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Practical HPLC Method Development, Second Editionby Lloyd R. Snyder, Joseph J. Kirkland and Joseph L. GlajchCopyright © 1997 John Wiley & Sons, Inc.
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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 predict-able 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 reten-tion 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
Practical HPLC Method Development, Second Edition by Lloyd R. Snyder, Joseph J. Kirkland and Joseph L. Glajch
Copyright 1997 John Wiley & Sons, Inc.
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 NPC
Group
Phenyl - B r - C H 3 - C H 2 -- C l - F -OCH3 - H * -C0 2 CH 3 - C N - C H O - O H - N H 2 -CONH2 -S02NH2
30% ACN
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
60% ACN
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
Hexane
13 0.7 1.2 1.0 0.7 0.8
24 (1.0)
390 310 410
1,400 6,700
90,000
CH2C12
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 polar-ity 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
APPENDIX III 731
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
Compound
2,6-Dimethylaniline 3,4-DimethylaniIine N, N-Diethy l-2-methy laniline 2-Methyl-4-n-butyIaniline
Normal Phase"
k a
2.8 9.5 3.4 0.3 5.1 17
Reversed Phase*
a
1.02
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 mo-bile phase
Compound k a
m-Iodoanisole 2.2 p-Iodoanisole 4.1 1.9 1-Methoxy naphthalene 4.6 2-Methoxynaphthalene 12.9 2.8 Phenanthrene 6.5 Anthracene 27.5 4.2
Source: Ref. 4.
732 APPENDIX HI
I - . A..
10 20 30 40 min (b)
1
4
m 20 15 10
Minutes (c)
15 10 5 Minutes
(d)
FIGURE in.l Comparison of isomer separations by reversed-phase vs. normal-phase 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 adsorp-tion 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 pro-vide 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 sepa-rated 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 re-solved. 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 sum-mary 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.