Ion-pairing RP chromatography

RP chromatography (C18 or amino stationary phase) or ion-pair RP chromatography are used for thiamine analysis. The mobile phases used are mainly methanol or acetonitrile within sodium acetate or phosphate acetate. 

Vitamin Bi (thiamin) is related to beriberi, a disease associated with a deficiency of this vitamin. In fact, thiamin is a coenzyme in different biochemical reactions. Pork, legumes, as well as liver and kidney products are regarded as excellent sources of this vitamin. Thiamin, as well as other water-soluble vitamins, is frequently found bound to proteins or carbohydrates or even phosphorylated. Therefore, prior to their analysis, a sample treatment to release the free forms of the vitamin is common. A t)q)ical extraction protocol for water-soluble vitamins includes autoclaving the sample with hydrochloric acid for the acid hydrolysis of the vitamin followed by an adjustment in the pH to values around 4.0—4.5, adequate for an enzymatic treatment. This vitamin can be, subsequently, separated by ion-pair RP chromatography and detected with a fluorescence detector after postcolumn oxidation to thiochrome. MS detection through electrospray ionization is also used, although the separation pH should be adjusted to maximize the ionization of the vitamin. 

RP and ion-pair RP chromatography on C18 stationary phases with UV—Vis [12,13] or fluorescence detection [6,8,10] are the LC methods most frequently employed other works report separations on C8 [14] and amide-C16 columns [15]. Free flavins in aqueous solution exhibit an... 

TABLE VIII. Comparison of the General Effect of Variables on Retention in Reversed-Phase Ion-Pair (RP-IPC) and Micellar Liquid Chromatography (MLC)... 

The choice of fhe LC mode for the analysis of water-soluble vitamins depends on the extraction procedure employed and the vitamin form to be quantified (Figure 18.1). The most popular LC modes are normal-phase (NP), reversed-phase (RP), ion-pair RP, ion-suppression RP, and ion-exchange chromatography. 

Although the FI A technique does not include a separation procedure, the simultaneous determination of 5 -mononucleotides essentially requires a separation step before detection of these compounds. Thus, high-performance LC (HPLC) techniques were employed for the quantitation of 5 -mononucleotides. The 5 -mononucleotides were quantified not to determine flavor enrichment but as components of nutritional or clinical importance, especially in infant formulas [16]. Three main modes of LC are applied for nucleotide analysis ion-exchange chromatography, reversed-phase LC (RP-LC), and ion-pair RP-LC [16]. 

The comprehensive review by Gocan et al. [25] focused specifically on lipophilic-ity measurements by liquid chromatography, including reversed phase, thin-layer, micellar, RP-ion-pair and countercurrent chromatography. 

Ion-pair chromatography separates ionic compounds using traditional RP stationary phases. A so-called counter-ion of opposite charge is added to the mobile phase. It forms a neutral ion-pair which can be easily separated under RP conditions. The mobile phase generally consists of water or buffer mixed with an organic modifier such as methanol or ACN. 

Acetonitrile shows in mixtnres with water, a better solnbility for salts. It is therefore recommended in ion-pair chromatography [52], Basic analytes also show better peak shapes in acetonitrile-buffer mixtures than with methanol. The proper selection, whether acetonitrile or methanol, should be used as the organic component in the mixtnre with a bnffer, however, the type of RP column used classical RP or a shielded RP column is also important. For demonstration with basic analytes, a standard mixture of anti depressives is used. Iso-eluotropic mixtures of methanol and acetonitrile are used. For standardization, the concentration of buffer components are also be kept constant. The analyte structures and the eluent mixtures are summarized in Table 2.2. As selectivity is worse in acidic eluents, a pH value of 7 has been used. Two phases... 

Since the 1970s numerous HPLC methods using lEC, RP and ion-pair chromatography have been proposed. In the last years, RP chromatography has become the most used method, thanks to its simplicity, sensitivity, and compatibility with different detection techniques. The stationary phases usually used are C18 or phenyl-bonded silica-based phases. More recently, alternative stationary phases, such as polar-embedded, polar endcapped, and perfluorinated phases, have been successfully tested for folate analysis [577]. The mobile phase is usually a mixture of phosphate or acetate buffer and acetonitrile or methanol. 

Herrmann et al. (24) used ion-pair chromatography for the determination of cyclamate. The efficiency of LiChrosorb RP-18 and Hypersil MOS 3 with a mobile phase of 5 mM tetrabutylammonium p-toluenesulfonate, pH 3.5, mixed with 12% methanol for the separation of cyclamate from other sweeteners was investigated. With the first column, cyclamate separated from saccharin, but the second was the recommended column for the analysis of cyclamate, saccharin, aspartame, and dulcin in a single run. 

The first successful attempt to use HPLC for the analysis of betalains was carried out by Vincent and Scholz (247). With the help of paired-ion chromatography (PIC) on an RP column (/zBon-dapak C, 8) they separated betacyanins and betaxanthins of red beets. The ion-pairing approach was adopted in order to exploit the highly ionic character of these pigments and the durability of the C18 columns. Using methanol-water at pH 7.5 and tetrabutylammonium as the PIC reagent,... 

Equation 1 describing a simple RP system (18,23) is analogous to that derived for the first time by Uekama et al (31) for determination of the stability constants of CD complexes with various ionic species by ion exchange chromatography. The analogous equations have been proposed by Horvath et al (32) for ion pair chromatography. 

---