Mobile phase buffers

The mobile phase supply system consists of a series of reservoirs normally having a capacity ranging from 200 ml to 1,000 ml. Two reservoirs are the minimum required and are usually constructed of glass and fitted with an exit port open to air. Stainless steel is an alternative material for reservoir construction but is not considered satisfactory for mobile phases buffered to a low pH and containing... 

All myosin derived proteins were dialized against mobile phase buffer consisting of 50 mM Na3P04 (pH 7.4), 0.2 M (NH4)2S04,... 

The citrate would be strongly retained by the stationary phase, so that the retention of the two acids would decrease. However, if mobile phase buffers are formed using polyvalent salts, there is a strong probability of complex formation, which will alter the predicted behaviour of the system. 

Ionisation suppression caused by co-elution of other analytes or natural matrix components or due to the presence of ion pairing (IP) agents or high salt concentrations in the mobile-phase buffer used [2-4],... 

Fig. 5. Effect of the flow rate on the separation efficiency. Separation of a protein mixture at six different flow rates (40,80,120,160,200 and 240 ml/min) normalized to the elution volume. Conditions Column 80 ml CIM DEAE Tube Monolithic Column Mobile phase buffer A 20 mM Tris-HCl buffer, pH 7.4 buffer B 20 mM Tris-HCl buffer + 1 M NaCl, pH 7.4 Gradient 0-100% buffer B in 200 ml Sample 2 mg/ml of myoglobin (peak 1), 6 mg/ml of conalbumin (peak 2) and 8 mg/ml of soybean trypsin inhibitor (peak 3) dissolved in buffer A Injection volume 1 ml Detection UV at 280 nm. (Reprinted with permission from Podgornik A, Barut M, Strancar A, Josic D, Koloini T (2000) Anal Chem 72 5693)...

Fig. 7. Semi-Preparative Anion Exchange Purification of a 16-mer Oligodeoxynucleotide on a CIM DEAE Disk Monolithic Column. Conditions Column 0.34 ml CIM DEAE Disk (3X12 mm ID) Instrumentation Gradient HPLC system with extra low dead volume mixing chamber Sample 16mer oligodeoxynucleotide from the reaction mixture - bold line, standards of 1,2,3,4,5,6,7,9,10,11,12,14,15,16mer- thin line Injection Volume 20 pL Mobile Phase Buffer A 20 mM Tris-HCl, pH 8.5 Buffer B Buffer A+ 1 M NaCl Gradient as shown in the Figure Flow Rate 4 ml/min Detection UV at 260 nm...

The series of regioisomeric amines 48-50, methamphetamine (29) and phentermine (31), can be identified in forensic screening analyses by RP-HPLC-UVD (254/280 nm dual accessory) using a Cis stationary phase and a mobile phase buffered at pH 3.0. The capacity factors and retention times increase in the order 48 < 49 < 29 < 31 < 50. Other methods for identifying these compounds failed for example, the base peak in MS is m/z = 58 for all five compounds, corresponding to a loss of a benzyl group from the molecular peak also their IR and UVV spectra are too similar to be useful for this... 

When chromatographic resolution of species based on modifications located at the protein surface is desired, it may be advisable to use conditions that favor retention of native conformation.17 Here, the standard acidic conditions described in the preceding text may be inappropriate, and mobile phases buffered near neutrality may be required. Buffers based on ammonium acetate, ammonium bicarbonate, and triethylammonium phosphate may prove more useful in resolving polypeptide variants with differing posttranslational modifications, amino acid substitutions, or oxidation and deamidation products. The addition of more hydro-phobic ion-pairing agents may be needed to obtain polypeptide retention, and a variety of alkyl sulfonates and alkyl amines have been described for specific applications.17... 

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. 

Homogenization with HPO3, centrifugation, filtration, dilution in mobile phase buffer Homogenization with 1% HPO3 with 0.5% oxalic acid centrifugation filtration... 

