Reversed phase media

Restricted access phases are another approach to exploiting the differences in characteristics of analytes. Large analytes are excluded from an internal surface on which an adsorptive stationary phase is present. A herbicide analysis for Metsulfuron methyl, Bentazone, Bromoxynil, methylchlorophenoxy acid, and Mecoprop in the presence of humic acid was performed on restricted access reversed phase media.52 The cytostatic compound epirubicin and its metabolites were separated from plasma using a Pinkerton GFF II column.53 Gradient separations of polymers on reversed phase and on normal phase represent an alternative to gel permeation chromatography. Polyesters of noncrystalline materials were separated on a variety of such phases.54... 

Counter-ions which are frequently used include tetrabutylammonium phosphate for the separation of anions and hexane sulphonic acid for cations. The appropriate counter-ions are incorporated in the solvent, usually at a concentration of about 5 mmol 1" and the separation performed on the usual reverse phase media. This ability to separate ionic species as well as non-polar molecules considerably enhances the value of reverse-phase chromatography. 

Jarmalaviciene, R. Komysova, O. Westerlund, D. Maruska, A. Nonparticulate (continuous bed or monolithic) restricted-access reversed-phase media for sample clean-up and... 

One of the earliest reports of SCILs in LC concerned the analysis of ephed-rines [54] and tropane alkaloids [55]. In these studies, the N,N-dialkylimid-azolium motif was covalently attached through a 3-mercaptopropylsilane linker. Retention of the analytes seemed to be governed predominantly through reversed-phase interactions. In comparison with conventional reversed-phase media, the IL-based stationary phase provided better peak shape and resolution while simultaneously allowing for reduction in the organic content of the mobile phase. 

As stated above, the utility of silica based stationary phases does not limit its use to organic mobile phases. For many years it has been commonplace in flash chromatography to use aqueous solvents to elute analytes from silica based media. Isocratic elution with mixtures of butanol, acetic acid and water is standard protocol for the separation of amino acids and a carefully prepared combination of methanol, chloroform and water is useful for general organic compounds. Peptides are also readily purified by gradient elution on normal phase silica, moving from acetonitrile to aqueous mobile phase 3,2l This technique is particularly useful for extremely hydrophilic peptides that are not strongly retained on reversed phase media. 

The eluents most commonly used in peptide purification by reversed phase HPLC are water and acetonitrile. These are often buffered with trifluoroacetic acid (0.1% v/v, TFA), ammonium acetate (0.05-0.1 mol/dm3 at pH 4-8) or phosphate (0.05-0.1 mol/dm3 sodium or potassium salt at pH 2-8). In addition, polymeric reversed phase media also performs well at high pH and is often buffered with ammonium hydroxide or ammonium bicarbonate (0.05-0.1 mol/dm3 at pH 8-9). 

FIGURE 1.3 Photomicrograph of a tapered fused-silica tip with an integral fit and packed with 5-p.m-diameter CIS reverse-phase media. The ID of the tip is 15 pm and the ID of the packed chromatography bed is 75 pm. (Photograph courtesy of New Objective, Inc.)... 

Figure 2.23 Ion-pairing agents used in reversed phase chromatography. Ion-pairing agents illustrated in pink enhance the interaction of charged groups with the hydrophobic surface. They also suppress the ionization of residual silanols on silica-based reversed-phase media.

Desilets CP, Rounds MA, Regnier FE, Semipermeable-surface reversed-phase media for high-performance liquid chromatography, 7. Chromatogr. A 1991 544 25-39. 

At the concentrations required for the analysis, many sample matrices (mineral oil, edible oil, fat extracted from foods) are not soluble in reversed-phase media. 

The development of reversed-phase chromatographic media has intensified during the last decade. Now, many manufacturers pride themselves on their high degree of the batch to batch reproducibility, both with respect to the retention properties of the stationary phase and also in regards to the quality of the packed bed. Recent years have seen the development of a variety of polar embedded and polar endcapped reveresed-phase materials. There is in reality no limitation placed on the type of reversed-phase material that could be used for reversed-phase applications, other than commercial availability. Therefore expansion of the available reversed-phase media that enter the market place continues, as chromatographers continually seek new ways to gain selectivity. 

Ion-exchange and reversed-phase media are most commonly used as stationary phases in the LC analysis of water-soluble vitamins. Aminopropyl-bonded silica columns (e.g., LiChrosorb NH2, /tBondapak NH2) have been used as weak anion exchangers for... 

Alternative dual-column analyses of the cytosine and uracil arabinosides (using reversed-phase then cation-exchange media), and of riboxamide (on silica then reversed-phase media both coated with hexadecyltrimethylammoniiim bromide), have been developed to enable the desired separation of these components from other constituents in biological samples. 

A particular problem with use of silica-supported reversed-phase media for trace analysis of surfactants is the presence of free Si-OH groups. These bind surfactants tightly, making complete recovery difficult, particularly for cationics (55). Phenyl-modified silica is excellent for SPE of NPE, but recoveries are low for AE, particularly more highly ethoxylated AE (55). 

The most common application of critical conditions in reversed-phase HPLC is the determination of PEG in ethoxylates. This is easily carried out with most reversed-phase media, using 95 5 methanol/water and refractive index or evaporative light scattering detection (90,91). PEG elutes prior to the surfactants. PPG can be determined in propoxy-lates in the same manner (92). 

Determination of alkyl chain distribution has been demonstrated by chromatography on dodecanol-saturated paper, with partial resolution by position of sulfonation (16). Presumably, this separation may also be performed with modem Ci reversed-phase media. 

The hydrotropes benzene sulfonate and p-toluene sulfonate are well resolved from anionic surfactants by TLC on dodecanol-impregnated silica (7) or polyamide (12) with an ammo-niacal developing solution. Presumably, modern reversed-phase media will give superior performance. Xylene and cumene sulfonates give orange fluorescence with pinacryptol yellow, while p-toluene sulfonate gives a red color (14). 

NPE, E = 9 isolation of homologs up to E = 22 Reversed-phase media Woelm alumina (acid grade), 1.0 mm 2-Butanone Modified Dragendorff reagent 106... 

FIG. 1 TLC analysis of an alkylpolyglycoside mixture on normal phase and reversed-phase media. (Reprinted with permission from Ref. 115.)... 

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