
relative retention of Benzo[a]pyrene (BaP) and 1,2:3,4:5,6:7,
8-tetrabenzonaphthalene (TBN) is a measure of columns’ shape
selectivity. The shape selectivity values obtained using this test have
been shown to be an indicator of columns’ bonding type (monomeric or
polymeric)2.
We tested 116 silica-based C18 (or similar) HPLC columns using the
test mixes previously described. Sample retention, selectivity and peak
shape for these probes reflect the hydrophobicity of the bonded phase,
the activity of the underlying silica, and the bonding type. Retention
and peak shape were compiled into charts, and shape selectivity data
was tabulated to simplify column comparisons.
Figure 1 - Test Probes used at pH 2.5 and pH 7
O
H
N
N
HN
NH2
O
Uracil
3-Butylpyridine
4-Phenylbutylamine*
OH
O
OH
4-Phenylbutyric acid**
O
OH
Phenol
Propylbenzene
O
N
O
Quinizarin
OH
N,N-Diethylmeta-toluamide
Butylbenzene
* 4-Phenylbutylamine was not used for testing at pH 2.5
** 4-Phenylbutyric acid was not used for testing at pH 7
Figure 2 - Shape Selectivity Test Probes
Benzo[a]pyrene,
BaP
1,2:3,4:5,6:7,8Tetrabenzonaphthalene,
TBN
Phenanthro[3,4-c]phenanthrene,
PhPh
Materials and Equipment
Instrumentation
–
–
–
–
On-Line Degassing System, Alltech Associates, Inc. (Deerfield, IL)
Alltech Model 525 HPLC Pump, Alltech Associates, Inc.
Alltech Model 580 Autosampler, Alltech Associates, Inc.
12-Position Electrically Actuated Valve, Valco Instruments, Co., Inc.
(Houston, TX)
– JB-1000 Column Oven, Jordi Associates, Inc. (Bellingham, MA)
– Linear Model UVis-205 Absorbance Detector, ThermoQuest Corp.
(San Jose, CA)
Data Collection
– Chrom Perfect™ Magellan software version 3.54, Justice Innovations
(Mountain View, CA)
Reagents and Samples
– HPLC Grade water was produced in-house using a Millipore
(Bedford, MA) Elix® purification system.
– HPLC Grade Acetonitrile was purchased from VWR Scientific Products
(S.Plainfield, NJ).
– Uracil, phosphoric acid, potassium phosphate, monobasic anhydrous,
and potassium phosphate, dibasic trihydrate were purchased from
Sigma (St. Louis, MO).
– N,N-diethyl-m-toluamide, quinizarin, propylbenzene, butylbenzene,
4-phenylbutyric acid, 4-phenylbutylamine, 3-butylpyridine, and phenol
were purchased from Aldrich Chemical Co. (Milwaukee, WI).
– Standard Reference Material® 869a (SRM 869a) was purchased from
National Institute of Standards & Technology (Gaithersburg, MD).
HPLC Columns
– Table 1 lists the columns used and their suppliers. All of the columns
had dimensions of 150mm (length) x 4.6mm (ID) except the Resolve™
C18 (3.9mm ID). The particle size of all the packings was 5µm with
the exception of Genesis® C18 (4µm) and µBondapak® C18 (10µm).
