Mobile phase buffer selection

Omura et al.21 used a reverse phase high performance liquTcT cEromatographic column, JASCO PACK SV-02-500, for macrolide antibiotics with methanol, M/15 acetate buffer pH 4.9, and acetonitrile (35 60 5) as solvent. A variable wavelength UV detector using the absorption of the individual compounds gave the required sensitivity. Alterations of buffer pH and the composition ratio of the mobile phase gave selectivity for separation of individual macrolide antibiotics. 

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. 

Mobile phases for SEC fall into two broad categories aqueous buffers for GFC and organic solvents for GPC. In SEC, the mobile phase is selected not to control selectivity but for its ability to dissolve the sample. In addition, the mobile phase should have a low viscosity and be compatible with the detector and column packing. For example, polar solvents such as methanol... 

Examples of optimizations in HPLC using the simplex approach can be found in [28,84]. In [28] the mobile phase composition for the chiral separation of (6/ )- and (65)-leucovorin on a BSA (bovine serum albumin) stationary phase is optimized by means of a variable-size simplex. Three factors were examined, the pH of the mobile phase buffer, the ionic strength of the buffer and the percentage of 1-propanol in the mobile phase. Table 6.19 shows the experimental origin, the initial step size and the acceptable limits for the factors. The criterion optimized is the valley-to-peak ratio (Section 6.2). The points selected and the results are pre.sented in Table 6.20 and... 

These workers reported data for the separation of a mixture of synthetic pitt>tides (avenge 1100) on an 8-nm-pore C, column with acetoni-trile/water mobile phases and various buffers (73). A mobile-phase buffer that minimized band broadening was selected (this also minimized nonideal... 

The capability of HSCCC was also studied for enrichment and determination of metal ions at trace levels. Separation of selected divalent metal ions was performed using a small coiled column. A hexane solution of EHPA was employed as the stationary phase. Loaded divalent metal ions such as Ni, Co, Cu, and Zn were chromatographicaUy eluted in the order of increasing extractability by passing a mobile phase buffered at a desired pH. Each metal ion showed good linearity between concentrations and chromatographic peak areas of absorbance, as detected by postcolumn reaction with 4-(2-pyridylazo)resorcinol (PAR). Metal ions enriched in the stationary phase from a sample solution were separated into individual metal ions. A trace quantity of Zn in natural mineral water was determined by enrichment separation through an HSCCC column. 

The other component of mobile phase is aqueous buffer. The buffer pH may be critical to some separation. pKa values of compounds of interest should be considered when selecting mobile phase pH. The buffer pH of a mobile phase should be outside pKa 2 to ensure method robustness. Table 1.3 lists the commonly used mobile phase buffers. The most commonly used mobile phase buffer is a trifluo-roacetic acid (TEA) aqueous solution. One issue with TEA is its low sensitivity at wavelength 210 nm. 

Different mobile phase organic solvents have different selectivity for neutral, acidic, and basic compounds. Therefore, different mobile phase organic solvents are tested as modifiers during method development. In addition, mobile phase buffer pH affects selectivity of acidic and basic compounds. Therefore, different buffer pHs are investigated during method development for acidic and basic compounds. 

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... 

For most free amino acids and small peptides, a mixture of alcohol with water is a typical mobile phase composition in the reversed-phase mode for glycopeptide CSPs. For some bifunctional amino acids and most other compounds, however, aqueous buffer is usually necessary to enhance resolution. The types of buffers dictate the retention, efficiency and - to a lesser effect - selectivity of analytes. Tri-ethylammonium acetate and ammonium nitrate are the most effective buffer systems, while sodium citrate is also effective for the separation of profens on vancomycin CSP, and ammonium acetate is the most appropriate for LC/MS applications. 

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. 

Factors may be classified as quantitative when they take particular values, e.g. concentration or temperature, or qualitative when their presence or absence is of interest. As mentioned previously, for an LC-MS experiment the factors could include the composition of the mobile phase employed, its pH and flow rate [3], the nature and concentration of any mobile-phase additive, e.g. buffer or ion-pair reagent, the make-up of the solution in which the sample is injected [4], the ionization technique, spray voltage for electrospray, nebulizer temperature for APCI, nebulizing gas pressure, mass spectrometer source temperature, cone voltage in the mass spectrometer source, and the nature and pressure of gas in the collision cell if MS-MS is employed. For quantification, the assessment of results is likely to be on the basis of the selectivity and sensitivity of the analysis, i.e. the chromatographic separation and the maximum production of molecular species or product ions if MS-MS is employed. 

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 ... 

Buffer Salt Solvent Strength and Selectivity Solvent strength and selectivity are influenced by the nature of the counterion i.e., its value. A change in buffer salt may also change the mobile phase pH. 

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