226 THE COLUMN
C
≡
N
OOHO
OH
O
OH
O
OH
O
OH
O
CH
3
C
1
C
3
C
5
C
8
C
18
C
30
(TMS) (ODS)
(a) Alkylsilica columns
X
OH O OH

simplified cartoons of Figure 5.19 (the—Si[CH
3
] group is omitted in Fig. 5.19a–d).
The ligand of a RPC column is often an alkyl group, for example, C
3
,C
8
,C
18
(Fig. 5.19a). Alternatively, the ligand may consist of phenylpropyl or phenylhexyl,
called phenyl columns (Fig. 5.19b). If the ligand is –C
3
–C≡N (Fig. 5.19c), we have a
cyano column. The alkyl group may also be substituted by other functional groups
X (Fig. 5.19d), and this gives rise to the additional column types listed at the bottom
of Figure 5.19. So-called embedded-polar-group (EPG) phases have been growing
in popularity, because of their compatibility with low %B mobile phases, their
reduced silanol interactions, and unique selectivity (Section 5.4.1); peak shape for
basic solutes is usually quite good with these columns. The ligands in these phases
contain amide, carbamate, urea (all of which are strong hydrogen-bond bases), or
other polar functional groups embedded within the ligand structure. Some EPG
5.4 COLUMN SELECTIVITY 227
packings tend to be less stable than comparable alkyl or aryl columns. The nature
of the ligand mainly determines column selectivity, which is the subject of following
Section 5.4.
5.4 COLUMN SELECTIVITY
Column selectivity can be important for different reasons. During method devel-
opment a change of column may be necessary to improve selectivity and increase
resolution (Sections 2.5.2, 5.4.3). For the latter application we must be able to iden-
tify a second column with quite different selectivity. When a routine RPC procedure

(e) cation-exchange or electrostatic interaction between a cationic solute and
an ionized silanol (–SiO
−
) within the stationary phase; also repulsion of
an ionized acid (e.g., R–COO
−
)
228 THE COLUMN
(f) dipole–dipole interaction between a dipolar solute group (a nitro group
in this example) and a dipolar group in the stationary phase (a nitrile
group for a cyano column)
(g, h) π –π interaction between an aromatic solute and either a phenyl group
(phenyl column) (g), or a nitrile group (cyano column) (h)
(a)
Hydrophobic interaction
OO
COCH
3
Hydrogen bonding
(acidic solute)
(d)
OO
X

O
C
OH
(e)
Cation exchange
OO

N
(b)
Steric exclusion
OOO
(f)
Dipole-dipole interaction
O
C
=
N
+
−
O
2
N
−
+
Figure 5.20 Solute-column interactions that determine column selectivity (figures omit the
connecting silane group [–Si(CH
3
)
2
–]).
5.4 COLUMN SELECTIVITY 229
O
O
M
++
N


18
columns (Inertsil ODS-3 and Stablebond C18)—however, values
of log k for aliphatic amides () and protonated strong bases ()fallbelow
the best fit to these data. These latter deviations are due to interactions of these
solute molecules with silanol groups (silanol interactions are more significant for
the StableBond C18 column). These and other smaller deviations δ log k from this
plot (see the expanded inset of Fig. 5.21) represent contributions to retention from
nonhydrophobic interactions b–e of Figure 5.20. It is possible to analyze values of
δ log k for the combination of different solutes and columns so as to separately
evaluate the five interactions of Figure 5.20a–e. For columns other than phenyl or
cyano (i.e., those for which only interactions a–e of Fig. 5.20 are significant), values
230 THE COLUMN
δlog k
2.0
1.5
1.0
0.5
0.0
−0.5
−1.0
−1.5
−1.0 −0.5 0.0 1.00.5 1.5
log k (Inertsil ODS-3)
lo
g
k (StableBond C18)
y = 0.21 + 1.01 x
r
2
= 0.995

Here k and k
EB
are values of the retention factor for a given solute and the
reference compound ethylbenzene (EB), respectively. Terms i–v of Equation (5.3)
correspond, respectively, to the interactions of Figure 5.20a–e. Quantities η