Table 1 - Listing of Columns Tested
The columns tested in this study and where they were obtained are listed below:
Alltech Associates, Inc. (Deerfield, IL)
Adsorbosil® C18, Adsorbosphere® C18, Adsorbosphere® HS C18, Adsorbosphere® UHS C18,
Adsorbosphere® XL C18, Adsorbosphere® XL C18-B, Allsphere™ ODS-1, Allsphere™ ODS-2, Alltima™ C18,
Alltima™ C18-LL, alphaBond™ C18, Spheri-5® ODS, Spheri-5® RP-18, Brownlee™ Validated™ C18,
Econosil™ C18, Econosphere™ C18, Exsil™ ODS, Exsil™ ODS-B, Hypersil® 100 C18, Hypersil® BDS C18,
Hypersil® ODS, Inertsil® ODS-2, Inertsil® ODS-3, Kromasil™ C18, LiChrosorb® C18, LiChrosorb® RP
Select B, LiChrospher® 100 RP-18 (endcapped), LiChrospher® 100 RP-18, LiChrospher® RP Select B,
Nucleosil® C18, Nucleosil® C18AB, Nucleosil® C18HD, Nucleosil® PROTECT, Partsil® ODS-2, Perkin Elmer
HS C18, Perkin Elmer Reduced Activity C18, Partisil™ ODS-3, Platinum™ EPS C18, Platinum™ C18,
Ultrasphere® C18, Waters Spherisorb® ODS-1, Waters Spherisorb® ODS-2, Partisphere™ RTF
Bio-Rad® Laboratories (Hercules, CA)
Bio-Sil® HL 90-5
Eichrom Technologies, Inc. (Darien, IL)
SynChropak® RPP-100, SynChropak® SCD-100
ES Industries (West Berlin, NJ)
AquaSep™ C8
Higgins Analytical, Inc. (Mountain View, CA)
CLIPEUS C18, HAISIL 100 C18, TARGA C18
Jones Chromatography (Lakewood, CO)
APEX® Basic, APEX® II ODS, APEX® ODS, Genesis® C18
Keystone Scientific, Inc. (Bellefonte, PA)
AQUASIL C18, BetaBasic® C18, BETASIL® C18, PRISM® RPN, PRISM® RP
MAC-MOD Analytical, Inc. (Chadds Ford, PA)
ACE® 5 C18, Eclipse® XDB-C18, HydroBond™ PS C18, ProntoSIL C18-H, ProntoSIL ODS-AQ, Zorbax®
Bonus RP, Zorbax® Extend C18, Zorbax® ODS, Zorbax® Rx C18, Zorbax® SB-C18
Table 1 - Listing of Columns Tested (Cont’d)
MetaChem Technologies (Torrance, CA)
Abzelute™ ODS DB, Carbosorb™ C18, MetaSil™ AQ C18, MetaSil™ Basic, MetaSil™ ODS, MetaSil™
MonoChrom™ C18, Polaris™ C18A
Phenomenex (Torrance, CA)
Columbus™ C18, Cosmosil™ C18AR, Cosmosil™ C18MS, Kingsorb™ C18, Luna® C18, Luna® C18(2),
PrimeSphere™ C18, Prodigy™ ODS-2, Prodigy™ ODS-3
Restek Corp. (Bellefonte, PA)
Allure™ C18, Ultra C18, Ultra IBD
The Separations Group, Inc. (Hesperia, CA)
Vydac® 201SP C18
SGE Incorporated (Austin, TX)
Wakosil™ C18RS
Supelco (Bellefonte, PA)
Discovery® Amide C16, Discovery® C18, Supelcosil™ ABZ+, Supelcosil™ LC18 DB, Supelcosil™ LC18,
Suplex pKb-100
Thermo Hypersil, Inc. (Runcorn, U.K.)
HyPURITY® Advance, HyPURITY® Elite
TosoHaas (Montgomeryville, PA)
TSK™- GEL ODS80TS
Varian, Inc. (Walnut Creek, CA)
ChromSpher 5 C18, Microsorb 100, OmniSpher C18
Waters Corp. (Milford, CT)
J’Sphere™ ODS M80, Novapak® C18, Resolve™ C18, Symmetry® C18, SymmetryShield™ C18,
µBondapak® C18, XTerra™ RP18, XTerra™ MS18, YMCbasic™, YMCPack ODS-A™, YMCPack ODS-AL™,
YMC Pack ODS-AM™, YMCPack ODS-AQ™, YMCPack PRO C18™
Experimental
Each column was subjected to three different chromatographic tests.
Test 1 used the mixture of acidic, basic, and neutral probes listed in
Figure 1 and a pH 2.5 potassium phosphate buffer/acetonitrile mobile
phase. Test 2 used a similar test mix and a pH 7.0 potassium phosphate
buffer/acetonitrile mobile phase. Test 3 used the SRM® 869 test mix for
shape selectivity determination. Test conditions are described in more
detail in the HPLC Conditions section.
