Stationary phase buffer selection

In the development of a SE-HPLC method the variables that may be manipulated and optimized are the column (matrix type, particle and pore size, and physical dimension), buffer system (type and ionic strength), pH, and solubility additives (e.g., organic solvents, detergents). Once a column and mobile phase system have been selected the system parameters of protein load (amount of material and volume) and flow rate should also be optimized. A beneficial approach to the development of a SE-HPLC method is to optimize the multiple variables by the use of statistical experimental design. Also, information about the physical and chemical properties such as pH or ionic strength, solubility, and especially conditions that promote aggregation can be applied to the development of a SE-HPLC assay. Typical problems encountered during the development of a SE-HPLC assay are protein insolubility and column stationary phase... 

The peak capacity is not pertinent as the separation was developed by a solvent program. The expected efficiency of the column when operated at the optimum velocity would be about 5,500 theoretical plates. This is not a particularly high efficiency and so the separation depended heavily on the phases selected and the gradient employed. The separation was achieved by a complex mixture of ionic and dispersive interactions between the solutes and the stationary phase and ionic, polar and dispersive forces between the solutes and the mobile phase. The initial solvent was a 1% acetic acid and 1 mM tetrabutyl ammonium phosphate buffered to a pH of 2.8. Initially the tetrabutyl ammonium salt would be adsorbed strongly on the reverse phase and thus acted as an adsorbed ion exchanger. During the program, acetonitrile was added to the solvent and initially this increased the dispersive interactions between the solute and the mobile phase. 

The selectivity (separation capability) of an HPLC system is dependent upon the combination of mobile and stationary phases. Since ions are being generated directly from the mobile phase by electrospray, its composition, including the identity and concentration of any buffer used, and its flow rate are important considerations. 

The selection of proper mobile phase in TLC exerts a decisive influence on the separation of inorganic ions. With a particular stationary phase, the possibility of separation of a complex mixture is greatly improved by the selection of an appropriate mobile phase system. In general, the mixed aqueous-organic solvent systems containing an acid, a base, or a buffer have been the most favored mobile phases for the separation of ionic species. The mobile phases used as developers in inorganic PLC include ... 

Table 7.89 lists the main characteristics of MDHPLC (see also Table 7.86). In MDHPLC the mobile-phase polarity can be adjusted in order to obtain adequate resolution, and a wide range of selectivity differences can be employed when using the various available separation modes [906]. Some LC modes have incompatible mobile phases, e.g. normal-phase and ion-exchange separations. Potential problems arise with liquid-phase immiscibility precipitation of buffer salts and incompatibilities between the mobile phase from one column and the stationary phase of another (e.g. swelling of some polymeric stationary-phase supports by changes in solvents or deactivation of silica by small amounts of water).

Mixtures of methanol or acetonitrile with either phosphate, TRIS or MES (Table 4.23) buffer solutions are the most frequently used, but many others are being investigated. The higher above 4 the buffer pH is, the greater the EOF generated and the faster the separation. The composition of the mobile phase can have dramatic effects on both the EOF and the selectivity of the separation via the sorption processes with the stationary phase. 

Membrane absorbers are continuous chromatographic supports, which circumvent some of the above-mentioned problems of particulate stationary phases. They were originally derived from membrane (filtration) technology. The immobilization of interactive (ionic, hydrophobic, or biospecific) groups on the surface of microfiltration membranes was found to increase the selectivity of certain separation procedure. Ideally such activated membranes, or membrane adsorbers, allow the selective adsorption of certain substances and substance classes, which may subsequently be eluted by means of a stepwise change of the mobile phase (elution buffer). More complete information on the various types of modern membrane technology can be found in some recent reviews [e.g., 31-33]. 

