Introduction to Modern Liquid Chromatography, Third Edition part 41 pps

356 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY
Time (min)
1
2
3
4
5
6
7
8
9
10
11
0 2 4 6 8 10 12
Figure 7.21 Separation of a mixture of carbohydrate standards by anion-exchange chro-
matography with amperometric detection. Sample: 1, myo-inositol; 2,
D-sorbitol; 3, lactitol;
4,
L-fructose; 5, rhamnose; 6, D-galactose; 7, D-glucosamine; 8, D-glugose; 9, D-mannose; 10,
D-fructose; 11, D-ribose. Conditions: 300 × 4-mm anion-exchange column (5-μm particles);
mobile phase, aqueous 5-mM NaOH + 1-mM Ba(OAC)
2
; ambient temperature; 1 mL/min.
Adapted from [68].
RPC
Mixed mode
1-3
4
+
6
5

7
10
9
12
345
6
7
8
910
11
12
11
12
024681012141618(min)
(a)
(b)
Figure 7.22 Separation of a nitrogen-mustard mixture by RPC (a) versus mixed-mode IPC
(b). Sample: a mixture of small, hydrophilic amines. Conditions in (b): 150 × 2.1-mm Prime-
sep 100 column (5-μm particles) (SIELC Technologies, USA); 40% acetonitrile/buffer (0.1%
TFA); 0.2 mL/min. Adapted from [70].
while better peak shapes for compounds 9 and 10 are also observed. Another example
of mixed-mode separation with a cation-exchange column (PrimeSep SIELC) has
been reported to give a ‘‘unique anthocyanin elution pattern’’ for the analysis of
grape juice [71], a pattern that facilitates peak identification and quantitation.
Retention in mixed-mode separations can be controlled by varying %B, pH, and
buffer or salt concentration. Separation conditions that affect selectivity in RPC or
IEC can be used to vary relative retention.
Mixed-mode separation also offers an answer to the problem of poor retention
in RPC of certain strong bases, as the latter compounds can be more strongly
retained by interaction with a negatively charged column [72]. An additional

advantage of mixed-mode columns in this respect is their higher loadability for
ionized samples. Finally, mixed-mode columns are virtually unique in being able to
simultaneously separate mixtures of anions, cations, zwitterions, and neutrals [73].
A mixed-mode cation-exchange column which is especially stable at low pH, while
maintaining exceptional efficiency, was reported shortly before the present book
was sent to the publisher [74]. Comparisons of separations by a conventional C
18
REFERENCES 357
Figure 7.23 Separations of a mixture of acids, bases, and three amino acids by means of
different columns. Conditions: 50 × 4.6-mm columns (5-μm particles), 1.0 mL/min; (a)
C
18
column, 10% ACN/aqueous 0.1% TFA, 40
◦
C; (b) commercial mixed-mode column
(PrimeSep 200), 10% ACN/aqueous 0.01% TFA, 40
◦
C; (c) experimental mixed-mode col-
umn, 24% ACN/0.02% TFA, 65
◦
C. Adapted from [75].
column, a commercial mixed-mode column (hydrophobic cation-exchanger), and
the latter column are shown in Figure 7.23. Note the stronger retention of bases in
Figure 7.23c, despite the higher temperature and stronger mobile phase (24%B), as
well as their better resolution—possibly the result of an expanded retention range.
Mixed-mode phases for solid phase extraction (SPE) have also been found useful for
sample preparation (Section 16.6.7.1).
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CHAPTER EIGHT
NORMAL-PHASE
CHROMATOGRAPHY
8.1 INTRODUCTION, 362
8.2 RETENTION, 363
8.2.1 Theory, 366
8.2.2 Solvent Strength as a Function of the B-Solvent and %B, 370
8.2.3 Use of TLC Data for Predicting NPC Retention, 373
8.3 SELECTIVITY, 376
8.3.1 Solvent-Strength Selectivity, 376
8.3.2 Solvent-Type Selectivity, 376
8.3.3 Temperature Selectivity, 380
8.3.4 Column Selectivity, 381
8.3.5 Isomer Separations, 382
8.4 METHOD-DEVELOPMENT SUMMARY, 385
8.4.1 Starting Conditions for NPC Method Development: Choice of
Mobile-Phase Strength and Column Type, 388
8.4.2 Strategies for Optimizing Selectivity, 389
8.4.3 Example of NPC Method Development, 390
8.5 PROBLEMS IN THE USE OF NPC, 392
8.5.1 Poor Separation Reproducibility, 392
8.5.2 Solvent Demixing and Slow Column Equilibration, 394
8.5.3 Tailing Peaks, 394
8.6 HYDROPHILIC INTERACTION CHROMATOGRAPHY, 395
8.6.1 Retention Mechanism, 396
8.6.2 Columns, 397
8.6.3 HILIC Method Development, 398
8.6.4 HILIC Problems, 401
Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder,
Joseph J. Kirkland, and John W. Dolan

