Liquid chromatography ion pair

Felice, L. Felice, J. and Kissinger, P. Determination of catecholamines in rat brain parts by reverse-phase ion-pair liquid chromatography. 

Fernandez et al. [9] used supercritical fluid extraction combined with ion pair liquid chromatography to determine quaternary ammonium in digested sludges and marine sediments. Carbon dioxide modified with 30% methanol was used as the extractant at an operating pressure of 380atm. Between 0.2 and 3.7g kg-1 surfactant was found in Swiss works effluent sludges, determined with a relative standard deviation of 7%. 

R. Loos, M.C. Alonso and D. Barcelo, Solid-phase extraction of polar hydrophilic aromatic sulfonates followed by capillary zone electrophoresis-UV absorbance detection and ion-pair liquid chromatography-diode array UV detection and electrospray mass spectrometry. J. Chromatogr.A 890 (2000) 225-237. 

Sulphonated azo dyes were separated and quantitated in various food products by ion-pair liquid chromatography with DAD and electrospray MS detection. The chemical structure of sulphonated azo dyes included in the investigation are shown in Fig. 3.36. Dyes were separated in an ODS column (125 X 2.0 mm i.d. particle size 5 pm) using gradient elution. An aqueous solution of 3 mM triethylamine (pH adjusted to 6.2 with acetic acid) and methanol... 

M.-R. Fuh and K.-J. Chia, Determination of sulphonated azo dyes in food by ion-pair liquid chromatography with photodiode array and electrospray mass spectrometry detection. Talanta, 56 (2002) 663-671. 

The selection of the counter-ion and its concentration are important for the separation of ionic compounds in reversed-phase and ion-exchange liquid chromatography. The addition of hydrophobic ions is an especially powerful method and several surfactants can be used as hydrophobic counter-ions. The theoretical column efficiency of ion-pair liquid chromatography is much better than that of an ion-exchange column, and the regeneration of a column is much faster. Thus, if we can control ion-pair liquid chromatography, we can solve a separation problem. (The important background sources in this area are listed at the end of the chapter.)... 

Two types of system are used for ion-pair liquid chromatography. When polar stationary phase materials, such as silica gel, are used an ion-pair partition mechanism is applied. When non-polar stationary phase materials, such as octadecyl-bonded silica gel and polystyrene gel, are employed a paired-ion adsorption mechanism is involved. The former is called normal-phase ion-pair partition liquid chromatography, and the latter is called reversed-phase ion-pair liquid chromatography. 

The following conditions should be satisfied to perform satisfactory ion-pair liquid chromatography. 

Example ion-pair liquid chromatography of amino acids. Amino acids are zwitterions. The amino group can form an ion-pair with an alkanesulfonate ion (such as octanesulfonate), and the carboxyl group can form an ion-pair with a tetrabutylammonium ion, depending on the pH of the solution. 

Table 4.4 Relationship between analytes and counter-ions in ion-pair liquid chromatography...

Increasing the concentration of the counter-ion further increased the retention, but the retention reached a plateau, as seen in Figure 4.14. A total separation of the amino acids by reversed-phase ion-pair liquid chromatography could be performed.11 A column switching technique reduced the total analysis time (Figure 4.15). 

Two mechanisms for retention in reversed-phase ion-pair liquid chromatography have been considered. One is the adsorption of the hydrophobic paired ion on the hydrophobic surface of stationary phase material. In the second mechanism, the hydrophobic counter-ion is held on the surface of the hydro-phobic stationary phase, and the analyte ion is retained by ion-ion interactions, as shown in Figure 4.16. In the latter case, of a dynamic ion-exchange... 

Figure 4.15 Ion-pair liquid chromatography of free amino acids using a column switching system. Column I, butyl-bonded silica gel, 50 x 4.6 mm i.d., 2, octyl-bonded silica gel, 50 x 4.6 mm i.d., and 3, octadecyl-bonded silica gel, 250 x 4.6 mm i.d. eluent, 0.01 m sodium acetate buffer (pH 5.6) containing 4 mM copper acetate and 0.8 mM sodium heptanesulfonate flow rate, 1 ml min-1 detection, UV 235 nm. Peaks 1, Tyr 2, Val 3, Met 4, His 5, Lys 6, lie, 7, Leu 8, Phe 9, Arg 10, Asp 11, Ser 12, Glu 13, Thr 14, Gly 15, Pro 16, Cys and 17, Ala. 1-9 were separated on column 1 and 10-17 were separated by a combination of columns 2 and 3.

Figure 4.16 Model of reversed-phase ion-pair liquid chromatography in which counterions are held on the stationary-phase surface.

