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
Trang 1Time (min)
1
4
5
6
9
Figure7.21 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
1 2
3 4 5 6
7
11
12
(a)
(b)
Figure7.22 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 C18
Trang 2Figure7.23 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)
C18column, 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)
REFERENCES
1 P J Twitchett and A C Moffat, J Chromatogr., 111 (1975) 149.
2 E P Kroef and D J Pietrzyk, Anal Chem., 50 (1978) 502.
3 L R Snyder, A Maule, A Heebsch, R Cuellar, S Paulson, J Carrano, L Wrisley,
C C Chan, N Pearson, J W Dolan, and J Gilroy, J Chromatogr A, 1057 (2004) 49.
4 J A Lewis, J W Dolan, L R Snyder, and I Molnar, J Chromatogr., 592 (1992) 197.
5 U D Neue, A M´endez, K Van Tran, and D M Diehl, in HPLC Made to Measure,
S Kromidas, ed., Wiley-VCH, Hoboken, NJ, 2006, pp 71–87
6 A P Schellinger and P W Carr, LCGC, 22 (2004) 544.
7 D V McCalley, Anal Chem., 75 (2003) 3404.
Trang 38 F Gritti and G Guiochon, J Chromatogr A, 1033 (2004) 43–; 1132 (2006) 51.
9 J B Li, LCGC, 10 (1992) 856.
10 J W Dolan LCGC, 8 (1990) 212.
11 L R Snyder, unreported data
12 D V McCalley, J Chromatogr A, 987 (2003) 17.
13 R F Doerge, ed., Wilson and Gisvold’s Textbook of Organic Medicinal and
Pharma-ceutical Chemistry, 8th ed., J B Lippincott, Philadepha, app B.
14 M Ros´es, J Chromatogr A, 1037 (2004) 283.
15 X Subirats, E Bosch, and M Ros´es, J Chromatogr A, 1121 (2006) 170.
16 D V McCalley, Adv Chromatogr., 46 (2008) 305.
17 J A Lewis, D C Lommen, W D Raddatz, J W Dolan, L R Snyder, and I Molnar, J.
Chromatogr., 592 (1992) 183.
18 I Canals, K Valk ´o, E Bosch, and M Ros´es, Anal Chem., 73 (2001) 4937.
19 P L Zhu, J W Dolan, L R Snyder, D W Hill, L Van Heukelem, and T J Waeghe, J.
Chromatogr A, 756 (1996) 51.
20 P L Zhu, J W Dolan, L R Snyder, N M Djordjevic, D W Hill, J.-T Lin,
L C Sander, and L Van Heukelem, J Chromatogr A, 756 (1996) 63.
21 C B Castells, C R `afols, M Ros´es and E Bosch, J Chromatogr A, 1002 (2003) 41.
22 C B Castells, L G Gagliardi, C R `afols, M Ros´es, and E Bosch, J Chromatogr A,
1042 (2004) 23
23 L G Gagliardi, C B Castells, C R `afols, M Ros´es, and E Bosch, J Chromatogr A,
1077 (2005) 159
24 S M C Buckenmaier, D V McCalley, and M R Euerby, J Chromatogr A, 1060
(2004) 117
25 J Pellett, P Lukulay, Y Mao, W Bowen, R Reed, M Ma, R C Munger, J W Dolan,
L Wrisley, K Medwid, N P Toltl, C C Chan, M Skibic, K Biswas, K Wells, and
L R Snyder, J Chromatogr A, 1101 (2006) 122.
26 N S Wilson, M D Nelson, J W Dolan, L R Snyder, and P W Carr, J Chromatogr.
A, 961 (2002) 195.
27 J Gilroy, J W Dolan and L R Snyder, J Chromatogr A, 1000 (2003) 757.
28 J J Gilroy, J W Dolan, P W Carr, and L R Snyder J Chromatogr A, 1026 (2004)
77
29 N S Wilson, M D Nelson, J W Dolan, L R Snyder, R G Wolcott, and P W Carr,
J Chromatogr A, 961 (2002) 171.
30 D Guo, C T Mant, and R S Hodges, J Chromatogr., 386 (1987) 205.
31 J L Glajch, J C Gluckman, J G Charikofsky, J M Minor, and J J Kirkland,
J Chromatogr., 318 (1985) 23.
32 R G Wolcott, J W Dolan, and L R Snyder, J Chromatogr A, 869 (2000) 3.
33 P Haber, T Baczek, R Kaliszan, L R Snyder, J W Dolan, and C T Wehr.,
J Chromatogr Sci., 38 (2000) 386.
34 S Pous-Torres, J R Torres-Lapasi ´o, J J Baeza-Baeza, and M C Garc´ıa- ´Alvarez-Coque,
J, Chromatogr A, 1163 (2007) 49.
35 S Pous-Torres, J R Torres- Lapasi ´o, J J Baeza-Baeza, and M C Garc´ıa- ´Alvarez-Coque,
J, Chromatogr A, 1193 (2008) 117.
36 F Vanbel, B L Tilquin, and P J Schoenmakers, J Chromatogr A 697 (1995) 3.
Trang 437 D V McCalley, LCGC, 17 (1999) 440.
38 S M C Buckenmaier, D V McCalley, and M R Euerby, Anal Chem., 74 (2002)
4672
39 D V McCalley, J Chromatogr A, 1075 (2005) 57.
40 D V McCalley, J Chromatogr A, 708 (1995) 185.
41 D V McCalley, J Chromatogr A, 738 (1996) 169.
42 D M Marchand, L R Snyder, and J W Dolan, J Chromatogr A, 1191 (2008) 2.
43 M A Stadalius, J S Berus, and L R Snyder, LCGC, 6 (1988) 494.
44 J Nawrocki, J Chromatogr A, 779 (1997) 29.
45 M T W Hearn, ed., Ion-pair Chromatography: Theory and Biological and
Pharmaceu-tical Applications, Dekker, New York, 1985.
