Diol
The OH groups in this phase make the product comparable to silica. It is of interest for compounds with which it can form hydrogen bonds and is particularly suitable for tetracyclines, steroids, organic acids and biopolymers (proteins), amongst other analytes. Figure 11.1 presents the separation of a
178
Practical High-Performance Liquid Chromatography,Fourth edition Veronika R. Meyer
#2004 John Wiley & Sons, Ltd ISBN: 0-470-09377-3 (Hardback) 0-470-09378-1 (Paperback)
nonionic, polar surfactant which consists of a mixture of polyethoxylated alkylphenols. The oligomers differ in the number of ethoxy units; Triton X-100 has an average chain length ofca. nẳ10.
Nitrile (Cyano)
As it has a lower polarity, this phase often gives separations in a similar way to silica, but thekvalues are smaller for the same mobile phase.1It is particularly selective towards components with double bonds and towards tricyclic antidepressants. An application from this latter field is presented in Fig. 11.2.
Amino
Sugar and glycoside analysis (Fig. 11.3) is the classical use for amino phases.
The amino function can act as both a proton acceptor and a proton donor in
Fig. 11.1 Separation of a non-ionic surfactant on a diol phase. (Reproduced by permission of Supelco.) Conditions: sample, Triton X-100 with averagenẳ10;
column, 25 cm4.6 mm i.d.; stationary phase, Supelcosil LC-Diol, 5mm; mobile phase: 1 ml min1, non-linear gradient from 15.5% dichloromethane þ 3%
methanol in hexane to 40% dichloromethane þ 10% methanol in hexane in 35 min; temperature, 35C; UV detector, 280 nm.
——————————
1An example of this was given by H. Pfander, H. Schurtenberger and V. R. Meyer,Chimia,34, 179 (1980).
hydrogen bonding, being both Brứnsted base and acid. Organic and inorganic anions (acetate, acrylate, glycolate, formate, nitrite, bromide, nitrate, iodate and dichloroacetate) can be separated on an amino phase with a phosphate buffer of ca. pH 3.2Figure 11.4 shows the analysis of cabbage-lettuce containing about 1000 ppm of nitrate and 300 ppm of bromide (from soil treatment with methyl bromide).
NH2 is readily oxidized, so peroxides (in diethyl ether, dioxane or tetrahydrofuran) should be strictly avoided. Ketones and aldehydes react to give Schiff bases.3Amino silicas are less stable towards hydrolysis than other bonded phases and it is recommended to use a pre-column (scavenger column) to saturate the mobile phase.4
Fig. 11.2 Separation of tricyclic antidepressants on a nitrile phase. (Repro- duced with permission from G. L. Lensmeyer, D. A. Wiebe and B. A. Darcey,J.
Chromatogr. Sci., 29, 444 (1991).) Conditions: sample, serum extract from a patient who received clomipramine; column, 25 cm4.6 mm i.d.; stationary phase, Zorbax Cyanopropyl, 5–6mm; mobile phase, 1.2 ml min1 water–
acetonitrile–acetic acid–n-butylamine (600 : 400 : 2.5 : 1.5); temperature, 45C;
UV detector, 254 nm. Peaks: Mẳ metabolites of clomipramine; 1 trimipramine (internal standard); 2ẳdes-methylclomipramine; 3ẳclomipramine.
——————————
2U. Leuenberger, R. Gauch, K. Rieder and E. Baumgartner,J. Chromatogr.,202, 461 (1980); H. J.
Cortes,J. Chromatogr.,234, 517 (1982).
3
For reactivation of an amino column that had become exhausted as a result of reaction with actone, see D. Karlesky, D. C. Shelly and I. Warner,Anal. Chem.,53, 2146 (1981).
4B. Porsch and J. Kra´tka´,J. Chromatogr.,5431 (1991).
Fig. 11.3 Separation of potato tissue glycoalkaloids on an amino phase. (Re- produced with permission from K. Ko- bayashi, A. D. Powell, M. Toyoda and Y.
Saito, J. Chromatogr., 462, 357 (1989).) Conditions: sample, extract from young potato plantlets after solid-phase extrac- tion pretreatment; column, 30 cm 3.9 mm i.d.; stationary phase, mBondapak NH2; mobile phase, ethanol–acetonitrile–
potassium dihydrogenphosphate (3 : 2 : 1);
UV detector, 205 nm. Peaks: 1ẳa-chaco- nine; 2ẳa-solanine.
Fig. 11.4 Separation of nitrate and bromide in cabbage- lettuce extract on an amino phase. Conditons: sample, 20ml of extract after homogenization, protein precipitation and filtration; column, 25 cm3.2 mm i.d.; stationary phase, LiChrosorb NH2, 5mm; mobile phase, 1 ml min11% KH2PO4
in water adjusted to pH 3 with H3PO4; UV detector, 210 nm.
Peaks: 1ẳnitrile (from nitrate converted during sample treatment); 2ẳbromide; 3ẳnitrate.
The amino group acts as a weak anion exchanger in aqueous acidic solutions (taking the form of primary ammonium ion, RNH3
+); hence retention in an aqueous mobile phase is a function of pH. 0.1 M ammonia solution can be used to regenerate to the RNH2 form.
Nitro
This phase is selective for aromatics and its effectiveness is demonstrated in Fig. 11.5. Normal coal-tar pitch (residue from commercial high-temperature coal-tar distillation) was analysed and around 90 components could be distinguished, including polycyclic aromatic hydrocarbons and related hetero- cyclic and oligomer systems. Twenty-four of these were clearly identified by their characteristic UV spectra and comparison with known reference spectra.
Fig. 11.5 Separation of normal coal-tar pitch on a nitro phase. (Reproduced with permission from G. P. Blu¨mer, R. Thoms and M. Zander, Erdo¨l Kohle, Erdgas, Petrochem.,31, 197 (1978).) Conditions: columns, two 20 cm4 mm i.d.
plus a guard column; stationary phase, Nucleosil NO2, 5mm; mobile phase, hexane–chloroform, gradient elution; UV detector 300 nm; sensitivity, 1 a.u.f.s.
and, after 30 min, 0.4 a.u.f.s.
12 Ion-Exchange Chromatography