Stationary Phase Specifications | Phases Compared According to Relative

Hydrophobicity | Phases Compared According to Relative Polarity | Categorization

of Phases According to Hydrophobicity and Polarity | Comparison of Column
Efficiency for a Neutral Compound | Comparison of Column Efficiency for
Basic Compounds | Phases Grouped According to Silanol Activity | Comparison of
Phases According to Metal Activity
Fourth Edition June 2008
Comparison Guide to C18
Reversed Phase HPLC Columns
Comparison Data on Commonly Used C18 Phases
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MAC-MOD Analytical
103 Commons Court
P.O. Box 587
Chadds Ford, PA 19317
Phone: 1.800.441.7508
Fax: 1.610.358.5993
E-mail: info@mac-mod.com
Web Site: www.mac-mod.com
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Comparison Guide to C18 Reversed Phase HPLC Columns
Introduction
Tere are so many diferent C18 columns to choose from,
that fnding the right column for a particular separation
can be very time consuming and expensive. Two apparently
similar C18 phases can give very diferent results. For
example, Figure 1 compares the separation of the same
sample mixture on a Hypersil HyPurity C18 and a
Symmetry C18 column under identical mobile phase
conditions. Even though both columns are packed with base
deactivated C18 stationary phases, the band spacing
(selectivity) between peaks is very diferent on the two
columns. Without more information, it is impossible to
predict how the performance of diferent stationary phases
will compare.
Tis Comparison Guide to C18 Reversed Phase HPLC
Columns provides basic comparison information on
commonly used C18 columns to help you more easily
identify similarities and diferences before investing time
and money in chromatographic testing. Hopefully, this infor-
mation will help you fnd the right column for your
application quicker.
Only silica based C18 bonded phases are evaluated in this
Guide. Other bonded phases, such as C8, CN, Phenyl and
polar embedded phases, are excluded.
Tis Guide does not identify an overall best column. Te
column that works best for one application will not
necessarily be the column that will work best for other
applications. And, there certainly is not a single column that
will work best for all applications. However, this Guide can
help you identify columns that are likely to perform well
so that at least you can narrow the number of columns for
chromatographic testing. You may fnd that this Guide helps
you identify several columns that provide good separations
and performance. It is always desirable to have more than
one column identifed for an application, especially if you are
running routine assays.
Increasingly, chromatographers are seeking to identify
alternate brands of HPLC columns suitable for their assays.
Having an alternate column choice for a method reduces
the risk of down time due to column problems such as a
change in selectivity from one manufactured lot to another
or slow supplier delivery. Finding an alternate or back-up
column that will provide acceptable selectivity and
performance when substituted into a method can be as
expensive and time consuming as fnding the right column
for developing an initial separation. It is our hope that this
Guide will make that job easier by identifying columns with
similar chromatographic characteristics.

Figure 1
Apparently Similar C18 Phases Can Give Very Different
Chromatographic Results
Mobile Phase: 60% CH
3
CN
40% 50mM KH
2
PO
4
, pH 3.2
Sample: 1. Uracil 5. N,N-Dimethylaniline
2. Pyridine 6. 4-Butylbenzoic acid
3. Phenol 7. Toluene
4. Dimethyl phthalate
Both Hypersil HyPURITY C18 and Symmetry C18 are base deactivated
phases. You would expect them to provide similar performance, and in some
cases they do. However, in the example given here you can see signifcant
differences in peak retention times, selectivity and even peak shape.
Tis Guide provides the following comparison data on
commonly used C18 phases:
Stationary Phase Specifcations
Specifcations provided by column manufacturers
Phases Compared According to Relative Hydrophobicity
Retention data for hydrophobic and neutral compounds
Phases Compared According to Relative Polarity