Three major difficulties have been generally met in directly combining LC with MS. The first concerns the ionization of nonvolatile and/or thermolabile analytes. The second is related to the mobile-phase incompatibility as result of the frequent use of nonvolatile mobile-phase buffers and additives in LC. The third is due to the apparent flow rate incompatibility as expressed in the need to introduce a mobile phase eluting from the column at a flow rate of 1 ml/min into the high vacuum of the MS. 

D. Sykora, E. Tesarova, and D. W. Armstrong, Practical Considerations of the Influence of Organic Modifiers on the Ionization of Analytes and Buffers in Reversed-Phase LC, LCGC 2002,20, 974 G. W. Tindall, Mobile-Phase Buffers. I. The Interpretation of pH in Partially Aqueous Mobile Phases, LCGC 2002,20, 102 S. Espinosa, E. Bosch, and M. Roses, Acid-Base Constants of Neutral Bases in Acetonitrile-Water Mixtures, Anal. Chim. Acta 2002,454, 157. 

Reverse-phase chromatography has been used extensively for the determination of saccharin. Smyly et al. (30) and Eng et al. (39) used /rBondapak Cl 8 and 5% acetic acid for the determination of saccharin. Based on this work, an Association of Official Analytical Chemists (AOAC) collaborative study was conducted, and the method using a mobile phase buffered to pH 3 with sodium acetate and modified with 3% isopropanol was adopted. Webb and Beckman (61) used this method successfully for the separation of saccharin from aspartame, caffeine, sodium benzoate, and artificial colors and flavors. Veerabhadrarao et al. (27) added methanol to the mobile phase (methanol acetic acid water, 4 1 1, v/v) for improved separation of saccharin from caffeine, benzoic and p-hydroxybenzoic acids, vanillin, aspartame, acesulfame-K, and dulcin. Saccharin was also determined using LiChrosorb Cl8 and 4 6 v/v methanol phosphate buffer,... 

Using a Zipax SAX column, these compounds were eluted in a reproducible pattern by increasing the ionic strength of a mobile phase buffered with 0.1 M Na2B407 (214). 

The first approach uses a suppressor device which is located between the analytical column and the detector cell. This device chemically removes the mobile-phase buffer counterions, thus reducing the background conductivity. This type of detector increases postcolumn dead volume and puts... 

HPLC-k correlation, cyanopropyl column, mobile phase buffered to pH 3, Hodson Williams... 

The mobile phase used for the CZE separations of the trypsin digests was 0.1 M tricine and 0.02 M morpholine adjusted to pH 8.15. The mobile phase composition for the linearity and precision data was 0.01 M tricine, 0.0058 M morpholine, and 0.02 M NaCl adjusted to pH 8.0. The mobile phase buffers used to determine peak shape and electrophoretic mobility vs. pH all contained 0.02 M NaCl and 0.01 M of the buffer (pH 3.0, citrate pH 4.0, formic acid pH 5.0, acetate pH 6.0, MES pH 7.0, MOPS pH 8.0, tricine pH 9.0, borate and pH 10.0, glycine) and were adjusted to the indicated pH with acid or base. The column was rinsed with mobile phase between injections or successively with 0.1 M sodium hydrooxide and mobile phase when the mobile phase composition was changed. 

A simple method of modifying the surface of capillaries utilizes mobile phase (buffer) additives. The objective is to negate the predominantly electrostatic interactions of protein with the walls of capillary. This can be accomplished in three ways ... 

Since monoclonal antibodies recognize a particular epitope on the antigen, they tend to be less cross-reactive than polyclonal antibodies in Western blotting and ELISAs. Also, because the interaction takes place with a defined affinity (dissociation constant), monoclonal antibodies are frequently preferred in the technique of im-munoaffinity chromatography (see Introduction to Experiment 2). The wide variety of antigen-antibody interactions that are present in a polyclonal antibody preparation makes it difficult to define a mobile-phase buffer that will promote the elution of the antigen of interest in good yield from an im-munoaffinity column prepared with polyclonal antibodies. 

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