PH of the mobile phase

In reverse-phase chromatography, which is the more commonly encountered form of HPLC, the stationary phase is nonpolar and the mobile phase is polar. The most common nonpolar stationary phases use an organochlorosilane for which the R group is an -octyl (Cg) or -octyldecyl (Cig) hydrocarbon chain. Most reverse-phase separations are carried out using a buffered aqueous solution as a polar mobile phase. Because the silica substrate is subject to hydrolysis in basic solutions, the pH of the mobile phase must be less than 7.5. 

In this experiment a theoretical model is used to optimize the HPLC separation of substituted benzoic acids by adjusting the pH of the mobile phase. An empirical model is then used... 

In the course of mixture separation, the composition and properties of both mobile phase (MP) and stationary phase (SP) are purposefully altered by means of introduction of some active components into the MP, which are absorbed by it and then sorbed by the SP (e.g. on a silica gel layer). This procedure enables a new principle of control over chromatographic process to be implemented, which enhances the selectivity of separation. As a possible way of controlling the chromatographic system s properties in TLC, the pH of the mobile phase and sorbent surface may be changed by means of partial air replacement by ammonia (a basic gaseous component) or carbon dioxide (an acidic one). 

The derivatives have an optimum fluorescence at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The adduct is relatively stable at a pH of 9-11 but it rapidly degrades to a non-fluorescent residue at low pH values. Consequently, when used as a pre-column derivatizing reagent the pH of the mobile phase should be kept fairly high, o-phthalaldehyde has been employed for derivatization in the analysis of dopamine (29), catecholamines (30) and histamines (31). 

In contrast to GC, in which, particularly at high temperatures, the stationary phase may give rise to a continuous background at the detector, this is not normally observed in HPLC unless the pH of the mobile phase is such that degradation of the stationary phase occurs. Under these circumstances, both an increased background and a reduction in chromatographic performance may be observed. 

Since the net charge on a protein is determined by the pH (see Chapter 3), sequential elution of proteins may be achieved by changing the pH of the mobile phase. Alternatively, a protein can be subjected to consecutive rounds of ion exchange chromatography, each at a different pH, such that proteins that co-elute at one pH elute at different salt concentrations at another pH. 

The CHI parameter approximates the percentage of organic modifier in the mobile phase for eluting the compounds and can be used for high-throughput determination of physicochemical properties (50-100 compounds per day). CHI is a system property index, and depends on the nature of the stationary phase and the organic modifier as well as the pH of the mobile phase for ionizable compounds. 

The HPLC elution pattern is affected to some extent by the pH of the mobile phase. Moderate pH adjustment to optimize the resolution between EMA and MEMA may be performed. Retention time can be affected greatly by the history of the HPLC column and also the buffer/methanol ratio. The mobile phase ratio should be adjusted to provide adequate separation and retention. Control and fortified samples should be run in the same analytical set with treated samples. 

For ionisable solutes, the pH of the mobile phase is an important factor in the control of retention and selectivity. 

If the pH of the mobile phase is changed, then we will alter the proportions of the ionised and unionised forms of each acid. Decreasing the pH will increase the amount of the unionised form of each acid and so reduce the retention. The stronger acid (with the smaller p.Ka) will be the more ionised and so will be retained longer by the stationary phase. 

Fig. 3.3e shows the separation of weak acids on a C-18 column with a mobile phase of methanol/water 50 50 + tetrabutylammonium phosphate. The pH of the mobile phase is about 7.5, as at this pH... 

Fig. 28. Effect of pH of the mobile phase on linear flow velocity (1) and electrical current (2) in the monolithic capillary column. (Reprinted with permission from [149]. Copyright 1998 American Chemical Society). Conditions monolithic capillary column 100 pm i. d. 30 cm, mobile phase 80 20 acetonitrile/5 mmol/1 phosphate buffer, pH adjusted by addition of concentrated NaOH, flow marker thiourea 2 mg/ml, UV detection at 215 nm, voltage 25 kV, pressure in vials 0.2 MPa, injection, 5 kV for 3 s...

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... 

As the pH of the mobile phase markedly influences the retention of ionizable compounds, it can be assumed that the separation capacity of RP-HPLC for ionizable analyses can be modified by changing the pH of the mobile phase. The theory of effect of pH gradient on the performance of RP-HPLC systems has been recently elaborated. The basic equation describing the dependence of the retention of the solute on the gradient of pH or organic modifier is ... 

The dependence of the retention factor on the pH of the mobile phase at constant concentration of organic modifier can be described for monoprotic acids as... 

When the pH of the mobile phase changes linearly in time the pH change can be... 

The pH dependence of the tailing of Dil was investigated in separate experiments. The experimental conditions were the same but the pH of the mobile phase was adjusted to different values by HC1. The effect of pH on the retention behaviour of the dye is illustrated by chromatograms in Fig. 3.113. The pH dependence of tailing was tentatively explained by the marked contribution of free silanol groups to the reversed-phase retention of the dye... 

In order to produce the ions necessary for analysis, the pH of the mobile phase often has to be modified. Volatile organic acids (formic acid, acetic acid, and trifluoroacetic acid) and volatile bases (ammonium hydroxide) are used to provide this modification. With the analysis of basic compounds, a lower pH mobile... 

The silanol induced peak tailing is also a function of the pH of the mobile phase. It is much less pronounced at acidic pH than at neutral pH. Therefore many of the older HPLC methods use acidified mobile phases. However, pH is an important and very valuable tool in methods development. The selectivity of a separation of ionizable compounds is best adjusted by a manipulation of the pH value. The retention factor of the non-ionized form of an analyte is often by a factor of 30 larger than the one of the ionized form, and it can be adjusted to any value in between by careful control of the mobile phase pH. This control must include a good buffering capacity of the buffer to avoid random fluctuations of retention times. 

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