, σ

,
β

, α

,andκ

refer to properties of the solute molecule: hydrophobicity (η

),
‘‘bulkiness’’ (σ

), hydrogen-bond (H-B) basicity (β

), H-B acidity (α

), and effective
ionic charge (κ

). Corresponding column parameters are of primary practical interest:
H, hydrophobicity; S*, steric interaction, or resistance by the stationary phase to
penetration by bulky solutes; A, H-B acidity; B, H-B basicity; and C, ion-exchange

.
Steric exclusion or ‘‘steric interaction’’ is illustrated in Figure 5.20b by the
retention of two polycyclic aromatic hydrocarbon (PAH) isomers: the narrow, long
naphthacene and the more ‘‘bulky’’ triphenylene. Naphthacene is better able to
squeeze between adjacent ligands, but if the spacing of column ligands is increased
(lower ligand concentration), it becomes easier for the bulky triphenylene to enter
the stationary phase. The column parameter S* measures the ‘‘tightness’’ of the
stationary phase or the difficulty that bulky solute molecules experience in squeezing
between the ligands; larger values of S* mean a ‘‘tighter’’ stationary phase and
relatively less retention of bulky solute molecules. Values of S* increase for longer
ligands, a higher concentration of the ligand (ligands closer together), and smaller
pore diameters. Solute bulkiness is measured by its value of σ

. Steric exclusion is a
somewhat complex phenomenon; see Section 5.4.1.2 below for further insights.
Hydrogen bonding of a non-ionized basic solute (e.g., pyridine) by a column
silanol is illustrated in Figure 5.20c. The hydrogen-bond acidity A of the column
is due to the presence of surface silanols, and therefore decreases when the column
is end-capped (due to the removal of some silanols and blocking of others; see the
example of Fig. 5.16d). The silanols of type-A columns are usually more acidic than
those present in type-B columns; therefore values of A tend to be larger for type-A
columns. The H-B basicity of the solute is measured by its value of α

; unprotonated
amines and amides are more basic and have larger values of α

, while nitriles and
nitro compounds are much less basic and have smaller values of α

. Most other polar


for a solute is approximately equal to its molecular charge (e.g., +1 for fully
protonated bases, −1 for fully ionized acids). The main difference in selectivity for
type-A versus type-B columns is determined by their low-pH values of C;type-B
columns have values of C < 0.25 at pH 2.8, while type-A columns have C
>
0.25.
For columns with values of C < 0.00 at low pH, it is believed that these columns
carry a net positive charge [63], presumably the result of protonated amine groups
that are introduced during the manufacturing process for some columns. Values of
H, S*, A,andB are assumed not to change with the pH of the mobile phase.
Values of the column-selectivity parameters H, S*, etc., have been measured
for over 400 different columns; see [64] for a partial listing, or for a current list of
values contact one of the authors (or />Average values of these column parameters are summarized at the top of Table 5.8
for several different kinds of RPC column. Within a given column type, there is
also a significant variation in values of H, S*, etc., as illustrated at the bottom of
Table 5.8 for several type-B C
18
columns. Consequently not all columns of a given
kind can be regarded as equivalent in terms of selectivity. Apart from values of H
and S*, for example, average retention as measured by values of k for ethylbenzene
(last column of Table 5.8) increases with the surface area of the particle.
5.4.1.2 Shape Selectivity
The following, minor digression examines two distinct forms of steric exclusion; for
now, the reader may prefer to skip to Section 5.4.2.
Two separate manifestations of steric exclusion have been described: steric
interaction, as measured by term ii of Equation (5.3), and shape selectivity [65].
Differences between these two phenomena are illustrated in Figure 5.22 for the
separation of two isomeric hydrocarbons on a polymeric column (Fig. 5.22a)and
a monomeric column (Fig. 5.22b). The basis of shape selectivity is illustrated in