Categorization of Phases According to Hydrophobicity
and Polarity
Comparison of Column Efciency for a Neutral
Compound
Comparison of Column Efciency for Basic Compounds
Also measures peak tailing
Phases Grouped According to Silanol Activity
Phases Compared According to Metal Activity

0 1 2 3 4 5 6 7 8 9 10 11
Time (min)
1
2
3 4
5
6
7
0 1 2 3 4 5 6 7 8 9 10 11
Tine (nin)
1
2
3 4
5
6
7
Comparison Guide to C18 Reversed Phase HPLC Columns | 1
Hypersil HyPURITY C18
Symmetry C18
0 1 2 3 4 5 6 7
Tine (nin)
1
2
3
Figure 3
Specifcations of C18 Stationary Phases
Particle Pore Surface Carbon High Purity
Stationary Phase Size (m) Size () Area (m2/g) Load (%) Endcapped Silica
ACE C18 5 100 300 15.5 yes yes
ACE C18-300 5 300 100 9.0 yes yes
ACE C18-HL 5 90 400 20.0 yes yes
Bondapak C18 10 125 330 10 yes no
Capcell Pak AG C18 5 120 300 15 yes no
Capcell Pak UG C18 5 120 300 15 yes yes
Develosil ODS-HG 5 140 300 18 yes yes
Develosil ODS-MG 5 100 450 15 yes yes
Develosil ODS-UG 5 140 300 18 yes yes
Exsil ODS 5 100 200 11 yes no
Exsil ODS1 5 100 200 11 yes no
Exsil ODSB 5 100 200 12 yes no
Gemini C18 5 110 375 14 yes yes
Hichrom RPB 5 110 340 14 yes yes
Hypersil BDS C18 5 130 170 11 yes no
Hypersil GOLD 5 180 200 10 yes yes
Hypersil HyPurity C18 5 180 200 13 yes yes
Hypersil ODS 5 120 170 10 yes no
Inertsil ODS 5 100 350 14 yes no
Inertsil ODS3 5 100 450 15 yes yes
Inertsil ODS2 5 150 320 18.5 yes yes
Kromasil C18 5 100 340 19 yes yes
LiChrosorb RP-18 10 100 300 17 no no
LiChrospher RP-18 5 100 350 21.6 no no
Luna 5 C18(2) 5 100 400 17.5 yes yes
Novapak C18 4 60 120 7.3 yes no
Nucleosil C18 5 100 350 15 yes no
Nucleosil C18 HD 5 100 - 20 yes yes
Nucleosil C18AB 5 100 350 24 yes no
Partisil ODS 10 85 350 5 no no
Partisil ODS2 10 85 350 15 yes no
Partisil ODS3 10 85 350 10.5 yes no
Prodigy ODS2 5 150 310 18.4 yes yes
Prodigy ODS3 5 100 450 15.5 yes yes
Purospher RP18-e 5 80 500 - yes yes
Resolve C18 5 90 200 10 no no
SunFire C18 5 100 340 16 yes yes
Symmetry C18 5 100 335 19 yes yes
TSK ODS-120T 5 120 - 22 yes no
TSK ODS-80TM 5 80 - 15 yes no
Ultrasphere ODS 5 80 - 12 yes no
Vydac 218MS 5 300 70 - yes no
Vydac 218TP 5 300 70 8 yes no
Vydac Selectapore 300M 5 300 70 - yes yes
Vydac Selectapore 300P 5 300 70 - yes yes
Vydac Selectapore 90M 5 90 250 - yes yes
Waters Spherisorb ODS1 5 80 220 6.2 no no
Waters Spherisorb ODS2 5 80 220 11.5 yes no
Waters Spherisorb ODSB 5 80 220 11.5 yes no
XTerra MS C18 5 125 - 15.5 yes
YMC JSphere ODS H80 4 80 510 22 yes no
YMC JSphere ODS M80 4 80 510 14 yes no
YMC ODS A 5 120 300 17 yes no
YMC ODS AM 5 120 300 17 yes no
YMC Pro C18 5 120 335 16 yes yes
Zorbax Extend C18 5 80 180 12.5 yes yes
Zorbax ODS 5 70 330 20 yes no
Zorbax Rx-C18 5 80 180 12 no yes
Zorbax SB-C18 5 80 180 10 no yes
Zorbax XDB-C18 5 80 180 10 yes yes
Figure 2
High Purity Silicas Provide Better
Peak Shape for Basic Compounds
High Purity Silica (ACE C18)
Acidic Silica
(Waters Spherisorb ODS2)
Mobile Phase: 60% CH
3
OH,
40% H
2
O
Sample: 1. Uracil
2. Pyridine
3. Phenol
Interaction between cationic compounds
and acidic silanol sites on the surface of
silica stationary phase supports can
contribute to retention and peak tailing.
Phases made with high purity silica
(less acidic silica) generally can be
expected to provide better peak shape
for basic compounds.
0 1 2 3 4 5
Tine (nin)
1
2
3
2 | Comparison Guide to C18 Reversed Phase HPLC Columns
Figure 4
C18 Phases Compared According to Relative Hydrophobicity
Stationary Phase Specifcations
Stationary phase specifcations provide basic information that can be helpful in deciding
which phases to select for evaluation. For example, phases with high surface area and high
carbon load will generally retain hydrophobic compounds longer than phases with low
surface area and low carbon load. If you are analyzing macromolecules, such as peptides
and proteins, a wider pore (200 300 ) phase usually provides better performance than
a phase with small pores. New high purity silicas usually provide better peak shape for basic
compounds than older, more acidic silicas (see Figure 2). Stationary phase specifcations,
however, will not give you enough information to accurately predict retention or band
spacing (selectivity). Tis is especially true when separating polar compounds.
