Introduction to Modern Liquid Chromatography, Third Edition part 31 pdf

256 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES
‘‘normal-phase’’ chromatography (NPC). As RPC involves a less polar column and
a more polar mobile phase, the two phases can be regarded as interchanged or
‘‘reversed.’’ The first RPC packings were made from silica particles that had been
reacted with (CH
3
)
2
SiCl
2
, so as to render the surface nonpolar. A nonpolar stationary
phase was then used to coat the particles, allowing their use for reversed-phase
liquid–liquid partition chromatography [2], a HPLC technique that is rarely used
today.
Bonded-phase columns represented the next major advance in ‘‘high-
performance’’ RPC. Early C
18
columns of this type were prepared by the covalent
attachment of polyoctadecylsiloxane to silica particles [3, 4]. This was followed a
few years later by the introduction of columns where individual C
18
groups (rather
than a C
18
polymer) were attached to the particle (Section 5.3.1). Beginning in the
early 1970s, RPC columns of the latter type were used increasingly because of their
many advantages. In the mid-1970s, the use of RPC underwent an explosive growth
in popularity; several hundred separations are cited in [5] for the period 1976 to
1979. Whereas earlier applications of RPC emphasized the separation of more
hydrophobic (‘‘lipophilic’’) samples, the classic paper by Horv
´

ath [6] demonstrated
that RPC could also be used for the separation of relatively polar compounds,
especially water-soluble samples of biochemical interest. A little later, RPC was
further adapted for the separation of ionizable compounds (including enantiomers)
by the addition of ion-pairing compounds to the mobile phase [7] (ion-pair
chromatography, Section 7.4). In the late 1970s, several groups reported the first
RPC separations of large peptides and proteins, using short-chain, wide-pore
column packings. Today, RPC is the dominant HPLC mode and accounts for a
substantial majority of all HPLC separations.
6.2 RETENTION
Retention in RPC was discussed briefly in Section 2.2. Because very polar molecules
interact more strongly with the polar mobile phase, these compounds are less
retained and leave the column first. Similarly less polar compounds prefer the
nonpolar stationary phase and leave the column last. Thus molecules of similar
size are eluted in RPC approximately in order of decreasing polarity. An example is
provided by Figure 6.1a, where it is seen that the more-polar benzonitrile (1) appears
in the chromatogram first, followed by the increasingly less polar anisole (2), and
finally toluene (3). A more detailed example of RPC retention as a function of solute
polarity is provided by Figure 2.7c; see also the related discussion of RPC column
selectivity in Section 5.4.
Retention in RPC is largely the result of interactions between a solute molecule
and either the mobile phase or the column (Section 2.3.2.1). An increase in %B
(volume-% of organic solvent in the mobile phase) makes the mobile phase less polar
(‘‘stronger’’) and increases the strength of interactions between solute and solvent
molecules. The result is decreased retention for all solute molecules when %B is
increased. This is illustrated by the separation of Figure 6.1b (with a mobile phase
of 60% B) compared to that of Figure 6.1a (40% B). An increase in temperature
weakens the interaction of the solute with both the mobile phase and column, and
6.2 RETENTION 257
C

18
column
40% ACN
30°C
C
18
column
60% ACN
30°C
C
18
column
40% ACN
70°C
Cyano column
40% ACN
30°C
(a)
(b)
(c)
(d )
C≡N (1)
OCH
3
(2)
CH
3
(3)
1
2