Mobile phase was prepared in one large batch for each test. Concentrated
stock solutions of the individual sample probes used in tests 1 and 2 were
prepared by dissolving reagents in mobile phase. Known amounts of
these stock solutions were mixed and diluted in mobile phase to make-up
the test mixes. The shape selectivity test mix (SRM® 869a) was obtained
pre-mixed and we added acetone as a void marker. The concentrations of
individual probes in all of the test mixes were varied such that we could
use peak areas to help in identifying peaks. Individual samples of the
test probes having the same concentrations as in the test mixes were also
prepared. We injected these individual test probes when necessary for
peak identification and to isolate peaks for tailing factor calculations.
All of the columns used in this study were previously unused and were
either freshly packed by Alltech Associates, Inc. or recently purchased.
Prior to each experiment, the columns were flushed with at least 15
column volumes of mobile phase. Each test mix was injected twice on
each column. Reproducibility of the injections was checked to confirm
that columns were fully equilibrated. The testing was done using an
automated system capable of unattended testing of 12 columns in
succession.
We calculated capacity factors using k´= (tr – t0)/t0, where tr is the
retention time of the peak of interest and t0 is experimentally determined
void time. We calculated tailing factors (T) using the United States
Pharmacopoeia formula, T = W0.05/2f, where W0.05 is the peak width at
5% peak height and f is the distance at 5% height from the leading edge
of the peak to a perpendicular drawn from the peak maximum to the
baseline. Shape selectivity or TBN/BaP was calculated as the ratio of
capacity factors k´TBN/k´BaP.
Test 1
HPLC Conditions
Mobile Phase:
Flowrate:
Column Temperature:
Detection:
Injection Volume:
Sample (mg/mL):
Acetonitrile:20mM Potassium Phosphate,
pH 2.5 (65:35)
1.0mL/min
30°C
UV at 254nm
10µL
Uracil (0.025), 3-butylpyridine (0.067),
phenol (0.90), 4-phenylbutyric acid (2.1),
N,N-diethyl-m-toluamide (0.66), quinizarin
(0.20), propylbenzene (5.0), and
butylbenzene (6.7) dissolved in mobile phase
Test 2
HPLC Conditions
Mobile Phase:
Flowrate:
Column Temperature:
Detection:
Injection Volume:
Sample (mg/mL):
Acetonitrile:20mM Potassium Phosphate,
pH 7.0 (65:35)
1.0mL/min
30°C
UV at 254nm
10µL
Uracil (0.05), 3-butylpyridine (0.02),
phenol (1.0), 4-phenylbutylamine (4.0),
N,N-diethyl-m-toluamide (1.0), quinizarin
(0.20), propylbenzene (4.0), and
butylbenzene (4.0) dissolved in mobile phase
Test 3
HPLC Conditions
Mobile Phase:
Flowrate:
Column Temperature:
Detection:
Injection Volume:
Sample:
Acetonitrile:Water (85:15)
2.0mL/min
25°C
UV at 254nm
10µL
SRM® 869a (contains: Benzo[a]pyrene,
1,2:3,4:5,6:7,8-tetrabenzonaphthalene, and
phenanthro[3,4-c]phenanthrene), and Acetone
(added as void marker)
Results and Discussion
Test 1
Figure 3a shows a chromatogram that is typical of those obtained with
base-deactivated columns. Figure 3b shows a chromatogram that is
typical of those obtained using columns developed before basedeactivation techniques were available. These two chromatograms differ
in 3 aspects: peak order, peak shape, and retention. In Figure 3a, the
basic probe, 3-butylpyridine, elutes before phenol. In Figure 3b,
3-butylpyridine elutes after phenol. Figure 3a shows sharp, symmetrical
peaks for all of the test probes. In Figure 3b, 3-butylpyridine and the
metal chelator, quinizarin, tail significantly. In Figure 3a, the overall
retention times are higher than the retention times in Figure 3b.
Peak order and peak shape of the polar compounds is related to the
activity of the underlying silica. In Figure 3a, 3-butylpyridine elutes
prior to phenol with good peak shape on the base-deactivated column.
