Practical HPLC Method Development, Second Edition

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

Copyright 1997 John Wiley & Sons, Inc.

APPENDIX III
RETENTION IN REVERSED-PHASE
AND NORMAL-PHASE HPLC AS
A FUNCTION OF SAMPLE
MOLECULAR STRUCTURE

In this book we recommend an approach to HPLC method development that

largely ignores the structures of individual sample compounds. One exception
is the case of samples that contain acidic or basic compounds, where predictable changes in retention can be created by a change in mobile phase pH
(Section 7.2). If ion-pair or ion-exchange chromatography is used for such
samples, it is useful to know whether the sample contains acidic or basic
compounds. Another exception occurs for "special" samples (Fig. 1.3), which
benefit from separation conditions that are generally different from those
chosen for "regular" samples; see Chapters 11 and 12.
If a separation method is required where the molecular structures of the
sample compounds are known, it is possible to estimate relative retention
for either reversed-phase (RPC) or normal-phase (NPC) separation. Such
predictions are usually quite approximate, but even rough estimates of retention may be helpful in some cases. See the further discussion of Section 10.5.1,
which describes a computer program for reversed-phase HPLC based on
this approach.

ffl.l

SUBSTITUENT EFFECTS

In Table III.l the effect on retention of adding a substituent group to an

aromatic molecule is shown for some representative RPC and NPC conditions.
For example, adding an alkyl carbon (methyl or mthylne group) to a sample
molecule increases its RPC retention 1.5- to 2.5-fold, but has less effect on
729

730

APPENDIX HI

TABLE UM Retention as a Function of Sample Molecular Structure for

Substituted Benzenes: Effect on k of Different Substituent Groups
Relative Value of kf
RPC
Group
Phenyl
-Br
-CH3
-CH2-Cl
-F
-OCH3
-H*
-C02CH3
-CN
-CHO
-OH
-NH2
-CONH2
-S0 2 NH 2

NPC

30% ACN

60% ACN

Hexane

CH2C12

12.3
2.8
2.5
2.2
2.3
1.3
1.1
(1.0)
0.9
0.5
0.4
0.2
0.2
0.1
0.1

3.2
1.7
1.5
1.5
1.5
1.0
1.0
(1.0)
0.8
0.6
0.6
0.3
0.4
0.2
0.2

13
0.7
1.2
1.0
0.7
0.8
24
(1.0)
390
310
410
1,400
6,700
90,000

1.5
0.6
0.9
0.7
0.4
0.7
3.5
(1.0)
13
10
13
60
180
1,800

Source: Refs. 1 and 2.

" Compared to benzene (H); C,g column for RPC [1), silica for NPC [2].
* Compound before substitution (benzene).

NPC retention (0.7- to 1.2-fold). Therefore, RPC is a better HPLC method

for the separation of homologs or other compounds, differing only in alkyl
carbon number. Similarly, adding a polar group such as hydroxyl to a sample
molecule decreases its RPC retention (by a factor of 0.2 to 0.3), whereas this
same change in molecular structure increases NPC retention (by a factor of 60
to 1400). If a very pronounced separation of a compound from a hydroxylated
derivative were required (as in preparative HPLC, Chapter 13, where large
values of a are preferred), NPC separation on a silica column would be
preferred over a similar separation by RPC.
Table III. 1 shows that retention decreases with increasing substituent polarity for RPC and increases for NPC. That is, more polar compounds will elute
first in RPC and last in NPC. The effect of a substituent group on retention
decreases for a stronger mobile phase (e.g., 60% ACN vs. 30% ACN (RPC)
or CH2C12 vs. hexane (NPC) in Table III.l). A corollary to this observation
is that a values usually decrease for higher values of % B, although there are
important exceptions to this rule (Section 6.3.1). For silica as column packing,
differences in solute functionality cause a larger change in retention than is
observed for RPC separation. Thus, other factors equal, NPC with silica will

731

APPENDIX III

give larger values of a for compounds differing in functionality. This increased

selectivity for silica vs. RPC is not as pronounced when polar-bonded-phase
packings are used for NPC separation. The RPC data of Table III.l are for
aromatic functional groups, but similar changes in retention are found for the
same functional groups as aliphatic substituents.

III.2 ISOMER SEPARATIONS

As noted in Chapter 6, NPC is usually better for separating achiral isomers
than RPC. This ability of NPC for isomer separations arises from two effects:
(1) the more rigid or "ordered" structure of most NPC column packings, and
(2) localization effects as illustrated in Fig. 6.216. The adsorption sites A in
Fig. 6.216 occupy fixed positions, and the polar solute groups X and Y will
differ in their ability to interact with these sites according to the positions of
groups X and Y within the sample molecule. Intramolecular electronic and

TABLE m.2 Examples of Isomer Selectivity in NPC Separation

(a) Separation of aniline isomers
Normal Phase"
Compound

2,6-Dimethylaniline
3,4-DimethylaniIine
N, N-Diethy l-2-methy laniline
2-Methyl-4-n-butyIaniline

2.8
9.5
0.3
5.1

Reversed Phase*

3.4

1.02

17

1.20

Source: Ref. 3.
* Cyano column with 0.2% 2-propanol as mobile phase.
b
Cg column, 60% MeOH-buffer as mobile phase.