3
3
2
1
1
2
3
0 2 4 6 8 10 12 14 (min)
0 2 4 6 8 10 12 14 (min)
0 2 4 6 8 10 12 14 (min)
0246810 12 14 (min)
Figure 6.1 Retention in RPC as a function of temperature and the polarity of the solute,
mobile phase and column. Sample: as indicated in figure. Conditions: (a-c) 150 × 4.0-mm
5-μm) Symmetry C
18
column, and (d) 150 × 4.6-mm (5-μm) Zorbax StableBond cyano
column; 2.0 mL/min; mobile phase is acetonitrile/water, with mobile-phase composi-
tion (%B) and temperature indicated in figure (bolded values represent changes from [a]).
Chromatograms recreated from data of [8, 9].
decreases retention; compare Figure 6.1c (70
◦
C) and Figure 6.1a (30
◦
C). Finally, a
decrease in column hydrophobicity weakens the interaction between the solute and
column, and reduces retention; compare Figure 6.1d (more-polar cyano column)
and Figure 6.1a (less-polar C
18
column).
6.2.1 Solvent Strength

As noted in Section 2.4.1, retention in RPC varies with mobile phase %B as
log k = log k
w
− Sφ (6.1)
where k
w
refers to the (extrapolated) value of k for 0% B (water as mobile phase), S
is a constant for a given solute when only %B is varied, and φ is the volume-fraction
of organic solvent B in the mobile phase (φ ≡ 0.01% B). The value of %B selected
258 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES
80% ACN
(k = 0.3)
R
s
= 0.0
(a)
(b)
(c)
1
2
3
4
1
2
3
4
0.2 0.4 0.6 0.8
Time (min)
t
0

1−4
50% ACN
R
s
= 1.0
(1.9 ≤ k ≤ 4.2)
40% ACN
R
s
= 1.4
(4 ≤ k ≤ 10)
0 4
Time (min)
2
6
0 2
Time (min)
Figure 6.2 Separation of a mixture of four nitro-substituted benzenes as a function of
solvent strength (%B). Sample: 1, nitrobenzene; 2, 4-nitrotoluene; 3, 3-nitrotoluene; 4,
2-nitro-1,3-xylene. Conditions: 100 × 4.6-mm (3-μm) Zorbax C8 column; mobile phase con-
sists of acetonitrile-water mixtures (varying %B); 35
◦
C; 2 mL/min. Chromatograms recreated
from data of [10].
for the final separation should provide values of k for the sample that are within a
desired range (e.g., 1 ≤ k ≤ 10), while at the same time maximizing solvent-strength
selectivity (Section 6.3.1).
A suitable value of %B can be obtained as described in Section 2.5.1: start with
80% B, and then reduce %B in steps until the desired retention range of 1 ≤ k ≤ 10
is obtained. Figure 6.2 provides an example of this approach. The first separation

with 80% B (Fig. 6.2a) provides very little retention (k = 0.3), so a change to 50%
B is tried for the next experiment (Fig. 6.2b). The retention range for the sample
is now reasonable (1.9 ≤ k ≤ 4.2), but the resolution is inadequate (R
s
= 1.0). A
further decrease in %B will usually (but not always) increase resolution; for samples
with molecular weights < 500 Da, a 10%B decrease will increase values of k by a
factor of about 2.5, which suggests a mobile phase of 40% B for the next experiment
(Fig. 6.2c). Resolution is increased moderately (R
s
= 1.4) but is still inadequate. As
a further decrease in %B will result in values of k
>
10, some other means of further
increasing resolution may be necessary: a change in selectivity (Section 6.3) or a
change in column conditions (Section 2.5.3).
It should be noted that Equation (6.1) is not an exact relationship but
an approximation. For example, values of log k for a representative solute
(4-nitrotoluene; compound-2 in Fig. 6.2) are plotted against %B in Figure 6.3
6.2 RETENTION 259
2.4
2.0
1.6
1.2
0.8
0.4
0.0
log k
020 6080
%B