Cabon tetrachloride, n-hexane, chloroform, ACN, acetone, THF, pyridine, acetic acid, and their various mixtures were applied as mobile phases for adsorption TLC. Methanol, 1-propanol, ACN, acetone, THF, pyridine and dioxane served as organic modifiers for RP-TLC. Distilled water, buffers at various pH (solutions of and dipotassium hydrogen phosphate or potassium dihydrogen phosphate) and solutions of lithium chloride formed the aqueous phase. Carotenoids were extracted from a commercial paprika sample by acetone (lg paprika shaken with 3 ml of acetone for 30 min), the solution was spotted onto the plates. Development was carried out in a sandwich chamber in the dark and at ambient temperature. After development (15 cm for normal and 7cm for HPTLC plates) the plates were evaluated by a TLC scanner. The best separations were realized on impregnated diatomaceous earth stationary phases using water-acetone and water-THF-acetone mixtures as mobile phases. Some densitograms are shown in Fig.2.1. Calculations indicated that the selectivity of acetone and THF as organic modifiers in RP-TLC is different [14],... 

Reversed-phase chromatography employs a nonpolar stationary phase and a polar aqueous-organic mobile phase. The stationary phase may be a nonpolar ligand, such as an alkyl hydrocarbon, bonded to a support matrix such as microparticulate silica, or it may be a microparticulate polymeric resin such as cross-linked polystyrene-divinylbenzene. The mobile phase is typically a binary mixture of a weak solvent, such as water or an aqueous buffer, and a strong solvent such as acetonitrile or a short-chain alcohol. Retention is modulated by changing the relative proportion of the weak and strong solvents. Additives may be incorporated into the mobile phase to modulate chromatographic selectivity, to suppress undesirable interactions of the analyte with the matrix, or to promote analyte solubility or stability. 

Interactions between proteins and salts in the binding buffer are also a major determinant of selectivity. Salts that are strong retention promoters in HIC are excluded from protein surfaces by repulsion from their hydrophobic amide backbones and hydrophobic amino acid residues.8,9 This causes the mobile phase to exert an exclusionary pressure that favors the association of proteins with the column, regardless of stationary-phase hydrophobicity.1(W2 Because this mechanism involves the entire protein surface, the degree of exclusion is proportional to average protein hydrophobicity, regardless of the distribution of hydrophobic sites. 

In the stationary phase test discussed here, we use naphthalene and acenaphthene as our hydrophobic reference compounds, propranolol and amitriptyline as the compounds with basic functional groups, and dipropylphthalate and butylparaben for the determination of other polar selectivities of a packing. The mobile phase is a pH 7.00 phosphate buffer (35.0%) mixed with methanol (65.0%). The details of the test procedure are discussed in References 34, 38 and 39. 

Additional modes of HPTC include normal phase, where the stationary phase is relatively polar and the mobile phase is relatively nonpolar. Silica, diol, cyano, or amino bonded phases are typically used as the stationary phase and hexane (weak solvent) in combination with ethyl acetate, propanol, or butanol (strong solvent) as the mobile phase. The retention and separation of solutes are achieved through adsorp-tion/desorption. Normal phase systems usually show better selectivity for positional isomers and can provide orthogonal selectivity compared with classical RPLC. Hydrophilic interaction chromatography (HILIC), first reported by Alpert in 1990, is potentially another viable approach for developing separations that are orthogonal to RPLC. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal phase retention behavior. Typical stationary phases include silica, diol, or amino phases. Diluted acid or a buffer usually is needed in the mobile phase to control the pH and ensure the reproducibility of retention times. The use of HILIC is currently limited to the separation of very polar small molecules. Examples of applications... 

In summary, the use of RPLC is ideal for pharmaceutical analyses because of the broad range of commercially available stationary phases because the most common RPLC mobile phases (buffers with acetonitrile or methanol) have low UV cut-off wavelengths, which facilitate high sensitivity detection for quantitation of low-level impurities and because selectivity can readily be controlled via mobile phase optimization. Additionally, the samples generated for selectivity screening (as detailed above) are typically aqueous based. In subsequent phases of pharmaceutical development, aqueous-based sample solvents are ideal for sample preparation and are, under limited constraints, compatible with MS detection required to identify impurities and degradation products. 

In summary, it can be stated that the stationary phase and the mobile phase (buffer) pH are the most important factors determining the generic selectivity of a CS. The organic modifier composition and the column temperature can influence the selectivity locally, i.e., when separating a specific mixture of rather similar compounds, e.g., a drug impurity profile. 

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