In Figure 3b, the same peak elutes later with poor peak symmetry.
From these results we can conclude that the base-deactivated column
(3a) is less active towards polar amines than the non base-deactivated
column (3b). Neither of the columns showed activity towards acids as
they both give good peak shape for the acidic probe (4-phenylbutyric
acid).
The retention and peak shape of the chelator, quinizarin, is related to
the amount of metal impurity in the underlying silica. Most basedeactivated phases have low levels of metal impurities, so the
quinizarin peak shape is symmetrical as in Figure 3a. Some of the
older type silicas have higher levels of metal impurities. These columns
tend to give poor peak shapes for quinizarin (see Figure 3b), or in
some cases irreversibly adsorb the compound.
Test 2
Figures 4a and 4b show chromatograms obtained at pH 7 with the
same base-deactivated and non-base-deactivated columns shown in
Figures 3a and 3b.
The chromatogram in Figure 4a in typical of what was observed for
base-deactivated type columns. Peak shapes were all symmetrical
except for 4-phenylbutylamine, which has an odd peak shape that we’re
unable to explain. The elution order is slightly different at pH 7.0 than
it was at pH 3.0. 3-Butylpyridine elutes later in the chromatogram,
indicating the increased silanol activity that is expected at higher pH.
The chromatogram in Figure 4b is typical of what was observed for
many of the non-base-deactivated column types. Quinizarin, the metal
chelator, did not elute from this column at pH 7.0, an indication that the
packing material contains a significant amount of metal impurities.
Also typical is the poor peak symmetry and longer retention of the basic
probes (3-butylpyridine, and 4-phenylbutylamine).
Column Comparisons Charts* based on Tests 1 and 2
Figure 5 is a graphical presentation of data from 25 of the columns we
tested at pH 2.5, while Figure 6 presents data from the same 25
columns at pH 7.0. On the x-axes are test probe capacity factors (k’),
while y-axes show the columns in descending order of butylbenzene
capacity factors (k’). The right-hand side of each chart lists test probes
that that did not elute and test probes that tailed (T  2.0) under our
test conditions.
These charts are helpful for choosing back-up or alternative columns.
We can choose a good backup column by finding columns with similar
retention and selectivity. Alternatively, if we are looking for a
replacement for a column that can not separate a sample adequately,
we can choose one with different retention and selectivity
characteristics.
*Note: Due space limitations, charts containing data for all 116 columns tested were not
included here. Charts containing data for all of the columns tested will be included in
reprints of this poster.
When choosing a column to separate a specific sample, it is important
to compare based on only those test probes that are similar to the
sample components. If the sample contains only non-polar
components, then compare columns based on the retention and
selectivity of the hydrophobic probes (propyl- and butylbenzene). If the
sample is a polar amine or acid then compare columns based on the
3-butylpyridine and DEET, or 4-phenylbutyric acid probes. Additionally,
it is best to compare based on the appropriate pH. If you intend to
work at acidic pH, then use the pH 2.5 comparison chart (Figure 5). If
you plan to work at neutral pH then use the pH 7 comparison chart
(Figure 6).
Test 3
We determined column shape selectivity from the results of test 3. A
shape selectivity value was calculated for each column using the
formula TBN/BaP= k´TBN/k´BaP, where TBN/BaP is shape selectivity, k´TBN is
the capacity factor of 1,2:3,4:5,6:7,8-tetrabenzonaphthalene, and k´BaP
is the capacity factor for Benzo[a]pyrene. Shape selectivity values for
all columns are listed in Table 2. Sander and Wise2 have shown that
these shape selectivity values are good indicators of C18 bonding phase
type. Phases with shape selectivity values  1.7 reflect monomeric-like
C18 bonding. An example is the Platinum™ C18 column with a shape
selectivity value of 1.84 (the chromatogram is shown in Figure 7a).
Phases with shape selectivity values  1 reflect polymeric-like C18
bonding. An example is the Nucleosil® C18AB column with a shape
selectivity value of 0.69 (the chromatogram is shown in Figure 7b).