40
MeOH
ACN
best linear
fit to ACN data
Figure 6.3 Variation of log k with %B. Sample is 4-nitrotoluene. Conditions: 250 × 4.6-mm
(5-μm) Zorbax C8 column; mobile phase consists of organic/water mixtures; 35
◦
C; 2 mL/min.
Created from data taken from [10].
for both acetonitrile (ACN, ) and methanol (MeOH,
•
) as the B-solvent. Whereas
Equation (6.1) predicts a linear plot, a slightly curved plot results for ACN as
B-solvent. The data for MeOH fall closer to the linear curve in Figure 6.3 that
is fitted to these data. This behavior is typical of other samples and experimental
conditions [11]; more linear plots are usually obtained for MeOH, compared to the
use of ACN or other organics as B-solvent. However, over the usual range in k
that is of interest (e.g., 1 ≤ k ≤ 10), Equation (6.1) is adequately accurate for either
MeOH or ACN as the B-solvent.
6.2.2 Reversed-Phase Retention Process
The following discussion provides further insight into the basis of RPC retention.
However, it is mainly of academic interest, with little practical value for the
application of RPC. A results-oriented reader may prefer to skip to following
Section 6.3.
The nature of RPC retention (the retention ‘‘mechanism’’) has been the subject
of a large number of research studies, as summarized in several reviews and research
publications [5, 12–16]. Some of the questions that this work has addressed include:
• positioning of the solute molecule within the stationary phase (adsorption
or partition?)

260 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES
• dependence of solute retention on mobile-phase composition (k vs. φ)
• conformation of the alkyl ligands that form the stationary phase (‘‘extended’’
vs. ‘‘collapsed’’)
The positioning of the solute molecule within the stationary phase might
occur in any of the ways pictured in Figures 6.4a–c for a C
8
column. Solvophobic
interaction (Fig. 6.4a) assumes that the solute molecule aligns with and is attached
to a ligand group (C
8
in this example). Adsorption (Fig. 6.4b)impliesthatthe
solute molecule does not penetrate into the stationary phase, but is retained at the
interface between the stationary and mobile phases. Partition (Fig. 6.4c) considers
the stationary phase to be similar to a liquid phase, into which the solute molecule
dissolves. Notice that the stationary phase consists of alkyl ligands plus an organic
solvent that is preferentially extracted from the mobile phase by the C
8
groups of the
stationary phase. In both solvophobic interaction and partition, the solute molecule
lies within the stationary phase.
When discussing the mechanism of RPC retention, Horv
´
ath’s solvophobic-
interaction model [5] is commonly cited: (relatively) hydrophobic solute molecules
prefer to adhere to the hydrophobic alkyl ligands—so-called hydrophobic retention.
Soon after the introduction of RPC for HPLC, it was observed that RPC retention
(values of k) correlates with partition coefficients P for the distribution of the solute
between octanol and water (Fig. 6.4d; [17]); this suggests that a partition process
best describes RPC retention. However, later studies showed that correlations of

log P versus log k, as in Figure 6.4d (for amino acids) are less pronounced when the
sample consists of molecules with more diverse structures, which makes the latter
argument on behalf of partition less compelling.
In the early 1980s [18] a surprising observation was made for the RPC
retention of various homologous series (CH
3
–[CH
2
]
n − 1
–X), where X represents a
functional group such as –OH or –CO
2
CH
3
. Plots of log k versus carbon-number
n were found to exhibit a discontinuity for a value of n that is approximately
equal to the carbon-number n

for the stationary-phase ligand (e.g., n

= 8for
C
8
≡ CH
3
–[CH
2
]
7

–). This anomalous behavior is illustrated in Figure 6.5a by a
hypothetical plot of log k versus n for a homologous series and a C
8
column; a
discontinuity in the expected linear plot (dashed line) is observed (arrow) when n
equals 8 for the solute (CH
3
–[CH
2
]
7
–X). It was concluded from this observation
that the contribution to retention for successive –CH
2
-groups in the solute becomes
slightly smaller when the length of the solute molecule just exceeds the length of the
alkyl ligand. The reason for this discontinuity in the plot of Figure 6.5a is visualized
in Figure 6.5b,wheren = 12 for solute ii exceeds the value of n

