Mobile phase acidic

The following are amongst the reagents that have been reported as being added to the mobile phase acids for quinine alkaloids [184], ninhydnn for amino acids [185 — 187], fluorescamine for biogenic amines [188] Fluorescein sodium [189], dichlorofluorescein [190], rhodamine 6G [191], ANS reagent [192] and bromine [193] have all been descnbed as additives to mobile phases... 

The effect of temperature on the acid base chemistry of the stationary phase can also play a role in separation. Free silanol groups on the stationary phase may exhibit changes in acid base chemistry with temperature [28]. Also, reverse phase columns with amine, amide, or acidic functional groups will be affected by the interaction of the temperature, the ionization state of the stationary phase, the mobile phase acidity, and the ionization state of the solute. Most non-linear van t Hoff plots can be rationalized in these terms, but it is difficult to predict a priori what the effects will be on a given system. Thus, it is important to characterize the system under study if a simple change in temperature produces unexpected effects. 

M. Roses and E. Bosch, Influence of mobile phase acid-base equilibria on the chromatographic behavior of protolytic compounds,/. Chromatogr. A 982 (2002), 1-30. 

Mobile phase acid acetonitrile acid acetonitrile acetonibri]e THF ... 

Mobile Phase Water Methanol Glacial Acetic Acid (69 28 3)... 

The concentrations of benzoic acid, aspartame, caffeine, and saccharin in a variety of beverages are determined in this experiment. A Gig column and a mobile phase of 80% v/v acetic acid (pH = 4.2) and 20% v/v methanol are used to effect the separation. A UV detector set to 254 nm is used to measure the eluent s absorbance. The ability to adjust retention times by changing the mobile phase s pH is also explored. 

This experiment focuses on developing an HPLG separation capable of distinguishing acetylsalicylic acid, paracetamol, salicylamide, caffeine, and phenacetin. A Gjg column and UV detection are used to obtain chromatograms. Solvent parameters used to optimize the separation include the pH of the buffered aqueous mobile phase, the %v/v methanol added to the aqueous mobile phase, and the use of tetrabutylammonium phosphate as an ion-pairing reagent. 

This experiment describes the quantitative analysis of the asthma medication Quadrinal for the active ingredients theophylline, salicylic acid, phenobarbital, ephedrine HGl, and potassium iodide. Separations are carried out using a Gi8 column with a mobile phase of 19% v/v acetonitrile, 80% v/v water, and 1% acetic acid. A small amount of triethylamine (0.03% v/v) is included to ensure the elution of ephedrine HGl. A UV detector set to 254 nm is used to record the chromatogram. 

The use of an amperometric detector is emphasized in this experiment. Hydrodynamic voltammetry (see Chapter 11) is first performed to identify a potential for the oxidation of 4-aminophenol without an appreciable background current due to the oxidation of the mobile phase. The separation is then carried out using a Cjg column and a mobile phase of 50% v/v pH 5, 20 mM acetate buffer with 0.02 M MgCl2, and 50% v/v methanol. The analysis is easily extended to a mixture of 4-aminophenol, ascorbic acid, and catechol, and to the use of a UV detector. 

In this experiment phosphate is determined by singlecolumn, or nonsuppressed, ion chromatography using an anionic column and a conductivity detector. The mobile phase is a mixture of n-butanol, acetonitrile, and water (containing sodium gluconate, boric acid, and sodium tetraborate). 

Students determine the concentrations of caffeine, acetaminophen, acetylsalicylic acid, and salicylic acid in several analgesic preparations using both CZE (70 mM borate buffer solution, UV detection at 210 nm) and HPLC (C18 column with 3% v/v acetic acid mixed with methanol as a mobile phase, UV detection at 254 nm). 

Otto and Wegscheider report the following capacity factors for the reverse phase separation of 2-aminobenzoic acid on a C18 column when using 10% v/v methanol as a mobile phase. ... 

In this experiment a theoretical model is used to optimize the HPLC separation of substituted benzoic acids by adjusting the pH of the mobile phase. An empirical model is then used... 

Fig. 12. Tryptic map of it-PA (mol wt = 66,000) showing peptides formed from hydrolysis of reduced, alkylated rt-PA. Separation by reversed-phase octadecyl (C g) column using aqueous acetonitrile with an added acidic agent to the mobile phase. Arrows show the difference between A, normal, and B, mutant rt-PA where the glutamic acid residue, D, has replaced the normal arginine residue, C, at position 275.

Fig. 2. Tic densitometer scans showing the resolution of isoproterenol on a hpflc siUca-gel plate obtained using a mobile phase consisting of 6.8 mM (1 R)-(—)-ammonium-10-camphorsu1fonic acid in 75 25 (v/v) methylene chioride methano1. (a) 254 nm, (b) 275 m, (c) 300 nm.

The dependence of chiral recognition on the formation of the diastereomeric complex imposes constraints on the proximity of the metal binding sites, usually either an hydroxy or an amine a to a carboxyHc acid, in the analyte. Principal advantages of this technique include the abiHty to assign configuration in the absence of standards, enantioresolve non aromatic analytes, use aqueous mobile phases, acquire a stationary phase with the opposite enantioselectivity, and predict the likelihood of successful chiral resolution for a given analyte based on a weU-understood chiral recognition mechanism. 

Mobile phases used with this stationary phase are typically 0.01 N perchloric acid with small amounts of methanol or acetonitrile. One significant advantage of these phases is that both configurations of the chiral stationary phase are commercially available and can be obtained from J. T. Baker Inc. and Chiral Technologies, Inc. (Crownpak CR). 

Achiral Columns Together with Chiral Mobile Phases. Ligand-exchange chromatography for chiral separation has been introduced (59), and has been appHed to the resolution of several a-amino acids. Prior derivatization is sometimes necessary. Preparative resolutions are possible, but the method is sensitive to small variations in the mobile phase and sometimes gives poor reproducibiUty. 

An hplc assay was developed suitable for the analysis of enantiomers of ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory dmg (NSAID), in plasma and urine (59). Following the addition of racemic fenprofen as internal standard (IS), plasma containing the KT enantiomers and IS was extracted by Hquid-Hquid extraction at an acidic pH. After evaporation of the organic layer, the dmg and IS were reconstituted in the mobile phase and injected onto the hplc column. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (chiral AD) with hexane—isopropyl alcohol—trifluoroacetic acid (80 19.9 0.1) as the mobile phase pumped at 1.0 mL/min. The enantiomers of KT were quantified by uv detection with the wavelength set at 254 nm. The assay allows direct quantitation of KT enantiomers in clinical studies in human plasma and urine after adrninistration of therapeutic doses. 

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