Shape selectivity values between 1 and 1.7 reflect C18 phases of
intermediate type bonding. These may be densely loaded monomeric
phases or lightly loaded polymeric phases. An example is the Inertsil®
ODS-2 column with a shape selectivity value of 1.36 (the
chromatogram is shown in Figure 7c).
Figures 3 - Chromatograms at pH 2.5
3a
Base-deactivated
9238
3b
Non base-deactivated
1.
2.
3.
4.
5.
6.
7.
8.
Uracil
3-butylpyridine
Phenol
4-phenylbutyric acid
N,N-diethyl-m-toluamide (DEET)
Quinizarin
Propylbenzene
Butylbenzene
Column: Alltima™ C18, 5m, 150 x 4.6mm
Column:
9239
Adsorbosphere™ C18, 5m, 150 x 4.6mm
Figures 4 - Chromatograms at pH 7
4a
Base-deactivated
9324
4b
Non base-deactivated
1.
2.
3.
4.
5.
6.
7.
8.
Uracil
Phenol
4-phenylbutylamine
N,N-diethyl-m-toluamide
(DEET)
3-butylpyridine
Quinizarin
Propylbenzene
Butylbenzene
Column: Alltima™ C18, 5m, 150 x 4.6mm
Column:
9325
Adsorbosphere® C18, 5m, 150 x 4.6mm
Figure 5 - Column Comparison at pH 2.5
Figure 6 - Column Comparison at pH 7
Table 2 - Shape Selectivity
Packing
Abzelute™ ODS DB
ACE® 5 C18
Adsorbosil® C18
Adsorbosphere® C18
Adsorbosphere® HS C18
Adsorbosphere® UHS C18
Adsorbosphere® XL C18
Adsorbosphere® XL ODS-B
Allsphere™ ODS-1
Allsphere™ ODS-2
Alltima™ C18
Alltima™ C18-LL
Allure™ C18
alphaBond™ C18
APEX® Basic
APEX® II ODS
APEX® ODS
AquaSep™ C8
AQUASIL C18
BetaBasic® C18
BETASIL® C18
Bio-Sil® HL 90-5
Brownlee™ Spheri-5® ODS
Brownlee™ Spheri-5® RP-18
Brownlee™ Validated™ C18
Carbosorb™ C18
ChromSpher 5 C18
CLIPEUS C18
Columbus™ C18
Cosmosil™ C18AR
Cosmosil™ C18MS
Discovery® Amide C16
Discovery® C18
Eclipse® XDB-C18
Econosil™ C18
Econosphere™ C18
Exsil™ ODS
Exsil™ ODS-B
Genesis® C18
Shape
Selectivity
1.98
1.95
1.60
1.78
1.99
1.11
1.57
1.76
1.54
1.76
1.83
1.82
1.74
1.94
0.70
1.90
1.78
1.73
1.66
1.69
1.72
1.82
1.55
1.94
1.85
1.99
1.39
2.00
2.14
1.47
1.93
1.62
1.75
2.11
1.57
1.94
1.76
0.76
1.96
Packing
HAISIL 100 C18
HydroBond™ PS C18
Hypersil® 100 C18
Hypersil® BDS C18
Hypersil® ODS
HyPURITY® Advance
HyPURITY® Elite
Inertsil® ODS-2
Inertsil® ODS-3
J'Sphere™ ODS M80
Kingsorb™ C18
Kromasil™ C18
LiChrosorb® C18
LiChrosorb® RP Select B
LiChrospher® RP Select B
Lichrospher® RP18
Lichrospher® RP18 (endcap)
Luna® C18
Luna® C18 (2)
MetaSil™ AQ C18
MetaSil™ Basic
MetaSil™ ODS
Microsorb 100 C18
MonoChrom™ C18
Novapak® C18
Nucleosil® C18
Nucleosil® C18 AB
Nucleosil® C18 HD
Nucleosil® PROTECT
OmniSpher C18
Partisil® ODS-2
Partisil® ODS-3
Partisphere™ RTF C18
PerkinElmer HS C18
PerkinElmer Red. Activity C18
Platinum™ C18
Platinum™ EPS C18
Polaris™ C18A
PrimeSphere™ C18MC
Shape
Selectivity
1.92
2.08
1.99
1.63
1.90
1.30
1.68
1.36
2.24
2.06
2.13
1.73
1.67
1.71
1.66
1.60
1.76
2.14
2.13
1.44
1.61
1.46
1.93
1.60
1.90
1.78
0.69
1.79
1.52