= 8 for the column
ligand (the situation for n = n

= 8 is illustrated by solute i). In the example of
Figure 6.5b with n = 12, the end of the molecule likely folds back onto or into the
stationary phase—rather than extending into the mobile phase as shown.
Presumably there is a decreased interaction with the column for solute
molecules that are too long to penetrate fully into the stationary phase (or attach to a
single column ligand), with a corresponding decrease in the retention of –CH
2
-groups

that ‘‘stick out of’’ the stationary phase. The contribution to retention of each
–CH
2
-group can be defined as α
CH2
= ratio of k values for successive homologs, and
this value is normally assumed constant (see discussion of Eq. 2.34). However, for
6.2 RETENTION 261
(a)
(c)
(b)
(d )
solvophobic
interaction
adsorption
partition
0
−1
−2
−3
−4
log P
−2 −101
lo
g
k
y = x − 2
sorbed mobile
phase
solute molecule

Figure 6.4 Different possibilities for the retention of a solute molecule in reversed-phase
chromatography. (a) Solvophobic interaction; (b) adsorption; (c) partition; (d) comparison
of RPC retention (k) with octanol-water partition P [17]; sample; eight amino acids; column:
C
8
; mobile phase: aqueous buffer (pH-6.7); 70
◦
C.
excluded –CH
2
-groups, the value of α
CH2
should be somewhat smaller. This ‘‘exclu-
sion’’ effect is demonstrated experimentally in Figure 6.5c for a C
18
column, where
α
CH2
is plotted versus n. There is a distinct break in the plot in the vicinity of
n = n

= 18 (dashed vertical line). Similar breaks are shown in the plots of
Figures 6.5d,e for the C
8
and C
6
columns, respectively, but no significant penetration
of the solute is possible for the C
1
column of Figure 6.5f . The data of Figures 6.5c–e

suggest that small solute molecules can penetrate the stationary phase of a C
6
,C
8
,
or C
18
column, so this would rule out a purely adsorption process (as in Fig. 6.4b)
for other small solutes. Figure 6.5 also supports the solvophobic-interaction model
of Figure 6.4a.
Figure 6.5 is suggestive of possible conclusions concerning retention in RPC,
but more complicated arguments have been offered concerning the adsorption and
partition processes [14, 15]. It should be noted that the nature of the stationary
phase (and presumably the retention process) varies with experimental conditions.
Increasing amounts of the B-solvent (e.g., acetonitrile) are taken up by the stationary
262 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES
(b)(a)
log k
n
n = n’ = 8
ii
i
X
X
C
18
5
10 15
20
25

1.40
1.38
1.36
α
CH
2
n
(c)
(d )
(e)
(f )
1.30
1.28
1.26
1.24
5
10
15 20 25
n
C
8
α
CH
2
1.28
1.24
1.20
C
6
510152025

n
α
CH
2
C
1
1.21
1.17
1.13
510152025
n
α
CH
2
Figure 6.5 Retention as a function of alkyl chain length (for both the solute and column).
(a) Illustrative plot of log k versus number of –CH
2
-groups n for a homologous series
CH
3
–(CH
2
)
n−1
–X;C
8
column; (b) illustration of the ‘‘overlapping’’ of alkyl chains in the
solute and column; (c–f) plots of experimental methylene selectivity α
CH2
versus carbon num-

ber n
c
for indicated columns of differing ligand length. Average data for several homologous
series; 90% methanol-water as mobile phase; 25
◦
C. Figures are adapted from [18].
6.3 SELECTIVITY 263
phase as %B increases. Likewise some solutes may interact with underivatized
silanols present on the particle surface (Section 5.4.1). For these and other reasons
the precise nature of the retention process is likely to vary with the column, the
solute, and experimental conditions. Horv
´
ath anticipated this situation early on
[17], in noting that ‘‘a clear distinction between partition and adsorption in RPC of
nonpolar [solutes] [and] with no apparent thermodynamic or practical significance
[so that] this issue may not be worth further investigation.’’ The authors of this
book find it difficult to argue with this conclusion.
Speculation concerning the nature of RPC retention has also been based on
retention as a function of mobile-phase composition (%B). While Equation (6.1) is
a purely empirical relationship, several theory-based equations for k as a function of
φ have been derived [11]. The resulting expressions for k versus φ are in some cases
slightly more reliable than Equation (6.1)[19], largely because of additional fitting
parameters. However, Equation (6.1) is generally adequate for practical application,
and is much more convenient to use. The major assumptions involved in all previous
theoretical derivations of k versus φ negate any value in their use for interpreting
the nature of RPC retention.
Stationary phase ligand conformation has been claimed to play a role in the
‘‘mechanism’’ of RPC retention. The use of mobile phases that are predominantly
aqueous (φ ≈ 0) can lead to greatly reduced sample retention—the opposite of
that predicted by Equation (6.1). When first observed, this reduced retention was