(b) Separation of aromatic isomers on an alumina column with 10% CH2CI2 as mobile phase
Compound
k
a
m-Iodoanisole
p-Iodoanisole
1-Methoxy naphthalene
2-Methoxynaphthalene
Phenanthrene
Anthracene
Source: Ref. 4.

2.2
4.1
4.6
12.9
6.5
27.5

1.9
2.8
4.2

APPENDIX HI

732

I-.

A..

10

20

30

40

min

(b)
1

20

15

10
Minutes
(c)

15

m
10

5
Minutes
(d)

FIGURE in.l Comparison of isomer separations by reversed-phase vs. normalphase HPLC. (a) RPC separation of aniline mixture; 20 x 0.44-cm Qg column, 80%
MeOH-buffer (pH 7.0), 0.75 mL/min [3]; (b) NPC separation of Cranilines from (a);
20 x 0.44-cm cyano column, 0.2% 2-propanol-isooctane, 0.75 mL/min [3]; (c) RPC
separation of five cis-trans isomers of retinol (bands 2 to 6); 15 x 0.46-cm Ci8 column,
80% MeOH-water, 1 mL/min, 40C [5]; (d) NPC separation of sample of (c); 25 x
0.4-cm silica column, 8% dioxane-hexane, 1 mL/min, 40C [5]. See the text for details.
(Reprinted with permission from Refs. 3 and 5.)

APPENDIX III

733

stehe effects will further affect the localization and interaction of individual
sample substituents X and Y. As a result, isomeric mixtures of compounds
are usually better separated by NPC than by RPC, due to differences in the
ability of different isomers to align their polar functional groups with adsorption sites (somewhat like a lock-and-key fit). Several examples in Table III.2
illustrate better isomer separations by NPC.
In Table III.2a the separation of some aniline isomers by NPC with a cyano
column is compared with RPC separation of these same compounds. In the
first example, NPC separates 2,6-dimethylaniline from the 3,4-isomer with
a = 3.4. The RPC separation of these two compounds results in almost
complete overlap (a = 1.02). In the second example of Table III.2a, two
isomeric C5-substituted anilines are very well resolved by NPC (a = 17),
whereas RPC separation is much poorer (a = 1.20).
In Table 111.2ft, NPC separation with alumina is shown for several aromatic
hydrocarbon isomers: m- and p-iodoanisole, 1- and 2-methoxynaphthalene,
and phenanthrene/anthracene. In each case, a large value of a results
(a > > 1), allowing the easy separation of these isomeric compounds. Inorganic
adsorbents such as silica and alumina are more ordered and rigid than their
polar-bonded-phase counterparts, and the inorganic adsorbents therefore provide generally better separations of isomers.
Figure III.l compares NPC and RPC separation of isomeric compounds
in two samples. In Fig. III. la, a mixture of alkyl-substituted anilines is separated by RPC. Compounds of the same carbon number (Co aniline, C\
methyl anilines, etc.) are unresolved as shown further by the data of Table
III.2a. However, compounds differing in carbon number are well separated
from each other. Figure III. lft shows the further separation of the C2 fraction
(circled in Fig. III. la) using NPC (cyano column); all eight isomers are resolved. Figure III.lc and d compare the separation of five cis-trans isomers
of retinol (bands 2-6) by (c) RPC and (d) NPC. The better separation of
these isomers in (d ) is apparent.
A vast number of studies have been reported that attempt to further relate
HPLC retention to molecular structure and separation conditions. For a summary of some of these approaches, see Refs. 6 to 8 for RPC retention and
Ref. 9 for NPC retention.

REFERENCES
1. R. M. Smith, J. Chromatogr., 656 (1993) 381.
2. L. R. Snyder, Principles of Adsorption Chromatography, Marcel Dekker, New York,
1968, p. 264.
3. L.-A. Truedsson and B. E. F. Smith, J. Chromatogr., 214 (1981) 291.
4. L. R. Snyder, / Chromatogr., 20 (1965) 463.
5. B. Stancher and F. Zonta, J. Chromatogr., 234 (1982) 244.

734

APPENDIX III

6. P. W. Carr, D. E. Martire, and L. R. Snyder, eds., "The Retention Process in

Reversed-Phase Liquid Chromatography," J. Chromatogr., 656 (1993).
7. T. Hamoir, D. L. Massart, W. King, S. Kokot, and K. Douglas, /. Chromatogr. Sei.,
31 (1993) 393.
8. K. Valko and P. Siegel, J. Chromatogr., 631 (1993) 49.
9. L. R. Snyder, Principles of Adsorption Chromatography, Marcel Dekker, New
York, 1968.