Buffered mobile phases

O. V. Olesen and B. Poulsen, On-line fully automated deteimination of clozapine and desmethylclozapine in human semm by solid-phase extraction on exchangeable car-ti idges and liquid cliromatography using a methanol buffer mobile phase on unmodified silica , 7. Chromatogr. 622 39-46 (1993). 

Berzas Nevado, J.J. et al., A reverse phase HPLC method to determine six food dyes using buffered mobile phase, Anal. Lett., 31, 2513, 1998. 

The pKa of basic compounds as well as the buffered mobile phase were influenced by the organic solvent and depended on its proportion in the soluhon. The results indicated that the stationary phases and mobile phases studied were not suitably optimized for the estimation of lipophilicity of basic compounds. 

Modern pump designs also include a means for flushing the piston with solvent behind the pump seal (not shown in Figure 13.4). The solvent for this is drawn in from a separate reservoir and pumped back into this same reservoir. The purpose is to continuously rinse the piston free of mobile phase residue such that abrasive solute crystals resulting from a mobile phase that has dried out on the piston will not deposit there. These solutes, such as the salts dissolved in the buffered mobile phases used in ion exchange chromatography, may otherwise crystallize on the piston and then damage the piston or the pump seal when the piston moves back and forth. Mobile phases that contain such solutes must be flushed from the system after use so that there is also no crystallization on the front side of the seal. 

Buffered mobile phases are used and the proportions of polar solvents (e.g. methanol, tetrahydrofuran) depend upon the type of derivative employed. Gradient elution is required for the resolution of complex mixtures and analysis times are less than 1 h. Ultraviolet, fluorescence or electrochemical detectors are used depending upon the nature of the amino acid derivatives. 

Ponceau 4R, E-124 and Erythrosine, E-127) using a buffered mobile phase. Separation of dyes was performed in an ODS column (150 X 3.9 mm i.d. particle size 3 pm). Components of the mobile phase were methanol (eluent A) and 0.1 M NaH2P04/Na2HP04 buffer (pH = 7). The gradient elution started with 20 per cent A and reached 100 per cent in 2 min, final hold 4 min. The flow rate was 2 ml/min and dyes were detected at 520 nm. The baseline separation of dyes in 6 min is illustrated in Fig. 3.34. Commercial samples were diluted and injected into the analytical column without any pretreatment. The amounts of dyes found in the samples are compiled in Table 3.20. It was concluded from the good validation parameters that the technique is specific, sensitive, accurate and rapid. Consequently, its application for the determination of these synthetic dyes in drinks was proposed [112],... 

Do not use aqueous buffered mobile phase older than 10 days because of the increased risk of bacterial growth, which can damage the HPLC column. ... 

Never let buffered mobile phases sit in the HPLC system due to the danger of precipitation. Run at low flow rate of O.lmL/min when idling. 

Buffered mobile phases are inherently used to adjust and control the adsorption-desorption process. These CSPs are especially useful for the separation of very polar charged analytes, such as sulphonic acids. Chiral anion-exchangers are the most successful CSPs and among them the cinchona alkaloids, quinine and quinidine (Figure... 

FIGURE 10 Separation of a neutraceutical mixture in phosphate- and formate-buffered mobile phases (courtesy of Waters Corp.). 

The pH of a buffered mobile phase is significant not only because retention of ionizable compounds is dependent upon pH but because bonded phase columns are made via an acid catalyzed reaction. Manufacturers recommendations are to avoid any system with a pH lower than 2 or accept the risk of reversing the bonding reaction. However, the effective pH experienced by a column in a water organic mixture is difficult to predict. We have operated at a pH of 1.5 in this laboratory with no adverse effects to the column, but exercise the simple precaution of not allowing the acidic mobile phase to remain on the column overnight or static for long periods of time (1 hr). We have particularly noticed that the CN column is stable at low pH s. 

Analysis of aqueous solutions of the polar compounds (DCP, TCP, CA, and DCB) at concentrations of 1-10 ppm was easily accomplished by direct aqueous injection liquid chromatography. The Hamilton PRP-1 reverse-phase column gave a better resolution of these compounds than the conventional reverse-phase columns. Acetonitrile/water mixtures have been found to be as effective as the buffered mobile phases recommended by the manufacturer (28). Analyses of the nonpolar compounds (BHC and DEHP) at concentrations of 25-100 ppb were achieved by XAD resin adsorption-desorption, concentration, and GC techniques. 

Reverse-phase and ion-exchange columns have been used for the separation of acesulfame-K. Veerabhadrarao et al. (27) and Hannisdal (62) separated acesulfame-K from other sweeteners and additives on reverse-phase Cl 8 columns using methanol acetic acid and methanol-.phosphate buffer mobile phase, respectively. However, most of the reverse-phase methods for the separation of acesulfame-K use acetonitrile phosphate buffer as the mobile phase (14,16,33,44,51,63). According to Prodolliet and Bruelhart (33), the use of acetonitrile in the mobile phase provides a better resolution for sweeteners than methanol. 

High-performance LC was also used for determination of TBZ after its extraction from marmalades and curds with ethyl acetate (13). The use of a buffered mobile phase improved the response of the UV detector, and column performance remained constant throughout 2 months of daily use with a detection limit of 100 ppb. Three detectors (UV, fluorimetric, and electrochemical) were used for the determination of OPP, BP, and TBZ in plant materials (45). The compounds were extracted with dichloromethane and separated on an RP-18 column with a methanolic formic acid buffer as eluent. It was not possible to determine TBZ using an electrochemical detector, although the extraction recovery varied between 80 and 95%. 

Kim H, Kaczmarski K, Guiochon G (2006) Isotherm parameters and intraparticle mass transfer kinetics on molecularly imprinted polymers in acetonitrile/buffer mobile phases. Chem Eng Sci 61(16) 5249-5267... 

Examination of the structure of the example compound clearly indicates that it possesses both a phenyl and an indole moiety and should therefore be amenable to UV detection. The compound has a tertiary amine, which is an ionizable functional group with a pAa of approximately 9, therefore, a buffered mobile phase will likely be required if HPLC is chosen as the analytical method. Since the compound does not contain a chiral center, there is... 

Buffer—Mobile phase modifier used to control pH. Usually salts of weak acids or bases, most effective at their pKa, where concentrations of ionized and unionized form are equal. 

Phosphate buffer, Mobile phase, System suitability solution, and Chromatographic system Proceed as directed in the Assay. 

Propyl, trimethylamino Si-O-Si-C -ch2ch2ch2-n+(ch3)2ci- Ion exchange Strong anion exchange phase for aqueous and biological samples suitable for weaker anions such as carboxylic acids properties may be modified or conditioned by proper formulation of buffer mobile phases (see the appropriate table in the Solution Properties chapter)... 

---