Practical HPLC Method Development, Second Edition

by Lloyd R. Snyder, Joseph J. Kirkland and Joseph L. Glajch

Copyright 1997 John Wiley & Sons, Inc.

APPENDIX V
CHARACTERIZING THE
DIFFERENCES AMONG C8 OR C
REVERSED-PHASE COLUMNS
FROM DIFFERENT SUPPLIERS

Columns of the same nominal type (e.g., C8 or Qg) from different companies
will often exhibit important differences in retention. These differences can
arise from the following column packing characteristics (Section 5.2.4):
Differences in the silica particle (e.g., acidity) (Table 5.4)
Choice of silane and bonding process (e.g., monomeric vs. polymeric
phases) (Section 5.2.3.1)
Concentration of bonded phase (fimoVm2, Section 5.2.3.1)
Presence or absence of endcapping
Particle surface area
As a result, a column from one supplier that is nominally equivalent to a
column from a different source (e.g., Qg columns in both cases) may not be
a suitable replacement in an HPLC assay. It is useful to be able to compare
different Cg or C18 columns in terms of sample retention. This allows columns
to be identified that can be expected to perform similarly or quite differently
in a given assay. Similar columns may be interchangeable for a given assay,
which gives the chromatographer a choice of usable columns. Quite different
columns can be used to test the sensitivity of an assay to the column; if the
assay "works" for two such columns, it is likely that a number of other columns
will also be usable.
Figure 5.9 provides useful retention data for a large number of commercial
columns. Relative retention data are provided for acid, base, and neutral
sample compounds. If two columns show similar retention for each compound
740

APPENDIX V

741

in this test mixture, it is more likely that these same two columns will be
interchangeable for some HPLC assay. If relative and absolute retention of
the test mixture is quite different for two columns, these two columns can
serve as a test of the sensitivity of the assay procedure to differences in
the column.
There are two additional ways of comparing C8 and/or C18 columns. Column
acidity has been noted as an important column characteristic, and several
relatively non-acidic columns were listed in Table 5.4. A ranking of some
other columns according to their relative acidity is given in Table V.l. Columns
that are close together in this table can be expected to be similar in terms of
acidity, and such columns should perform similarly for the separation of basic
compounds. The data in support of Table V.l were collected between 1983 and
1987, which raises the question of whether these data are still representative
of columns produced today. Since manufacturers try to maintain the same
separation characteristics for their columns over time, it seems likely that the
ranking of Table V.l is still useful. Several silicas claimed to be less acidic
and highly purified have been made available by manufacturers in recent years
(see Table 5.4). However, insufficient data are available to place these silicas
in the ranking of Table V.l.

TABLE V.1 Ranking of C$ or C u Columns

According to Relative Acidity
Column
(less acidic)

(more acidic)
Source: Ref. 1.

Zorbax RX
Vydac
Nucleosil
Supelcosil DB
/iBondapak
Novapak
Partisil
RSil
Polygosil
Spherisorb
Lichrosorb
Chrompack
Rainin
IBM
Hypersil
Perkin-Elmer
Supelcosil
Zorbax
Micropak

ft

J. T. Baker

Bakerbond C18
Wide-Pore
Chromspher
PAH
Bio-Rad RP 318
Supelcosil LCPAH
Vydac 201TP
Spherisorb PAH
Erbas C18 H

ES Industries BF-C18
LiChrospher 100 RP-18
Bakerbond C18
Erbas C18 M
LiChrospher 60 RPselect B
PartisiJ 5 ODS-2
Partisil 5 ODS
Spherisorb ODS-1
Brownlee ODS 5A
Sepralyte C18
Spherisorb ODS-2

Column

Whatman
Whatman
Phase Separations
ABI
Analytichem
Phase Separations

ES Industries
E. Merck
J. T. Baker
Carlo Erba
E. Merck

Manufacturer
Erbasil C18 L
Pecospher 5 Cr C18
Partisphere C18
Zorbax ODS
Serva C18
Partisil 5 ODS-3
Hypersil ODS (HP)
Microsorb C18
J&W Accuphase ODS 2
Novapak C18
Ultrasphere ODS
Capcell C18 SG120
Supelcosil LC-18
IBM ODS
Brownlee Spheri 5
RP-18
ODS Hypersil
Cosmosil C18-P
J&W Accuphase ODS
YMC 120 A "A"
Adsorbosphere C18 H5
Supelcosil LC-18-DB

Column

Shandon
Nacalai Tesque
J&W Scientific
YMC
Alltech
Supelco

Carlo Erba
Perkin-Elmer
Whatman
Mac-Mod (distributor)
Serva
Whatman
Shandon
Rainin
J&W Scientific
Waters
Beckman
Shiseido
Supelco
IBM
ABI

Manufacturer

1.7 < (*TBN B.P ^ 2.2

Source: Ref. 2.
' Selectivity coefficients were determined using 85% acetonitrile/water mobile phase at 2 mL/min at ambient temperature (25 2C). Within each category,
columns are listed (from top to bottom) in order of increasing C*TBN BP values; however, because these values may vary with different column lots, individual
TBN BP values are not listed. The ranges shown above should not be construed to reflect lot-to-lot variability.

Separations Group
Phase Separations
Carlo Erba

Bio-Rad
Supelco

Chrompack

Manufacturer

Column

1.0 < TBN B>P < 1-7

Ranking of C M Columns According to Shape Selectivity"

0.5 < TBN BP ^ 0.9

TABLE XJ.

APPENDIX V

743

A second way of characterizing the column is in terms of shape selectivity

[2]. It has been shown that the separation of certain polyaromatic hydrocarbons
(PAHs) is quite sensitive to column source, and it has been suggested that
this is caused by "slots" in the stationary phase that are sensitive to the shape
of sample molecules. It is therefore reasonable that this column characteristic
will affect the retention of other samples as well. Table V.2 summarizes the
results from a large number of commercial columns. Three different groups
are defined according to the value of a for a particular pair of PAHs. Columns
in the same group are more likely to give similar results than columns in
different groups. Polyfunctional-silane bonded phases (e.g., Vydac 201TP)
have low values of the selectivity factor OTN/BSP. while monofunctional-silane
bonded phases (e.g., Zorbax ODS) have high values. The classification of
Table V.2 thus distinguishes these two types of packing.
The concentration of the bonded phase (/xmol/m2) can affect both absolute
retention and selectivity. A higher concentration of a particular ligand (e.g.,
Cu) generally gives greater retention, other factors being equal. A higher
bonded-phase concentration can also affect shape selectivity [3]. Endcapping
mainly affects the retention of basic compounds, which are retained more
strongly for non-endcapped phases. Values of k for all compounds increase
in proportion to packing surface area, but selectivity is not affected by small
differences in surface area.
APPENDIX V
1. M. A. Stadalius, J. S. Berus, and L. R. Snyder, LC/GC, 6 (1988) 495.
2. L. C. Sander and S. A. Wise, LC/GC, 8 (1990) 378.
3. L. C. Sander and S. A. Wise, Anal. Chem., 67 (1995) 3284.