46 L R Snyder, J J Kirkland, and J L Glajch, Practical HPLC Method Development, 2nd
ed., Wiley-Interscience, New York, 1997, ch 7
47 J H Knox and R A Hartwick, J Chromatogr., 204 (1981) 3.
48 M W Dong, J Lepore, and T Tarumoto, J Chromatogr., 442 (1988) 81.
49 A Bartha, G Vigh, and Z Varga-Puchony, J Chromatogr., 499 (1990) 423.
50 A Bartha, G Vigh and J St ˚ahlberg, J Chromatogr., 485 (1989) 403.
51 A P Goldberg, E Nowakowska, P E Antle, and L R Snyder, J Chromatogr.,
316 (1984) 241
52 J M Roberts, A R Diaz, D T Fortin, J M Friedle, and Stanley D Piper, Anal Chem.,
74 (2002) 4927
53 J Flieger, J Chromatogr A, 1113 (2006) 37.
54 J Flieger, J Chromatogr A, 1175 (2007) 207.
55 J W Dolan and L R Snyder, Troubleshooting HPLC Systems, Humana Press, Clifton,
NJ, 1989, pp 429–435
56 S Levin and E Grushka, Anal Chem., 58 (1986) 1602.
57 E Rajakl ¨a, J Chromatogr., 218 (1981) 695.
58 H Small, T S Stevens, and W C Bauman, Anal Chem., 47 (1975) 1801.
59 P R Haddad and P E Jackson, Ion Chromatography, Elsevier, Amsterdam, 1990.
60 J S Fritz and D T Gjerde, Ion Chromatography, 3rd ed., Wiley-VCH, Weinheim, 2009.
61 J Weiss, Handbook of Ion Chromatography, 3rd ed., Wiley, 2005.
62 M More, M I H Helaleh, Q Xu, W Hu, M Ikedo, M.-Y Ding, H Taoda, and K
Tanaka, J Chromatogr A, 1039 (2004) 129.
63 J Qiu, J Chromatogr A, 859 (1999) 153.
64 Y Baba and G Kura, J Chromatogr A, 550 (1991) 5.
65 J E Madden and P R Haddad, J Chromatogr A, 850 (1999) 29.
66 J St ˚ahlberg, J Chromatogr A, 855 (1999) 3.
67 P J Andralojc, A J Keys, A Adam, and M A J Parry, J Chromatogr A, 814 (1998)
105
68 T R Cataldi, C Campa, M Angelotti, and S A Bufo, J.Chromatogr A, 855 (1999)
539
69 E Landberg, A Lundblad, and P P ˚ahlsson, J Chromatogr A, 814 (1998) 97.
70 H.-C Chua, H.-S Lee, and M.-T Sng, J Chromatogr A, 1102 (2006) 214.
Trang 571 J L McCallum, R Yang, J C Young, J N Strommer, and R Tsao, J Chromatogr A,
1148, (2007) 38
72 N H Davies, M R Euerby, and D V McCalley J Chromatogr A, 1138 (2007) 65.
73 J Li, S Shao, M S Jaworsky, and P T Kurtulik, J Chromatogr A, 1185 (2008) 185.
74 H Luo, L Ma, Y Zhang, and P W Carr, J Chromatogr A, 1182 (2008) 41.
75 H Luo, L Ma, C Paek, and P W Carr, J Chromatogr A, 1202 (2008) 8.
Trang 6CHAPTER 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
8.4.1 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
Trang 78.1 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 t0, 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
Trang 8(Section 5.3.3), chemically similar to those used in RPC, were introduced for NPC during the 1970s: (1) cyano columns, where –(CH2)3–C≡N groups are bonded to silica particles, (2) diol columns bonded with –(CH2)3–O–CH2–CHOH–CH2OH groups, and (3) amino columns with –(CH2)3–NH2ligands 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% CHCl3-hexane as mobile phase; the more-polar solvent CHCl3
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 (–NH2), benzoic acid (–COOH), and benzamide (–CONH2) 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 (r2= 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
Trang 90 2 4 6 8 10 12
Time (min)
–Cl –H
–SH –OCH3
–CO2CH3 + –CHO
silica column 20% CHCl3
(a)
(b)
–OH
–COCH3
–NH2
2
800
Time (min)
2.0
1.0
0.0
–1.0
log k (NPC)
log k (RPC)
y = 1.6 – 3.4x
r2= 0.76
–OH
–COCH3
2 CH3 –C N
–NO2
–OCH3
Figure8.1 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% CHCl3-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 Cn), 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 C8column and 60% MeOH
as mobile phase As the value of Cnincreases, retention increases for RPC (but not for NPC) Isomeric solutes of identical alkyl-carbon number (e.g., C1, consisting
of o-, m-, and p-methylanliline) are seen to be bunched together, while solutes of
differing carbon number (e.g., C1 vs C2) 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)
Trang 10(e)
average retention time (min) range in k RPC (a) NPC (b-d) RPC (a) NPC (b-d)
C0
C4
C5
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)
Time (min)
1
6 7
8 9 10 11
12 13
14
C0 aniline
C1
C2
(4-n-butylaniline)
C5
2-methyl-4-n-butylaniline
RPC
NPC
Time (min)
Figure8.2 Comparison of NPC separation (a) with RPC separation (b–d) for a
mix-ture of alkyl-substituted anilines Conditions: 150× 4.6-mm C8column (5-μm
parti-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 and 2.0 mL/min in (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 C1, C2, and C3 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