Inertsil ODS3
LiChrospher RP-18
YMC JSphere ODS H80
Nucleosil C18
Kromasil C18
Develosil ODS-MG
Partisil ODS2
LiChrosorb RP-18
Symmetry C18
Inertsil ODS
Develosil ODS-UG
Purospher RP18-e
YMC Pro C18
Prodigy ODS3
Zorbax ODS
Nucleosil C18HD
SunFire C18
YMC ODS AM
YMC ODS A
Inertsil ODS2
Cosmosil C18-AR
Prodigy ODS2
TSK ODS-80TM
ACE C18
Luna C18(2)
Genesis C18
Nucleosil C18AB
Waters Spherisorb ODS2
Zorbax Extend C18
Develosil ODS-HG
Resolve C18
Zorbax RX C18
Zorbax XDB C18
Waters Spherisorb ODSB
Hypersil BDS C18
Hypersil HyPurity C18
Ultrasphere ODS
TSK ODS-120T
Partisil ODS3
Hichrom RPB
Waters Spherisorb ODS1
Bondapak C18
Exsil ODS
Capcell Pak AG C18
YMC JSphere ODS M80
Capcell Pak UG C18
Zorbax SB C18
Exsil ODSB
XTerra MS C18
ACE C18-300
Hypersil ODS
Hypersil GOLD
Novapak C18
Exsil ODS1
Vydac SelectaPore 90M
Vydac SelectaPore 300P
Vydac 218TP
Partisil ODS
Vydac 218MS
Vydac SelectaPore 300M


Mobile Phase: 90% CH
3
OH, 10% H
2
O
Elution Order: 1. Dimethyl phthalate
2. Toluene
3. Biphenyl
4. Phenanthrene
Column Temperature: 24C
Comparison Guide to C18 Reversed Phase HPLC Columns | 3
lner:s|| ODS3
Furospner RF-!8e
Kronas|| C!8
YMC 'Spnere ODS H80
ACE C!8-HL
Deve|os|| ODS-MC
Synne:ry C!8
Frod|gy ODS3
SunF|re C!8
Zorbax ODS
L|Cnrospner RF-!8
Luna C!8(2)
Zorbax Ex:end C!8
lner:s|| ODS2
Nuc|eos|| C!8
Cen|n| C!8
Nuc|eos|| C!8 HD
Zorbax XDE-C!8
Frod|gy ODS2
Deve|os|| ODS-UC
Zorbax Rx C!8
lner:s|| ODS
\a:ers Spner|sorb ODS2
Far:|s|| ODS2
ACE C!8
YMC ODS A
YMC Fro C!8
YMC ODS AM
XTerra MS C!8
Capce|| Fa| AC C!8
U|:raspnere ODS
Reso|ve C!8
Zorbax SE-C!8
TSK ODS-80TM
\a:ers Spner|sorb ODSE
H|cnron RFE
L|Cnrosorb RF-!8
Deve|os|| ODS-HC
Exs|| ODS
Nuc|eos|| C!8AE
Hypers|| ODS
Hypers|| EDS
YMC 'Spnere ODS M80
TSK ODS-!20T
Far:|s|| ODS3
Hypers|| HyFURlTY C!8
\a:ers Spner|sorb ODS!
Vydac Se|ec:apore 90M
Novapa| C!8
Hypers|| COLD
Exs|| ODS!
ACE C!8-300
Eondapa| C!8
Exs|| ODSE
Vydac 2!8MS
Vydac 2!8TF
Vydac Se|ec:apore 300F
Vydac Se|ec:apore 300M
Far:|s|| ODS
0.0 0.5 1.0
k (ToIuene)
1.5 2.0 2.5
Figure 5
C18 Phases Ranked According to Retention for Toluene
Mobile Phase: 80% CH
3
OH
20% 25mM KH
2
PO
4
, pH 6.0
Sample: Toluene
Temperature: 24C
Phases Compared According to Relative
Hydrophobicity
Hydrophobicity is measured as the retention
of a hydrophobic solute, phenanthrene. Figure 4
gives a comparison of hydrophobicity with the
C18 phases listed according to hydrophobicity.
Notice, however, that the retention for dimethyl
phthalate, the least hydrophobic solute in the
mixture, cannot always be predicted from
the hydrophobicity ranking. Some low
hydrophobicity phases actually have greater
retention for dimethyl phthalate than some
high hydrophobicity phases. We fnd that this
is not unusual when separating polar
compounds. Phases that are signifcantly more
retentive for hydrophobic analytes may show
only slightly more retention for polar
compounds than low hydrophobicity phases,
and sometimes they show less.

Alternative Test for Hydrophobicity
Toluene can also be used as a probe to measure
hydrophobicity. Notice that the ranking of
C18 phases according to retention for toluene
(Figure 5) is slightly diferent from the ranking
according to retention for phenanthrene
(Figure 4).