The molecular absorption intensity of polar compounds is usually small, but highly sensitive detection can be obtained after pre- or post-column derivatiza-tions. The use of ultraviolet absorption or fluorescence-active counter-ions makes it possible to achieve highly sensitive detection of polar compounds and enhance the capability of ion-pair liquid chromatography. For example, N,N-dimethylprotriptyline has been used as a counter-ion for carboxylic acids12 and picric acid for quaternary amines13 in normal-phase ion-pair partition liquid chromatography. Phenethylammonium, cetylpyridinium, l-phenethyl-2-pyco-linium, and naphthalene-2-sulfonic acid have been used for sulfonic acid and alkyl amines detection.14,15 Ion-pair post-column extraction was applied on-line for fluorescence detection.16... 

Ion-pair liquid chromatography can be applied to compounds separated by ion-exchange liquid chromatography, and mixtures of ionic and non-ionic compounds are easily separated. The latter separation is difficult by ion-exchange liquid chromatography. Anions can be separated by reversed-phase ion-pair liquid chromatography (Figure 4.18). 

Figure 4.18 Analysis of anions in water using ion-pair liquid chromatography. Column, octadecyl-bonded silica gel, 15 cm x 4.6 mm i.d. eluent, 2 mM tetrabutyl-ammonium hydroxide (pH 5.3) in 3% acetonitrile-water flow rate, 1 ml min- detection, UV200 nm. Peaks 1, Br 2, N03 and3,1. ...

Ion-pair liquid chromatography can be applied to the separation of a wide variety of compounds. However, the flexibility of the selection of eluent components can confuse the operation. Trouble-shooting solutions are summarized in Table 4.6. 

B. A. Bidlingmeyer, S.N. Deming, W.P. Price, Jr., B. Sachok, and M. Petrusek, Retention mechanism for reversed-phase ion-pair liquid chromatography , J. Chromatogr., 1979,186, 419. 

S.O. Jansson, Effect of counter-ions in ion-pair liquid chromatography of hydrophobic amines on non-polar bonded phases , J. Liq. Chromatogr., 1982, 5, 677. 

In liquid chromatographic methods, reversed-phase columns are usually employed due to hydrophobic interaction of the amphenicols molecules witli the Cs or Ci8 stationary phases. Ion-pairing liquid chromatography has been also described for the separation of florfenicol and florfenicol amine (62) or chloramphenicol and deacetylchloramphenicol residues (46) using heptanesulfonate or octylsulfate-pairing ions, respectively. 

Liquid chromatographic separation of sedatives and -blockers is usually performed using reversed-phase columns. The preferred type of reversed-phase material is Cig-bonded silica (Table 29.16), but phenyl-bonded silica has also been employed for separation of xylazine and its major metabolite (525). Ion-pair liquid chromatography has also been suggested for separation of carazolol and xylazine residues, by addition to the mobile phase of dodecyl sulfate (522) or heptanesulfonate (520) pairing ions, respectively. 

Altenbach, and W. Giger, Determination of benzenesulfonates and naphthalenesulfonates in waste-water by solid-phase extraction with graphitized carbon-black and ion-pair liquid-chromatography with UV-detection , Anal. Chem., 67, 2325-2333 (1995). 

G Varela-Moreiras, E Seyoum, J Selhub. Combined affinity and ion pair liquid chromatographies for the analysis of folate distribution in tissues. J Nutr Biochem 2 44-53, 1991. 

J Selhub. Determination of tissue folate composition by affinity chromatography followed by high-pressure ion pair liquid chromatography. Anal Biochem 182 84-93,1989. 

JP Comet, GV Haar. The determination of some additives by ion pair liquid chromatography. Chromatogr Sci 20 343-361, 1982. 

K Takeba, K Fujinuma, T Miyazaki, H Nakazava. Simultaneous determination of /3-lactam antibiotics in milk by ion-pair liquid chromatography. J Chromatogr A 812 205 -211, 1998. 

R. El Harrak, M. Calull, R. M. Marce and F. Borrull, Determination of naphthalene-sulphonates in water by on-line ion-pair solid-phase extraction and ion-pair liquid chromatography with diode-array UV detection , Int. J. Environ Anal. Chem. 69 295-305 (1998). 

E. Pocurull, C. Aguilar, M. C. Alonso, D. Barcelo, F. Borrull and R. M. Marce, On-line solid-phase extraction-ion-pair liquid chromatography-electrospray mass spectrometry for the trace determination of naphthalene monosulphonates in water , J. Chromatogr. 854 187-195 (1999). 

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