Copyright © 2010 John Wiley & Sons, Inc.
361
362 NORMAL-PHASE CHROMATOGRAPHY
8.1 INTRODUCTION
In the early 1900s, when chromatography was first developed (Section 1.2), columns
were packed with polar, inorganic particles such as calcium carbonate or alumina.
The mobile phase used in these experiments was a less-polar (water-free) solvent
such as ligroin (a saturated hydrocarbon fraction from petroleum). For the next 60
years, this procedure continued to be the most common (‘‘normal’’) way in which
chromatography was carried out. For this reason the use of a polar stationary phase
(with a less-polar mobile phase) is today referred to as normal-phase chromatography
(NPC). Another term used to describe NPC is adsorption chromatography,in
recognition of the fact that retained solute molecules are attached to (or adsorbed
onto) the surface of particles within the column (Section 8.2).
After the introduction of high-performance reversed-phase chromatography
(RPC) in the 1970s, the use of NPC for HPLC analysis became increasingly less
common. This was in part the result of the greater convenience of RPC, as well as its
advantages for the separation of many samples of biological origin and/or medical
interest. Some problems that are common to NPC (Section 8.5) have also played a
role in its declining popularity compared with RPC.
Today NPC is useful mainly for (1) analytical separations by thin-layer chro-
matography (TLC, Section 1.3.2), (2) the purification of crude samples (preparative
chromatography and sample preparation, Chapters 15, 16), (3) the separation of
very polar samples that are poorly retained and separated by RPC, or (4) the reso-
lution of achiral isomers (Section 8.35). NPC may also prove beneficial occasionally
for other samples, by virtue of its unique characteristics; for example, samples that
contain very nonpolar compounds that are of no interest to the analyst. The latter
sample constituents would be strongly retained by RPC, necessitating either long run
times, sample preparation, or the use of gradient elution; with NPC, very nonpolar
compounds elute near t

0
, and do not create a problem for isocratic separation (e.g.,
see Section 8.4.3). In any case, it is often best to postpone the use of NPC until after
RPC has been tried and found wanting.
Prior to 1970 a wide variety of inorganic packings were used for NPC: alumina,
magnesia, magnesium silicate (Florisil), and diatomaceous earth (Celite, kieselguhr),
to name a few examples. By the advent of HPLC, however, synthetic (unbonded)
silica had become the column packing of choice for both column chromatography
and TLC. The advantages of silica for NPC include:
• a more neutral, less active surface, with less likelihood of undesirable sample
reactions during separation
• strong particles of controlled size and porosity that can withstand the high
pressures required in HPLC
• a generally higher surface area, allowing larger weights of injected sample
for either increased detection sensitivity or increased yields in preparative
chromatography
• greater purity and reproducibility, permitting more repeatable separations
• reasonable cost and availability
While a preference for silica has continued to the present day, other column
options for NPC have emerged over time. Three polar-bonded-phase packings
8.2 RETENTION 363
(Section 5.3.3), chemically similar to those used in RPC, were introduced for NPC
during the 1970s: (1) cyano columns, where –(CH
2
)
3
–C ≡N groups are bonded to
silica particles, (2) diol columns bonded with –(CH
2
)