4 | Comparison Guide to C18 Reversed Phase HPLC Columns
YMC Fro C!8
Deve|os|| ODS-UC
Luna C!8(2)
Cen|n| C!8
Exs|| ODSE
Zorbax XDE-C!8
Deve|os|| ODS-MC
ACE C!8
Capce|| Fa| UC C!8
Deve|os|| ODS-HC
H|cnron RFE
ACE C!8-300
SunF|re C!8
Frod|gy ODS2
Hypers|| HyFur|:y C!8
lner:s|| ODS3
Kronas|| C!8
Nuc|eos|| C!8 HD
ACE C!8-HL
Hypers|| COLD
Frod|gy ODS3
Zorbax Ex:end C!8
lner:s|| ODS
XTerra MS C!8
lner:s|| ODS2
Far:|s|| ODS2
Synne:ry C!8
YMC ODS AM
YMC ODS A
Nuc|eos|| C!8AE
Capce|| Fa| AC C!8
Far:|s|| ODS3
Vydac Se|ec:apore 90M
Nuc|eos|| C!8
U|:raspnere ODS
Vydac Se|ec:apore 300M
\a:ers Spner|sorb ODSE
Furospner RF!8-e
TSK ODS-!20T
YMC 'Spnere ODS M80
YMC 'Spnere ODS H80
Zorbax Rx-C!8
Zorbax SE-C!8
L|Cnrosorb RF-!8
Novapa| C!8
TSK ODS-80TM
Eondapa| C!8
Vydac 2!8MS
\a:ers Spner|sorb ODS2
Exs|| ODS
L|Cnrospner RF-!8
Vydac Se|ec:apore 300F
Vydac 2!8TF
Hypers|| EDS C!8
Far:|s|| ODS
Zorbax ODS
Exs|| ODS!
\a:ers Spner|sorb ODS!
Reso|ve C!8
Hypers|| ODS
0 1 2
k (Fyridine) / k (FhenoI)
3 4 5
High
FoIarity
Low
FoIarity
Figure 6
C18 Phases Ranked According to Polarity
Phases Ranked According to Relative
Polarity
Tere have been several chromatographic tests
suggested for measuring polarity of stationary
phases. Although there is no one test that we
think provides a defnitive measurement, we
have chosen to use the ratio of k values for
pyridine and phenol as our measure of relative
polarity for these C18 phases.
Figure 6 ranks stationary phases according to
relative polarity using the test conditions given.
In this ranking, there is not necessarily a
signifcant diference between consecutive
listings. If a diferent mobile phase condition
was used for the test, e g., a lower mobile phase
pH, or if diferent probes were used, the
ranking may be somewhat diferent. However,
phases at the high polarity end of the ranking
and phases at the low polarity end of the
ranking are likely to test that way under most
polarity tests conditions. Terefore, this ranking
can be used to identify relative diferences and
similarities in polarity that can afect selectivity
for polar compounds
Since silanol activity is a major contributor to
phase polarity, the test conditions used here to
measure polarity have also been used by some
chromatographers as an indication of silanol
activity. Tis seems consistent with the fact that
most phases at the high polarity end of the
ranking use more acidic silicas as stationary
phase supports where phases at the low
polarity end of the ranking use less acidic (high
purity) silicas. However, there are other factors
that contribute to the retention of pyridine and
phenol that prevent us from using their
relative retention as a reliable measure of silanol
activity. For example, Inertsil ODS has
moderate polarity but shows signifcant silanol
activity in other tests. Also, we see that Prodigy
ODS2 tests with similar polarity as the ACE
C18, but in tests for silanol activity, the ACE
C18 shows signifcantly less silanol activity
(see Figures 10 and 13).
Mobile Phase: 60% CH
3
OH
40% H
2
O
Polarity = k Pyridine / k Phenol
Temperature: 24C
Comparison Guide to C18 Reversed Phase HPLC Columns | 5
High Polarity/ High Polarity/ High Polarity/
Low Hydrophobicity Moderate Hydrophobicity High Hydrophobicity
Exsil ODS1 Exsil ODS LiChrosorb RP-18
Hypersil ODS Hypersil BDS C18 LiChrospher RP-18
Novapak C18 Resolve C18
Partisil ODS TSK ODS-80TM
Vydac 218MS Bondapak C18
Vydac 218TP Waters Spherisorb ODS1
Vydac Selectapore 300P Waters Spherisorb ODS2
Zorbax ODS
Moderate Polarity/ Moderate Polarity/ Moderate Polarity/
Low Hydrophobicity Moderate Hydrophobicity High Hydrophobicity
Vydac Selectapore 300M Capcell Pak AG C18 ACE C18-HL
Vydac Selectapore 90M Inertsil ODS Nucleosil C18
Xterra MS C18 Inertsil ODS2 Partisil ODS2
Nucleosil C18 AB Symmetry C18
Partisil ODS3 YMC JSphere ODS H80
Prodigy ODS3
Purospher RP18-e
TSK ODS-120T
Ultrasphere ODS
Waters Spherisorb ODSB
YMC JSphere ODS M80
YMC ODS A
YMC ODS AM
Zorbax Extend C18
Zorbax Rx-C18
Zorbax SB-C18
Low Polarity/ Low Polarity/ Low Polarity/
Low Hydrophobicity Moderate Hydrophobicity High Hydrophobicity
ACE C18-300 ACE C18 Develosil ODS-MG
Exsil ODSB Capcell Pak UG C18 Inertsil ODS3
Hypersil GOLD Develosil ODS-HG Kromasil C18
Develosil ODS-UG
Gemini C18
Hichrom RPB
Hypersil HyPurity C18
Luna C18(2)
Nucleosil C18 HD
Prodigy ODS2
SunFire C18
YMC Pro C18
Zorbax XDB-C18
Figure 7
C18 Phases Grouped According to Hydrophobicity and Polarity
Phases are listed in alphabetical order by category.
