Reversed phase RPC

A variety of micropellicular packing materials has been developed for the analysis of both small and large molecules by various HPLC modes, including ion exchange (lEC), metal interaction (MIC), reversed phase (RPC) [4], and affinity chromatography (AC) [5]. Besides analytical applications, other possible utilization of micropellicular stationary phases includes fundamental kinetic and thermodynamic studies of the retention mechanisms on a well-defined surface. Nevertheless, a relatively limited variety of micropellicular... 

By Chromatographic Modes Normal-phase (NPC), reversed-phase (RPC), ion-exchange (IEC), size-exclusion (SEC)... 

Reversed phase (RPC) and hydrophobic interaction chromatography (HIC) are based on very similar types of interactions they have been regarded as different... 

Reversed phase (RPC) Particles of well-defined size, coated witii hydrophobic ligands C4—Cig Water, buffers of low molarity, d oig c solvents fa elution Presence of salts problematic above 10 mM Indicated for neutral and uncharged molecules, soluble in aqueous/org c mixtures Excellent fa analytical HPLC, seldom preparative (proteins denatured by o g c solvents)... 

Reversed-phase chromatography rehes on significantly stronger-hydrophobic interactions than in HIC, which can result in unfolding and exposure of the interior hydrophobic residues, i.e., leads to protein denaturation and irreversible inactivation as such, RPC depends... 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

BPC = bonded-phase chromatography RPC = reverse-phase chromatography... 

The popularity of reversed-phase liquid chromatography (RPC) is easily explained by its unmatched simplicity, versatility and scope [15,22,50,52,71,149,288-290]. Neutral and ionic solutes can be separated simultaneously and the rapid equilibration of the stationary phase with changes in mobile phase composition allows gradient elution techniques to be used routinely. Secondary chemical equilibria, such as ion suppression, ion-pair formation, metal complexatlon, and micelle formation are easily exploited in RPC to optimize separation selectivity and to augment changes availaple from varying the mobile phase solvent composition. Retention in RPC, at least in the accepted ideal sense, occurs by non-specific hydrophobic interactions of the solute with the... 

Figure 4.27 Flow chart for coluwi selection based on sample type (m - molecular weight). PLC precipitation-liquid chromatography SEC = size-exclusion chromatography lEC - ion-exchange chromatography HIC hydrophobic interaction chromatography LSC liquid-solid chromatography RPC - reversed-phase liquid chromatography BPC (polar) bonded-phase chromatography and IPC - ion-pair chromatography.

The TLC process is an off-line process. A number of samples are chromatographed simultaneously, side-by-side. HPTLC is fast (5 min), allows simultaneous separation and can be carried out with the same carrier materials as HPLC. Silica gel and chemically bonded silica gel sorbents are used predominantly in HPTLC other stationary phases are cellulose-based [393]. Separation mechanisms are either NPC (normal-phase chromatography), RPC (reversed-phase chromatography) or IEC (ion-exchange chromatography). RPC on hydrophobic layers is not as widely used in TLC as it is in column chromatography. The resolution capabilities of TLC using silica gel absorbent as compared to C S reversed-phase absorbent have been compared for 18 commercially available plasticisers, and 52 amine and 36 phenolic AOs [394]. 

In reversed-phase chromatography (RPC), the mobile phase modulator is typically a water-miscible organic solvent, and the stationary phase is a hydrophobic adsorbent. In this case, the logarithm of solute retention factor is commonly found to be linearly related to the volume fraction of the organic solvent. 

Hydrophobic interaction chromatography (HIC) occupies a unique niche in the field of analytical chromatography. A particular advantage of HIC is its unique selectivity. Whereas ion-exchange chromatography (IEC) principally reveals differences based on the surface charge of native proteins, HIC reveals differences based principally on their surface hydrophobicity. HIC is complementary to reversed-phase chromatography (RPC) in a different sense. Whereas HIC discriminates primarily on the basis of surface hydrophobicity, RPC principally reveals differences based on total hydrophobicity of all the hydrophobic residues of denatured proteins. 

Reversed-phase chromatography is the predominant technique in HPLC, and chemically bonded silica gel supports are made specifically for the nonpolar stationary phase. In the last decade, as many as 60% of the published LLPC techniques refer to RPC. The reasons for this involve the significantly lower cost of the mobile liquid phase and a favorable elution order that is easily predictable based on the hydrophobicity of the eluate. 

Determination of various analgesic and antipyretic pharmaceuticals on reversed phase has included not only the analysis of serum levels of aspirin, salicylic acid and salicyluric acid using acidified acetonitrile (557), or methanol (338), but also suUinpyrazone under isocratic conditions (339), and 6-chloro-a-methylcarbazole-2-acetic acid (340). The polar thiol metabolites of acetaminophen were analyzed by RPC and the method was found to be superior to other chromatographic techniques used in this analysis (341). 

If the size of the literature is a reliable indicator, the analysis of compo-uents fotmd In nvironmfntnl samples has not been developed t the same extent as clinical applications of re versed-phase chromatography. More attention has been paid to the analysis of volatile species by gas phase chromatography. This is due in part to the difficulty in identifying large molecular weight complex molecules which are present in water at trace levels. However, determination of a variety of analytes in water, soil, or other matrices has been reported and the wider use of RPC in the evaluation of water quality especially can be expected. The apolar phases used in RPC may be a boon in the determination of dilute analytes. Frei (4M) has discussed how relatively unpolar compounds dissolved in water can be concentrated at the top of a reversed-phase column and then eluted as a narrow band with an appropriate solvent. This technique can be used for the analysis of environmental samples in which the analyte of interest is in exceedingly low concentration. 

Water-soluble vitamins in formulations have been determined by use of ion-pair chromatography. The vitamins include several B vitamins as well as niacin, folic acid, and ascorbic acid (565). Vitamins D and Da were rapidly separated on reverse phase columns (247) as are vitamins A, D, and E in multivitamin tablets (564). Addition of silver ions to the mobile phase has been shown to increase the flexibility inherent in RPC by complexing with the unsaturated bonds and thereby decreasing the retention factor. This effect is also observed with other unsaturated drug molecules including steroids (247). Vitamin A and related compounds have... 

Many other analytical methods which are not readily included in the above categories use RPC and have been reported in the literature. For instance, various metal complexes can be conveniently analyzed by this technique. Transition mettds have been separated on reverse phase as... 

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