Mobile phase in RP-HPLC

Figure 15-1. Dependence of retention volnme on pH of an aqueous mobile phase in RP-HPLC for drug product A on a 150 x 3.0-nun column using a 0.7-mL/min flow rate.

M.F. Borgerding and W.L. Hinze, Characterization and Evaluation of the Use of Nonionic Surfactant Micellar Mobile Phases in RP-HPLC, Anal Chem., 57 2183 (1985). 

Figure 1 compares the principles of RP-HPLC and HILIC RP-HPLC employs hydrophobic stationary phases such as alkyl or phenyl derivatized silica, and retention is achieved by interactions of the analyte with the hydrophobic column material and partitioning between the mobile and the stationary phases. Typically, the mobile phase in RP-HPLC contains a low concentration of the organic modifier in the aqueous buffer at the beginning,... 

HPLC methods can be ntilized for the pre-concentration of aromatic amines from polluted waters on silica gel or octadecyl silica (ODS) colnmns [55], The determination is then performed by RP HPLC using ODS packings as the stationary phases and a mixture of methanol, isopropanol, and water as the mobile phase [55], RP HPLC with diode array detector (DAD) methods coupled on-line with a continnons seqnential anaerobic/aerobic reactor system have been employed in wastewaters treatments [56], A continnons monitoring of the possible presence of aromatic amines in azo dyes wastes is based on indncing in the waste, the reaction of a reduction of the dye, followed by HPLC/ UV or HPLC/MS analysis [57-59], The redncing agent solutions are sodium dithionite or tin(II) chloride in an aqneons acidic medinm at 70°C, followed by SPE [58,59], LLE [60,61], or SEE [60-62],... 

As opposed to normal-phase HPLC, reversed-phase chromatography employs mainly dispersive forces (hydrophobic or van der Waals interactions). The polarities of mobile and stationary phases are reversed, such that the surface of the stationary phase in RP HPLC is hydrophobic and mobile phase is polar, where mainly water-based solutions are employed. 

The mobile phases in RP-TLC and HPLC are mixtures of water or buffer with organic modifiers. With an octanol-coated stationary phase, no organic modifier is added to the mobile phase, whereas in lAM chromatography, the organic modifier is necessary only in the case of highly retained solutes. 

Sigma Chemical Co. Standard protein solutions were prepared in water. Standard solutions were made for each protein of 10 mg dissolved in 1 L with water. The water was filtered with HA 0.5 xm membranes (Division of Millipore, Waters Co., Milford, Massachusetts, U.S.A.) and deionized prior to use. The extra pure grade solvent of ACN was purchased from Duksan Pure Chemicals Company (Incheon, Korea). TEA was purchased from Sigma Chemical Co. The sample for injection was filtered with 0.45 p.m polyvinylidene fluoride (PVDF) (Waters Co.). Sodium chloride and hydrochloric acid were purchased from Duksan Pure Chemicals Company. Piperazine was purchased from Sigma Chemical Co. For the anion exchange membrane, buffer A was 20 mm piper-azine-HCl, pH 6.4, and buffer B was made by addition of 1 M NaCl to buffer A. The mobile phases for RP-HPLC were as follows buffer A, 0.1% TEA in water and buffer B, 0.1% TEA in ACN. 

Reversed-phase high performance liquid chromatography (RP-HPLC)—an HPLC technique where the mobile phase is more polar than the solid phase. In RP-HPLC compounds are separated based on their hydrophobic character. 

In RP-HPLC the stationary phase is less polar than the mobile phase and the interaction between analyte and the stationary phase has a predominantly hydrophobic (apolar) character. The most commonly used stationary phase in RP-HPLC is silica gel in which octadecyl silica chains are covalently bound to the free hydroxyl groups, indicated as a C18 phase. The typical surface of such a phase is shown in Fig. 8. 

Fraction volumes are on the order of 200 mL to 1 L or more. A low 0.1% level impurity, with poor sample loadability and a complicated sample matrix can aggravate the situation with regards to project turnaround times and total solvent costs. Impurity fraction solution stability issues exacerbate the situation even more. For example, substituted benzylic alcohols can dehydrate under acidic HPLC conditions, or carboxylic esters can hydrolyze in aqueous mobile phase. A RP-HPLC isolation can yield solvent costs on the order of 50-200 with a turnaround time of approximately 1 week to yield the quality sample necessary for NMR analysis. 

As a method of research, has been used high-performance liquid chromatography in reversed - phase regime (RP HPLC). The advantage of the present method is the following the additional information about AIST and FAS composition (homologous distribution) simple preparation of samples (dilution of a CS sample of in a mobile phase). 

Early [1, 2] it was reported about RP-HPLC the separation of amino derivatives of 3-chloro-l,4-naphtoquinone with methanol mobile phase. In some cases changing organic modificator in eluent leads to the progress in effectiveness of sepai ation. In present work the compaiison was performed for separation of some amino derivatives of 3-chloro-I,4-naphtoquinone by RP-HPLC with methanol and acetonitrile eluent. It has been shown that certain differences exist for vaiious derivatives mentioned above. 

Global LSER calculations have also been applied to the study of the retention of ioniz-able analyses in RP-HPLC. While the retention of neutral analyses does not depend on the pH of the mobile phase the retention of analyses with one or more ionizable substructures considerably depends on the pH even at the same concentration of organic modifier in the eluent. The relationship between the retention and pH of the mobile phase and pK value of the analyte can be described by... 

The retention of analyses in RP-HPLC markedly depends on the adsorption of the organic constituent of the mobile phase on the surface of the stationary phase. The excess adsorption isotherms of ACN, THF and methanol were measured on silica support modified with C, C6, C8, C10, C12 and C18 monomeric phase and a model was developed for the description of the retention of solutes from the binary mobile phase. The dependence of the retention factor on the partition coefficient can be described by... 

Grumbach et al. [100] recommended the use of acetonitrile with bare silica columns, with concentration not greater than 95% or less than 70%. At least 5% of the mobile phase should be water to allow for the formation of the aqueous layer and to allow solubility of buffer, if one is used. In some cases, methanol can be used to form the polar layer. It was noted that while bare silica can be used at pH < 1 (no bonded ligands to hydrolyze, as in RP-HPLC) it is more susceptible to dissolution at intermediate pH (presumably since it not protected by a C18 layer), and should not be used above pH 6. Buffers such as ammonium acetate at pH 5 and ammonium formate at pH 3 were recommended at 5-20 mM concentrations. They reported the elution strength of various solvents using silica and HlLlC conditions as... 

Petrick and Wilson [137] recently developed an HPLC method using both UV-Vis and ESI-MS detection able to separate and detect 15 basic dyes and 13 disperse dyes. Separation was carried out in RP-mode using an acidified (formic acid) water-acetonitrile mobile phase in gradient mode. The method enabled the discrimination of fibers with the same apparent colour based on their different chromatographic and mass spectrometric profile. 

P.H.J. Schoeiunakers, A Systematic Approach to Mobile Phase Effects in RP-HPLC, thesis. Delft 1981. 

Although the silica-based columns are the most widely used in RP-HPLC separations of peptides, the use of polymeric carriers (polystyrene divinylbenzene) and composite materials (silica particles with a polymeric coating), which are more chemically stable in that they do not break down at pH values higher than 8 as silica does, is gaining currency (54,55). The mobile phase usually consists of a mixture of water and an organic solvent, generally acetonitrile, methanol, or... 

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