Figure 8
Chromatographic Comparison of Stationary
Phases from Different Categories
ACE C18
Low Polarity/Moderate Hydrophobicity
Hypersil BDS C18
High Polarity/Moderate Hydrophobicity
Symmetry C18
Moderate Polarity/High Hydrophobicity
Mobile Phase: 80% CH
3
OH,
20% 25mM KH
2
PO
4
, pH 6.0
Sample:
1. Norephedrine 4. Imipramine
2. Nortriptyline 5. Amitriptyline
3. Toluene
Categorization of phases according to hydrophobicity and polarity.
Te hydrophobicity and polarity data can be used to group phases with
similar characteristics into categories. Te following criteria was used for
the categories:
Hydrophobicity
High > 2.0
Moderate 1.30 to 1.99
Low < 1.30
High > 1.00
Moderate 0.50 to 0.99
Low < 0.50
k pyridine
k phenol
Polarity
k for phenanthrene
Figure 8 provides an example of how columns
from diferent polarity/hydrophobicity
categories will compare. In this separation of
antidepressants, Symmetry C18 (high
hydrophobicity) is slightly more retentive than
Hypersil BDS-C18 (moderate hydrophobicity),
and the band spacing of ACE C18 (low
polarity) is more similar to Symmetry C18
(moderate polarity) than it is to Hypersil BDS
C18 (high polarity).
6 | Comparison Guide to C18 Reversed Phase HPLC Columns
N/Meter (ToIuene)
Exs|| ODS!
ACE C!8
YMC Fro C!8
ACE C!8-300
Zorbax SE-C!8
ACE C!8-HL
Nuc|eos|| C!8
\a:ers Spner|sorb ODS!
YMC ODS A
Kronas|| C!8
H|cnron RFE
Zorbax XDE-C!8
Hypers|| ODS
Exs|| ODS
Synne:ry C!8
Deve|os|| ODS-UC
\a:ers Spner|sorb ODSE
SunF|re C!8
\a:ers Spner|sorb ODS2
Hypers|| COLD C!8
Zorbax Rx-C!8
Luna C!8(2)
Nuc|eos|| C!8AE
Zorbax ODS
Deve|os|| ODS-HC
Nuc|eos|| C!8 HD
YMC ODS AM
Exs|| ODSE
Vydac Se|ec:apore 300M
Zorbax Ex:end C!8
L|Cnrospner RF-!8
Vydac Se|ec:apore 90M
U|:raspnere ODS
Vydac 2!8MS
Hypers|| EDS C!8
Cen|n| C!8
L|Cnrosorb RF-!8
lner:s|| ODS
Hypers|| HyFURlTY C!8
Deve|os|| ODS-MC
YMC 'Spnere ODS H80
Vydac 2!8TF
Frod|gy ODS3
TSK ODS-80TM
lner:s|| ODS3
Novapa| C!8
YMC 'Spnere ODS M80
Capce|| Fa| UC C!8
TSK ODS-!20T
Vydac Se|ec:apore 300F
Far:|s|| ODS3
Capce|| Fa| AC C!8
Frod|gy ODS2
Far:|s|| ODS
XTerra MS C!8
Reso|ve C!8
Furospner RF!8-e
Far:|s|| ODS2
Eondapa| C!8
lner:s|| ODS2
0.0 20,000 40,000 60,000 80,000 100,000 120,000
Figure 9
Comparison of Column Effciency for a Neutral Compound
Column effciency reported as Plates per meter (N/Meter)
Comparison of Column Effciency for a
Neutral Compound
Column efciency is reported as plates per
meter (N/Meter). Using a neutral compound
(toluene) for the measurement greatly reduces
the efects of secondary retention on the
measurement of N and allows us to obtain
data that is a better indication of the
following factors:
Particle size
Smaller average packing particle size =
Larger N
Particle size distribution
Broader particle size distribution =
Smaller N
Packing efciency
Better packing procedures = Larger N
Mobile Phase: 90% CH
3
OH
10% H
2
O
Sample: Toluene
Temperature: 24C
Comparison Guide to C18 Reversed Phase HPLC Columns | 7
N/Meter (Fyridine)
ACE C!8
Deve|os|| ODS-UC
YMC Fro C!8
ACE C!8-300
SunF|re C!8
Hypers|| COLD
Deve|os|| ODS-MC
H|cnron RFE
Zorbax XDE-C!8
Deve|os|| ODS-HC
Capce|| Fa| UC C!8
Exs|| ODSE
Cen|n| C!8
lner:s|| ODS3
\a:ers Spner|sorb ODSE
lner:s|| ODS
ACE C!8-HL
YMC ODS A
Zorbax Ex:end C!8
Luna C!8(2)
Nuc|eos|| C!8 HD
YMC ODS AM
Vydac Se|ec:apore 300M
XTerra MS C!8
TSK ODS-!20T
Kronas|| C!8
Zorbax Rx-C!8
Far:|s|| ODS3
Frod|gy ODS3
Eondapa| C!8
Vydac Se|ec:apore 90M
Furospner RF!8-e
lner:s|| ODS2
YMC 'Spnere ODS M80
Hypers|| HyFURlTY C!8
Nuc|eos|| C!8
Zorbax SE-C!8
Far:|s|| ODS
Frod|gy ODS2
Novapa| C!8
TSK ODS-80TM
Vydac Se|ec:apore 300F
L|Cnrosorb RF-!8
YMC 'Spnere ODS H80
Vydac 2!8MS
Vydac 2!8TF
Far:|s|| ODS2
Nuc|eos|| C!8AE
Capce|| Fa| AC C!8
Synne:ry C!8
U|:raspnere ODS
\a:ers Spner|sorb ODS2
Hypers|| EDS C!8
Exs|| ODS!