attributed to ‘‘phase collapse,’’ whereby alkyl ligands clump together and tend
toward a horizontal rather than vertical alignment with the particle surface. This
retention behavior was subsequently shown to arise not from phase collapse but
rather from exclusion of mobile phase and sample molecules from particle pores
as a result of surface-tension effects (‘‘de-wetting,’’ Section 5.3.2.3 and [2, 21]).
More recent studies [22, 22a] suggest that ligand conformation does not change as a
function of either mobile-phase composition or the relative coverage of the particle
surface by ligands.
6.3 SELECTIVITY
The most effective way to improve the resolution (or speed) of a chromatographic
separation is to initiate a change in relative retention (selectivity). For the separation
of non-ionic samples by RPC, changes in selectivity can be achieved by a change
in solvent strength (%B), temperature, solvent type (e.g., ACN vs. MeOH as the
organic solvent), or column type (e.g., C
18
vs. cyano). The relative effectiveness of a
change in conditions for a change in selectivity varies roughly as
temperature (least effective) < %B < solvent type ≈ column type (most effective)
However, each of the four conditions above for changing selectivity can be useful
for different samples or separation goals, as discussed next.
6.3.1 Solvent-Strength Selectivity
In the examples of Figures 6.1 and 6.2, relative retention does not change when
solvent strength (%B) is varied. As %B decreases (and k increases), the resolution
264 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES
0
2
Time (min)
1
2
+

3
4
5
6
7
8
50% ACN
2 ≤ k ≤ 6
R
s
= 0.0
(a)
45% ACN
3 ≤ k ≤ 6
R
s
= 1.5
(c)
024
Time
(
min
)
1
2
3
4
5
6
7

8
4
40% ACN
4 ≤ k ≤ 14
R
s
= 1.0
(b)
02468
Time (min)
1
2
5
6
7
8
3
Figure 6.6 Separation of a moderately irregular sample (mixture of eight nitro-aromatic
compounds) as a function of solvent strength (%B). Sample: 1, nitrobenzene; 2,
2,6-dinitrobenzene; 3, benzene (shaded peak); 4, 2-nitrotoluene; 5, 3-nitrotoluene; 6, toluene;
7, 2-nitro-1,3-xylene; 8, 1,3-xylene. Conditions: 100 × 4.6-mm (3-μm) Zorbax C8 column;
mobile phase consists of acetonitrile/water mixtures; 35
◦
C; 2 mL/min. Chromatograms recre-
ated from data of [10].
of all peaks improves, but their relative spacing stays essentially the same. In
Section 2.5.2.1 we defined samples as in Figures 6.1 and 6.2 as regular. Figure 6.6
shows the separation of a sample where %B is varied, but relative retention does
not remain the same. An initial separation with 50% B (Fig. 6.6a) shows a complete
overlap of peaks 2 and 3 (shaded). When the mobile phase is changed to 40% B