3
–O–CH
2
–CHOH–CH
2
OH
groups, and (3) amino columns with –(CH
2
)
3
–NH
2
ligands. The differing properties
of these bonded-phase columns for NPC are discussed in Section 8.3.4, and some
reasons for their use in place of unbonded silica can be inferred from the discussion
of Section 8.5, which deals with problems associated with the use of silica columns
in NPC.
During the 1990s silicas of higher purity (type-B; Section 5.2.2.2) became
commercially available, and these materials gradually displaced the less pure type-A
silica used previously for analytical NPC separations. Some advantages of type-B
silica for NPC are discussed in Section 8.5. The latest version of NPC is so-called
hydrophilic interaction chromatography (HILIC; Section 8.6), also called aqueous
NPC. HILIC column-packings consist of either (a) silica particles that are bonded
with polar hydrophilic groups such as amides or (b) bare silica. For either kind of
HILIC column, the mobile phase is a mixture of water and organic solvent—as
opposed to the water-free mobile phases that have traditionally been used for
NPC. HILIC provides some of the convenience that is characteristic of RPC, while
minimizing other problems associated with the use of silica columns and nonaqueous
mobile phases (Section 8.5).
In the present chapter, unless noted otherwise, we will assume the use of

unbonded, type-B silica columns. The surface of a silica particle is covered with
silanol groups ≡Si–OH (Section 5.2.2.2) which are mainly responsible for its
chromatographic properties. These silanol groups are relatively strong proton donors
that can interact with and retain solute molecules that contain hydrogen-bond
acceptor groups (any molecule with available electrons or a dipole moment). The
silica surface also strongly attracts small polar molecules such as water, which can
lead to certain problems discussed in Section 8.5. For further details on the role of
the column in NPC separation, see Section 8.3.4 (column selectivity).
8.2 RETENTION
Because the column in NPC is more polar than the mobile phase, more-polar solutes
will be preferentially retained or adsorbed—the opposite of RPC. This is illustrated
in Figure 8.1a for the separation of several mono-substituted benzenes, using a silica
column with 20% CHCl
3
-hexane as mobile phase; the more-polar solvent CHCl
3
is the B-solvent and the less-polar hexane is the A-solvent. Here the less-polar
solutes benzene (–H) and chlorobenzene (–Cl) leave the column first, while the
more-polar aniline (–NH
2
), benzoic acid (–COOH), and benzamide (–CONH
2
)
leave the column last. This retention behavior can be contrasted with RPC retention
(Fig. 2.7c), where retention decreases with increasing solute polarity. Figure 8.1b
compares retention (log k) in NPC and in RPC for several mono-substituted
benzenes. As expected, there is a negative correlation of retention for NPC over
RPC—corresponding approximately to a reversal of retention order. While the
correlation of Figure 8.1b is moderately strong (r
2

= 0.76), there is also significant
scatter of the data. That is, NPC separation cannot be regarded as the exact opposite
of RPC retention. Keep in mind that relative retention in both NPC and RPC can
364 NORMAL-PHASE CHROMATOGRAPHY
024681012
Time (min)
–Cl
–H
–SH
–OCH
3
–NO
2
–C N
–CO
2
CH
3
+
–CHO
silica column
20% CHCl
3
(a)
(b)
–OH
–COCH
3
–NH
2

–COOH
–CONH
2
800400 1200
Time (min)
–0.2 0.0 0.2 0.4 0.6 0.8
2.0
1.0
0.0
–1.0
log k
(NPC)
lo
g
k
(
RPC
)
y = 1.6 – 3.4x
r
2
= 0.76
–OH
–COCH
3
–CHO
–CO
2
CH
3