Exs|| ODS
\a:ers Spner|sorb ODS!
Zorbax ODS
Hypers|| ODS
L|Cnrospner RF-!8
Reso|ve C!8
0 10,000 20,000 30,000 40,000 50,000 60,000 70,000 80,000
ACE
Hypersil
HyFUPlTY
SynneLry
Figure 10
Comparison of Column Effciency for a Basic Compound: Pyridine
Figure 11
Comparison of Peak Shape
Mobile Phase: 60% CH
3
OH
40% H
2
O
Sample: Pyridine
Temperature: 24C
ACE C18 gave the best peak shape and highest column
effciency for pyridine.
Comparison of Column Effciency for a
Basic Compound
Measuring column efciency using a neutral
compound is not very useful in predicting
column performance when separating ionic
compounds. Interaction between polar solutes
and silanol sites on the stationary phase can
cause tailing peaks and poor column efciency.
To gain a better understanding of column
performance with basic compounds, columns
were tested using pyridine and amitriptyline as
probes. Although columns are ranked somewhat
diferently on the two tests, phases at the higher
end of the ranking scale can be expected to
give better peak shape and higher resolution
for basic compounds than phases at the lower
end of the scale. Not surprisingly, stationary
phases that use high purity silicas exhibit better
peak shape and higher column efciency than
stationary phases that use more acidic silicas as
their stationary phase supports.
Mobile Phase: 60% CH
3
OH
40% H
2
O
Sample: Pyridine
Temperature: 24C
8 | Comparison Guide to C18 Reversed Phase HPLC Columns
Figure 12
Comparison of Column Effciency For a Basic Compound: Pyridine
ACE C18
65,200pl/m
ACE C18-300
52,100pl/m
SunFire C18
46,500pl/m
Zorbax XDB C18
39,400pl/m
Gemini C18
36,100pl/m
Inertsil ODS3V
35,900pl/m
Luna C18(2)
21,900pl/m
XTerra MS C18
18,600pl/m
Zorbax SB-C18
10,000pl/m
Symmetry C18
3,100pl/m
Hypersil BDS C18
600pl/m
Zorbax ODS
100pl/m
Column effciency measured at 10% pyridine peak height to account for peak tailing effects
Column Dimensions: 250 x 4.6mm, 5m
Sample: 1) uracil 2) pyridine 3) phenol
Mobile Phase: 60:40 MeOH/H
2
O
Temperature: 24C
Flow: 1.0ml/min
Comparison Guide to C18 Reversed Phase HPLC Columns | 9
N/Meter (AmitriptyIine)
ACE C!8-300
ACE C!8
Hypers|| COLD
YMC 'Spnere ODS M80
Cen|n| C!8
ACE C!8-HL
YMC Fro C!8
SunF|re C!8
Deve|os|| ODS-UC
Luna C!8(2)
Frod|gy ODS3
H|cnron RFE
Vydac Se|ec:apore 300M
Nuc|eos|| C!8 HD
lner:s|| ODS3
Deve|os|| ODS-MC
Frod|gy ODS2
Capce|| Fa| UC C!8
Hypers|| HyFURlTY C!8
XTerra MS C!8
Hypers|| EDS C!8
Synne:ry C!8
Deve|os|| ODS-HC
Furospner RF!8-e
Kronas|| C!8
Nuc|eos|| C!8AE
YMC ODS A
Vydac Se|ec:apore 90M
Zorbax XDE-C!8
YMC ODS AM
Zorbax Ex:end C!8
Vydac Se|ec:apore 300F
Vydac 2!8MS
YMC 'Spnere ODS H80
Exs|| ODS!
Zorbax SE-C!8
lner:s|| ODS2
lner:s|| ODS
Exs|| ODSE
Novapa| C!8
TSK ODS-80TM
\a:ers Spner|sorb ODSE
TSK ODS-!20T
Vydac 2!8TF
L|Cnrospner RF-!8
\a:ers Spner|sorb ODS2
Far:|s|| ODS
Far:|s|| ODS3
L|Cnrosorb RF-!8
Eondapa| C!8
Nuc|eos|| C!8
Zorbax Rx-C!8
U|:raspnere ODS
Capce|| Fa| AC C!8
Exs|| ODS
Hypers|| ODS
Far:|s|| ODS2
Reso|ve C!8
\a:ers Spner|sorb ODS!