(Fig. 6.6b), peak 3 moves toward peak 4 and partially overlaps it. From these two
experiments it can be seen that an intermediate value of %B should result in an
improved separation, which is observed for the separation of Figure 6.6c (45% B).
Samples such as this, where relative retention changes with solvent strength, are
referred to as irregular. Regular samples are often composed of structurally similar
molecules; for example, in the separation of Figure 6.2 the sample is a mixture of
mono-nitro alkylbenzenes. The sample of Figure 6.6, on the other hand, exhibits a
somewhat greater molecular diversity: it is a mixture of alkylbenzenes that contain
0, 1, or 2 nitro-substituents.
Changes in %B often lead to significant changes in relative retention, with
maximum resolution occurring for an intermediate value of %B. Despite this obvious
fact, practical workers often overlook solvent-strength selectivity as a useful tool
6.3 SELECTIVITY 265
for optimizing relative retention and resolution. To take maximum advantage of
solvent-strength selectivity, the allowable retention range can be expanded from
1 ≤ k ≤ 10 to 0.5 ≤ k ≤ 20. When conditions are varied so as to change selectivity,
it is important to keep track of which peak is which. The numbering of each peak in
the chromatogram (as in Fig. 6.6) may not be obvious; in such cases peak tracking
will be required (Section 2.6.4).
The remainder of this section, which expands on the discussion above of
regular and irregular samples, is somewhat detailed; the reader may prefer to skip
(or skim) this discussion and go on to Section 6.3.2.
The dependence of retention on %B for regular as opposed to irregular samples
is further illustrated in Figure 6.7. Figure 6.7a shows plots of log k against %B for
a regular sample; a mixture of nine herbicides of similar molecular structure (see
Fig. 2.6 for the separation of several of these compounds as a function of %B). The
slope of each plot increases for more retained solutes (in the order 1 < 2 < 3 ).
For plots of log k versus %B for regular samples, this results in a characteristic,
‘‘fan-like’’ appearance—with no intersection of one plot by another (no change
in relative retention). Values of α and resolution increase continuously for regular

samples as %B is decreased. Another way to describe the behavior of regular
samples is in terms of Equation (6.1). For regular samples, the slopes S of plots as
in Figure 6.7a are highly correlated with extrapolated values of log k for water as
mobile phase (log k
w
). Figure 6.7b shows such a plot for the data of Figure 6.7a;
an excellent correlation is noted (r
2
= 1.00). Corresponding correlations of log k
w
versus S for the regular samples of Figures 6.1a,b and 6.2 give r
2
= 0.99 for each.
A similar treatment as in Figures 6.7a,b for a regular sample is shown in
Figures 6.7c,d for the irregular sample of Figure 6.6. In Figure 6.7c, plots of log
k versus %B frequently intersect (marked by
•
), unlike the behavior of the regular
sample in Figure 6.7a. As a result several peak reversals occur over this range in
%B (e.g., peaks 3–4). Similarly a plot of S versus log k
w
for the irregular sample
in Figure 6.7d shows a somewhat poorer correlation (r
2
= 0.87). Samples that
contain acidic and/or basic compounds (unlike the examples of Fig. 6.7) tend to be
more irregular; However, most samples—whether ‘‘ionic’’ or ‘‘neutral’’—exhibit
some degree of irregularity and solvent-strength selectivity can be a useful tool for
improving the resolution of such samples.
6.3.2 Solvent-Type Selectivity

Changes in %B may fail to achieve adequate resolution of a given sample. An
illustration is shown in Figure 6.8 for the separation of a mixture of substituted
benzenes. In this example, a change in mobile phase from 46 to 34% ACN results in
some change in relative retention (e.g., peaks 2–3, 8–9), but that has no significant
effect on the separation of overlapped peaks 3 and 4. Consequently some other
change in conditions that affect selectivity will be necessary—for example, a change
from ACN to MeOH as the B-solvent. When 61% MeOH is used in place of
46% ACN (Fig. 6.9a), solvent strength is about the same (similar run times), but
several further changes in relative retention are seen due to solvent-type selectivity.
Although previously unresolved peaks 3 and 4 are now well separated, peaks 1 and
2 (which were well resolved with ACN as B-solvent) are overlapped. With results
such as this for two mobile phases with different B-solvents, a mixture of the two