1.38
1.32
1.94
1.10
2.04
1.52
1.84
1.21
1.72
1.83
Packing
PRISM® RP
PRISM® RPN
Prodigy™ ODS-2
Prodigy™ ODS-3
ProntoSIL C18-H
ProntoSIL ODS-AQ
Resolve™ C18
Supelcosil™ ABZ+
Supelcosil™ LC18
Supelcosil™ LC18 DB
Suplex pKb 100
Symmetry® C18
SymmetryShield™ C18
SynChropak® RPP-100
SynChropak® SCD-100
TARGA C18
TSK™-GEL ODS80TS
Ultra C18
Ultra IBD
Ultrasphere® C18
Vydac® 201SP C18
Wakosil™ C18 RS
Waters Spherisorb® ODS-1
Waters Spherisorb® ODS-2
XTerra™ MS18
XTerra™ RP18
YMCbasic™
YMCPack ODS-AL™
YMCPack ODS-AM™
YMCPack ODS-AQ™
YMCPack ODS-A™
YMCPack PRO C18™
Zorbax® Bonus RP
Zorbax® Extend C18
Zorbax® ODS
Zorbax® Rx C18
Zorbax® SB C18
µBondapak® C18
Shape
Selectivity
1.58
1.28
1.93
2.14
2.03
1.62
1.86
1.10
1.91
1.94
1.17
1.60
1.49
1.99
1.57
2.07
2.12
1.76
1.23
1.90
1.98
2.09
1.39
1.80
2.03
1.74
1.82
1.82
1.94
2.08
1.94
2.13
1.84
1.93
1.77
1.54
2.16
1.88
Figures 7 - Shape Selectivity Chromatograms
7b
7a
Platinum™ C18,
5µm, 150 x 4.6mm
Nucleosil® C18 AB,
5µm, 150 x 4.6mm
9321
1.
2.
3.
4.
7c
9320
Inertsil® ODS-2,
5µm, 150 x 4.5mm
Acetone
Benzo[a]pyrene, (BaP)
Phenanthro[3,4-c]phenanthrene, (PhPh)
1,2:3,4:5,6:7,8-Tetrabenzonaphthalene, (TBN)
9326
Conclusion
One-hundred and sixteen silica-based C18 (or similar) columns were
compared using 3 different chromatographic tests. The
chromatographic data was compiled into charts and tables for easy
comparison of columns. These charts and tables simplify selection of
backup or alternative columns based on chromatographic performance
rather than on the physical characteristics of the packing material.
Acknowledgements
Alltech Associates, Inc. acknowledges that this technical communication
represents a considerable investment in time and effort on the part of
many dedicated professionals.
Author(s):
Laboratory Contribution(s):
Editorial Contribution(s):
Graphics Preparation:
Charles H. Jersild
Charles H. Jersild, Michelle Cornelius
Raymond J. Weigand, Robert J. Ziegler
Kimberly Volk, Elizabeth Fisher, and
Julia Poncher
The trademarks referred to herein are the property of their respective owners.
References
1. R.J. Steffeck, “A Comparison of Silica-Based C18 and C8 HPLC
Columns to Aid Column Selection”, LC•GC 13 (9), 720-726 (1995).
2. Sander and Wise, “Evaluation of Shape Selectivity in Liquid
Chromatography”, LC•GC, 8 (5), 378-390 (1990).
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Figure 5 - Column Comparison at pH 2.5
Figure 5 - Column Comparison at pH 2.5 (cont’d)
Figure 5 - Column Comparison at pH 2.5 (cont’d)
Figure 5 - Column Comparison at pH 2.5 (cont’d)
Figure 6 - Column Comparison at pH 7
Figure 6 - Column Comparison at pH 7 (cont’d)
Figure 6 - Column Comparison at pH 7 (cont’d)
Figure 6 - Column Comparison at pH 7 (cont’d)