Zorbax ODS
0 10,000 20,000 30,000 40,000 50,000 60,000 70,000
Figure 13
Comparison of Column Effciency for a Basic Compound: Amitriptyline
Figure 14
Top 10 Columns Ranked According to Peak
Shape and Effciency
Sample: Amitriptyline
Mobile Phase: 80% CH
3
OH
20% 0.025M Phosphate, pH 6.0
Sample: Pyridine
Mobile Phase: 60% CH
3
OH
40% H
2
O
Note: All columns are packed with 5 micron
size particles.
Column ranking does differ in the two tests of column eff-
ciency for a basic compound (Figures 10 and 13). However,
of the 14 columns that ranked in the top 10 on at least one
of the tests, 6 ranked in the top 10 on both tests.
Plate count is measured at 10% of peak height
to include peak tailing in the calculation. Both
tests use mobile phases at neutral pH to encour-
age interaction between the basic probes and
silanols on the stationary phase.
Mobile Phase: 80% CH
3
OH
20% 0.025M KH
2
PO
4
,
pH 6.0
Sample: Amitriptyline
Temperature: 24C
0 10,000 20,000 30,000 40,000 50,000 60,000 70,000
ACE C!8-300
ACE C!8
Hypers|| COLD
YMC 'Spnere ODS M80
Cen|n| C!8
ACE C!8-HL
YMC Fro C!8
SunF|re C!8
Deve|os|| ODS-UC
Luna C!8(2)
ACE C!8
Deve|os|| ODS-UC
YMC Fro C!8
ACE C!8-300
SunF|re C!8
Hypers|| COLD
Deve|os|| ODS-MC
H|cnron RFE
Zorbax XDE-C!8
Deve|os|| ODS-HC
N/Meter (AmitriptyIine)
0 10,000 20,000 30,000 40,000 50,000 60,000 70,000
N/Meter (Fyridine)
0 10,000 20,000 30,000 40,000 50,000 60,000 70,000
ACE C!8-300
ACE C!8
Hypers|| COLD
YMC 'Spnere ODS M80
Cen|n| C!8
ACE C!8-HL
YMC Fro C!8
SunF|re C!8
Deve|os|| ODS-UC
Luna C!8(2)
ACE C!8
Deve|os|| ODS-UC
YMC Fro C!8
ACE C!8-300
SunF|re C!8
Hypers|| COLD
Deve|os|| ODS-MC
H|cnron RFE
Zorbax XDE-C!8
Deve|os|| ODS-HC
N/Meter (AmitriptyIine)
0 10,000 20,000 30,000 40,000 50,000 60,000 70,000
N/Meter (Fyridine)
10 | Comparison Guide to C18 Reversed Phase HPLC Columns
Figure 15
Comparison of Column Effciency For a Basic Compound: Amitriptyline
ACE C18-300
65,300pl/m
ACE C18
63,300pl/m
Gemini C18
48,500pl/m
SunFire C18
45,000pl/m
Luna C18(2)
43,200pl/m
Inertsil ODS3V
30,500pl/m
Hypersil HyPURITY C18
24,800pl/m
XTerra MS C18
24,400pl/m
Hypersil BDS C18
22,700pl/m
Symmetry C18
21,800pl/m
Zorbax SB-C18
4,900pl/m
Waters Spherisorb ODS2
1,300pl/m
Column effciency measured at 10% amitriptyline peak height to account for peak tailing effects
Column Dimensions: 250 x 4.6mm, 5m
Sample: 1) norephedrine 2) nortriptyline 3) toluene 4) imipramine 5) amitriptyline
Mobile Phase: 80:20 MeOH/0.025M KH
2
PO
4
, pH 6.0
Temperature: 24C
Flow: 1.0ml/min
Comparison Guide to C18 Reversed Phase HPLC Columns | 11
Feak Asymmetry
Higher
5iIanoI
Activity
Lower
5iIanoI
Activity
1.03
8.99
8.3
6.50
3.19
3.07
3.05
3.05
2.74
2.21
2.07
1.93
1.88
1.78
1.61
1.52
1.26
1.12
ACE C!8
XTerra MS C!8
lner:s|| ODS3
Luna C!8(2)
Hypers|| E||:e C!8
D|scovery C!8
\a:ers Spner|sorb ODS!
lner:s|| ODS2
Synne:ry C!8
Hypers|| EDS-C!8
Far:|s|| ODS2
Zorbax XDE-C!8
Novapa| C!8
Zorbax SE-C!8
Nuc|eos|| C!8
Vydac Se|ec:apore 300M
Cosnos|| C!8 AR-ll
Eondapa| C!8
1 2 4 6 8 10
Feak Asymmetry
Higher
MetaI
Activity
Lower
MetaI
Activity
1.07
3.07
2.56
2.42
2.41
2.27
1.46
1.43
1.37
1.34
1.31
1.27
1.26
1.24
1.16
1.12
1.09
1.07
ACE C!8
Zorbax XDE-C!8
Hypers|| E||:e C!8
XTerra MS C!8
Cosnos|| C!8 AR-ll
lner:s|| ODS3
Hypers|| EDS-C!8
lner:s|| ODS2
Synne:ry C!8
Vydac Se|ec:apore 300M
D|scovery C!8
Zorbax SE-C!8
Luna C!8(2)
\a:ers Spner|sorb ODS!