–C N
–NO
2
–OCH
3
–H
–Cl
Figure 8.1 Example of normal-phase retention as a function of solute polarity. Sample:
mono-substituted benzenes (substituents indicated for each peak; e.g., –H is benzene, –Cl
is chlorobenzene). Conditions: 150 × 4.6-mm silica (5-μm particles); 20% CHCl
3
-hexane
mobile phase; ambient temperature; 2.0 mL/min. (a) Chromatogram is recreated from data
of [1]; (b) retention of (a) compared with RPC retention from Figure 2.7c for benzenes substi-
tuted by the same functional group (50% acetonitrile-water as RPC mobile phase).
also vary significantly with changes in the column, mobile phase, or temperature, all
factors that contribute to the scatter of retention plots as in Figure 8.1b.
Apart from the approximately inverted retention order for the sample in NPC
as opposed to RPC, there are two additional differences in NPC retention that are
related to (1) the number n of alkyl carbons in the solute molecule (its carbon
number C
n
), and (2) isomeric solutes. These two general characteristics of NPC
versus RPC are illustrated in the separations of Figure 8.2. Figure 8.2a shows the
RPC separation of 17 alkyl-substituted anilines with a C
8
column and 60% MeOH
as mobile phase. As the value of C
n
increases, retention increases for RPC (but not

for NPC). Isomeric solutes of identical alkyl-carbon number (e.g., C
1
, consisting
of o-, m-, and p-methylanliline) are seen to be bunched together, while solutes of
differing carbon number (e.g., C
1
vs. C
2
) are well separated. As summarized in
Figure 8.2e, average retention times in RPC increase regularly as the carbon number
increases (by an average 1.4-fold per additional carbon in this example).
8.2 RETENTION 365
(a)
(b)
(c)
(d)
(e)
average retention time (min) range in k
RPC (a) NPC (b-d) RPC (a) NPC (b-d)
C
0
C
1
1.1 2.0
C
2
1.2 3.4
C
3
1.2 3.0

C
4
C
5
1.2 7.5 (not shown)
1.5 6.6
1.9 5.4
2.6 4.6
4.5 8.7 (not shown)
6.5 5.1 (not shown)
468
Time (min)
468 468
Time (min)
Time (min)
C
1
C
2
C
3
1
2
3
4
5
6
7
8
9

10
11
12
13
14
0246
C
0
aniline
C
1
C
2
C
3
C
4
(4-n-butylaniline)
C
5
2-methyl-
4-n-butylaniline
RPC
NPC
Time (min)
Figure 8.2 Comparison of NPC separation (a) with RPC separation (b–d)foramix-
ture of alkyl-substituted anilines. Conditions: 150 × 4.6-mm C
8
column (5-μmparti-
cles) in (a), 150 × 4.6-mm cyano column (5-μm particles) in (b–d); mobile phase is 60%

methanol–pH-7.0 buffer in (a), and 0.2% isopropanol-hexane in (b); ambient temperature
and2.0mL/minin(a)and(b). Sample (peak numbers): 1–3, 2-, 3- and 4-methylaniline;
4, 2,6-dimethylaniline; 5, 2-ethylaniline; 6, 2,5-dimethylaniline; 7, 2,3-dimethylaniline;
8, 2,4-dimethylaniline; 9, 3-ethylaniline; 10, 4-ethylaniline; 11, 3,4-dimethylanilne; 12,
2,4,6-trimethylaniline; 13,2-i-propylaniline; 14,4-i-propylaniline. Chromatograms recon-
structed from data of [2].
Figure 8.2b–d illustrates the further separation of fractions C
1
,C
2
,andC
3
from Figure 8.2a by NPC (using a cyano column with a mobile phase of 0.2%
isopropanol/hexane). It is seen that there is no consistent change in retention time
for NPC as the number of alkyl carbons increases (see the summary of Fig. 8.2e). That
is, NPC can separate solutes of differing functionality (as in Fig. 8.1a), but differences
in solute carbon number have much less effect on retention. For this reason NPC has
been used in the past for compound-class separations of petroleum-related materials
[3] and lipid samples [4]. NPC permits the group-separation of petroleum samples
into saturated hydrocarbons, olefins, benzenes, and various polycyclic aromatic