(type-B) 0.41 −0.08 −0.08 0.02 0.04 0.66 1.2
C
3
(type-B) 0.60 −0.12 −0.08 0.04 −0.08 0.81 2.8
C
8
(type-B) 0.84 0.00 −0.12 0.02 −0.03 0.25 5.4
C
18
(type-B) 0.99 0.01 −0.01 0.00 0.00 0.24 8.8
C
18
(type-B, wide-pore) 0.95 0.01 −0.05 0.01 0.22 0.31 3.2
C
18
(type-B, monolith) 1.01 0.02 0.12 −0.02 0.11 0.31 3.2
C
18
(type-B, hybrid) 0.98 0.01 −0.14 −0.01 0.13 0.05 6.3
C
18
(polar end-capped) 0.90 −0.04 −0.02 0.02 −0.02 0.40 7.4
C
18
(type-A) 0.94 −0.05 0.14 0.01 0.79 1.18 6.4
C
30
(type-B) 1.05 −0.01 0.09 −0.02 −0.08 0.45 13.0
Embedded-polar-group 0.74 0.00 −0.22 0.12 −0.27 0.53 5.9
Phenyl (type-B) 0.63 −0.12 −0.20 0.02 0.13 0.68 2.7

h
1.06 0.04 −0.10 −0.02 −0.08 0.32 11.0
ACE 5 C18
i
1.00 0.03 −0.10 −0.01 0.14 0.10 7.9
Chromolith
j
1.00 0.03 0.01 −0.01 0.10 0.19 3.1
Luna C18(2)
k
1.00 0.02 −0.12 −0.01 −0.27 −0.17 9.6
Gemini C18 110A
k
0.97 −0.01 0.03 0.01 −0.09 0.19 8.0
Discovery C18
l
0.98 0.03 −0.13 0.00 0.18 0.15 4.8
Hypurity C18
m
0.98 0.03 −0.09 0.00 0.19 0.17 5.6
Hypersil GOLD
m
0.88 0.00 −0.02 0.04 0.16 0.48 3.9
Symmetry C18
n
1.05 0.06 0.02 −0.02 −0.30 0.12 9.8
Xterra MS C18
n
0.98 0.01 −0.14 −0.01 0.13 0.05 6.3
Sunfire C18

Thermo/Hypersil;
n
Waters;
o
Tosoh Bioscience.
234 THE COLUMN
(a)(b)
(min)
0
10
20
30
4
≥≥
k 16
(c)
10
15
≥≥
5 k 7
(d)
Solute minimum cross-section
(width)
O
OH
OOOH
Si
Si-OH
O
OH

for the separation of Figure 5.22c exhibits greater shape selectivity and therefore
provides a much greater differentiation (and better separation) of these different
isomeric C
22
PAHs, versus the corresponding separation in Figure 5.22d with a
monomeric column (where shape selectivity is minimal). Long, narrow molecules
(compared to those that are short and wide) are preferentially retained when shape
selectivity is more important, while short, wide solute molecules (of similar molecular
weight) are more retained when steric interaction is dominant. As a rule, we can
say that shape selectivity is more important when C
30
or polymeric columns are
used, and sample molecules are both large and have very different ratios of length to
width. Most RPC separations are carried out with monomeric columns other than
C
30
, in which case steric interaction and values of S* largely define the effect of steric
5.4 COLUMN SELECTIVITY 235
exclusion on column selectivity. For further details on the practical utility of shape
selectivity, see Section 6.3.5.2.
5.4.2 Column Reproducibility and ‘‘Equivalent’’ Columns
Column manufacturers try to ensure that each column (e.g., Waters Symmetry C18)
has similar properties and will perform satisfactorily and reproducibly in a routine
RPC assay. Consequently the plate number N and column pressure drop for each
column usually is measured prior to its sale (Section 5.7); columns whose values of
N fall below some minimum value are discarded. Similarly other tests are carried out
by the manufacturer (Section 5.7) to ensure that column selectivity stays the same
from one batch to the next of the column packing (similar to the measurements of
values of H, S*, etc.). An example is shown in Figure 5.23 for several successive
batches of Zorbax

CH
3
N(CH
3
)
2
Figure 5.23 Monitoring different batches of column packing for possible changes in selectiv-
ity. Sample: dimethylaniline and toluene. Conditions: 150 × 4.6-mm Zorbax Rx-C
18
columns;
50% acetonitrile-water plus pH-7 phosphate buffer; 1.6 mL/min; 22
◦
C. Adapted from [66].