Nuc|eos|| C!8
Novapa| C!8
Eondapa| C!8
Far:|s|| ODS2
1.0 1.5 2.0 2.5 3.0 3.5
Figure 16
Comparison of Metal Activity Using the NIST Test: Asymmetry for Quinizarin
Mobile Phase: 80% CH
3
OH
20% 5mM Potassium Phosphate, pH 7.0
Sample: Quinizarin
Flow Rate: 2 mL/min
Temperature: 23C
Figure 17
Comparison of Silanol Activity Using the NIST Test: Asymmetry
for Amitriptyline
Mobile Phase: 80% CH
3
OH
20% 5mM Potassium Phosphate, pH 7.0
Sample: Amitriptyline
Flow Rate: 2 mL/min
Temperature: 23C
Additional Comparison Tests
Tere are numerous suggestions from diferent
scientifc groups about how to best characterize
stationary phases. Most of these tests have
merit, but the fact that the ranking of columns
will often difer among the diferent tests shows
the difculty in devising a defnitive test that
will predict column behavior in all, or even most
circumstances. Te National Institute of
Standards & Technology (NIST) has developed
test conditions (Standard Reference Material
870) that do a particularly good job of
characterizing stationary phases according to
metal activity and silanol activity.
Te presence of metals on the surface of
stationary phases can have a signifcant efect
on chromatographic performance. Even trace
levels of metal impurities can contribute to
peak tailing of some compounds. In addition,
subtle lot-to-lot variations in the amount of
trace metals are another cause of poor column
reproducibility. Te NIST test uses peak
asymmetry of quinizarin, a strong metal
chelating agent, to measure metal activity.
Figure 16 ranks stationary phases according to
metal activity using the NIST test.
To test silanol activity, the NIST test uses
amitriptyline, as does the test used to generate
the data in Figure 13. However, the NIST test
specifes a mobile phase pH of 7.0 rather than
6.0, and measures peak asymmetry rather than
plate count to determine silanol activity. Te
lower the asymmetry value of the amitriptyline
peak (less tailing) the less silanol activity.
Figure 17 ranks stationary phases according
to silanol activity using the NIST test.
12 | Comparison Guide to C18 Reversed Phase HPLC Columns
Figure 18
Grouping of C18 Columns According to
Silanol Activity
Material
ACE C18
ACE C18-300
Develosil ODS-MG
Hypersil GOLD
Hypersil HyPURITY C18
Inertsil ODS3
Luna C18(2)
Nucleosil C18 HD
SunFire C18
XTerra MS C18
YMC Pro C18
ACE C18-HL
Capcell Pak UG C18
Develosil ODS-HG
Develosil ODS-UG
Gemini C18
Hichrom RPB
Inertsil ODS2
Kromasil C18
Prodigy ODS2
Prodigy ODS3
Purospher RP18-e
Symmetry C18
YMC ODS A
YMC ODS AM
Zorbax Extend C18
Zorbax XDB-C18
Capcell Pak C18 SG
Exsil ODSB
Hypersil BDS C18
Inertsil ODS
Nova-Pak C18
Nucleosil C18AB
Partisil ODS3
Synchropak CR101
TSK ODS-120T
TSK ODS-80TM
Bondapak C18
Vydac 218MS
Vydac 218TP
Vydac Selectapore 300M\
Vydac Selectapore 300P
Vydac Selectapore 90M
Waters Spherisorb ODSB
YMC JSphere ODS H80
YMC JSphere ODS M80
Zorbax Rx-C18
Zorbax SB-C18
Capcell Pak C18 AG
Exsil ODS
Exsil ODS1
Hypersil ODS
LiChrosorb RP-18
LiChrospher RP-18
Nucleosil C18
Partisil ODS
Partisil ODS2
Resolve C18Ultrasphere ODS
Waters Spherisorb ODS1
Waters Spherisorb ODS2
Zorbax ODS
Phases Grouped According to Silanol Activity
Amitriptyline and pyridine are both good test probes to use for measur-
ing silanol activity of stationary phases. Even a small amount of silanol
exposure by the stationary phase can cause measurable peak broadening
and peak asymmetry on one or both of these compounds. Chromato-
graphic tests using these two probes are the primary measurements used
to group these C18 phases according to silanol activity. In general, phases
identifed as having very low silanol activity will give the highest
column efciency in the pyridine and amitriptyline tests (Figures 10, 13
and 17).
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Comparison Guide to C18 Reversed Phase HPLC Columns | 13
MAC-MOD Analytical
103 Commons Court
P.O. Box 587
Chadds Ford, PA 19317
Phone: 1.800.441.7508
Fax: 1.610.358.5993
E-mail: info@mac-mod.com
Web Site: www.mac-mod.com
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LC497