Buffers in the mobile phase

APCl is more tolerant to the presence of buffers in the mobile phase stream than is ESI. 

Fig. 4.3.2. Influence of the use of different buffers in the mobile phase on the formation of adduct formation in LC-ESI-MS of APEO mixtures.

In true thermospray, charging of the droplets is due to the presence of a buffer in the mobile phase. Both positively and negatively charged droplets are formed due to the statistical fluctuation in anion and cation density occurring when the liquid stream is disrupted. As with the interfaces previously described, involatile buffers are not recommended as blocking of the capillary is more likely to occur if temperature control is not carefully monitored and for this reason ammonium acetate is often used. 

Despite the distinct advantages of pneumatic nebulizers, ultrasonic nebulizers may alternatively be used, in some instances, with success. In a recent application, a variation of ultrasonic nebulizer called spray nozzle-rotating disk FTIR interface was successfully applied to confirm the presence of methyltestosterone, testosterone, fluoxymesterone, epitestosterone, and estradiol and testosterone cyp-ionate in urine, after solid-phase extraction and reversed-phase LC separation (151). Using a commercial infrared microscopy spectrometer, usable spectra from 5 ng steroid deposits could be readily obtained. To achieve success with this interface, phosphate buffers in the mobile phase were not used because these nonvolatile salts accumulate on the collection disk and their spectra tend to swamp out small mass deposits. Another limitation of the method was that only nonvolatile analytes could be analyzed because volatile compounds simply evaporated off the collection-disk surface prior to scanning. 

The CLND is limited, of course, to mobile phases that do not contain nitrogen. Acetonitrile and amine modifiers, commonly used in HPLC, are therefore precluded. In addition, the CLND is not readily amenable to non-volatile buffers in the mobile phase. However, it is still possible to determine RRF values for samples run under these non-CLND-compatible HPLC conditions. In such cases, a two-step process is used. First, a CLND-compatible mobile phase (e.g., methanol/water/trifluoroacetic acid) is used to separate the compounds of interest and determine RRF values under those conditions (RRF ). Separately, the UV peak areas obtained using both the CLND-compatible and non-compatible HPLC conditions are compared by analyzing a common sample by both sets of HPLC conditions (apart from the CLND). The peaks of interest must, of course, be tracked to avoid misassignment (e.g., through UV spectra comparison). The relative response factor (RRF ) obtained for the CLND-compatible method can then be used to determine the relative response factor (RRF2)... 

The aqueous mobile phases used in RPLC allow the use of buffers in the mobile phase. This may lead to improved selectivity and efficiency. Secundary (ionic) equilibria other than acid-base dissociation may also be used (see section 3.3.2). 

In order to optimize the LC/MS/MS system, the authors investigated the effects of methanol content, ammonium acetate concentration, and the percentage of acetic/formic acids in the mobile phase on the ESI response (m/z 744 to 495 transition) (Figure 7-19). They found that the best ESI response was obtained at -80% (v/v) of methanol (Figure 7-20A).They also discovered that the ammonium acetate (5 mM) could be applied as a buffer in the mobile phase to achieve better reproducible separation between ET-743 and the internal standard. It is well known that the addition of acetic acid or formic acid in the mobile phase can suppresse the ionization of residual silanols on silica-based reversed-phase columns for LC/MS analysis (Figure 7-19C). In positive ion mode, however, the acids can form an ion pair with the MH+... 

The introduction of phases that have mixed mode character has allowed the use of low-pH buffers in the mobile phase without any loss in EOF. Theses phases are a combination of reversed phase and sulphonic acid chemistries. Fig. 4.6 shows an EOF vs. pH plot on a capillary packed with a Cfi/SCX mixed mode phase (Hypersil. Runcorn). This provides a good EOF over the whole pH range and was also found to have a more predictable retention mechanism based on revensed-phase retention characteristics. 

Sometimes other variables must be investigated such as the pH and/or the ionic strength of the buffer in the mobile phase or the concentration of additives in the mobile phase such as for instance tensio-active substances in micellar chromatography. In such a case the first step in an optimization is to screen these factors and to identify the most important ones for the subsequent optimization. The screening (Section 6.4.2) leads to a definition of the experimental domain in which the optimum is probably situated. This is somewhat similar to the retention optimization step. It is followed by an optimization step (Sections 6.4 and 6.7), in which the most important variables are changed, often according to an experimental design. Similar methods are used in capillary zone electrophoresis. 

The compatibility of an organic solvent with a buffer in the mobile phase must be checked as the buffer salts can be easily precipitated when using on-line mixing, thus causing frit blockage, check valve malfunction, and other problems. 

Nevertheless, Figure 5 demonstrates that the enantiomeric separation using a phosphate buffer in the mobile phase can be coupled via the PSS approach on-line to an LC/MS moving belt interface. Other examples of the PSS approach or similar procedures with other compounds and other LC/MS interfaces have been described (2, 9-14). Besides the actual phase-system switching, which enables the choice of the most favorable solvent for a particular interface, the PSS approach offers some other features as well. The desorption flow-rate used can be adjusted to the capabilities of the LC/MS interface applied. While in the present example with the moving belt a flow-rate of 0.4 ml/min of methanol was used, desorption has also been demonstrated with 1.2 ml/min for a thermospray interfaced),... 

The use of buffers in the mobile phase is required for any samples containing acidic or basic analytes (see Chapter 2). Table 2.3 summarizes the common buffers with their respective pKa, UV cutoffs, and compatibility with mass spectrometer (MS). 

Figure 8. Chromatograms obtained using a 15-cm Zorbax C-18 column with solvent programming and UV detection which show (A) a blank profile run (B) a chromatogram of a mixture of standards containing (1) 40 ng aniline (2) 70 ng 2-amino 5-chlorophenol (3) 40 ng p-chloroaniline (4) 75 ng p-bromoaniline (5) 60 ng m-chloroaniline (6) 90 ng 2-chloroaniline (7) 180 ng 3,4-dichloroaniline (C) the gradient profde showing the percent buffer in the mobile phase (15)...

Chromatography Separations have usually been carried out using reversed phase HPLC with mixtures of THF or acetonitrile and water. Some authors have included acid or used acidic buffers in the mobile phase to maximize resolution. 

Several variables should be considered in the development of an MLC procedure the nature of surfactmit and modifier, their concentrations, and pH. When a surfactant solution is used as mobile phase, the retention of solutes can be adequately controlled through the addition of a small amount of alcohol, and through variation of pH. The alcohol usually also improves the efficiency of the chromatographic peaks. Other variables that affect the retention and efficiency are temperature and ionic strength. However, most procedures are performed at room temperature, and the ionic strengfti is given by the combination of the surfactant and buffer in the mobile phase, and is not studied as a separate variable. 

Of the sorbents available on prepared layers, alumina is one of the most unique. It can be made into different forms so that it not only has the basic character, but also neutral and acidic versions. This allows a great versatility for selectivity where the acid, neutral, or basic character need not be controlled with buffers in the mobile phase, certainly a disadvantage if doing TLC-MS work lest a buffer interfere in some manner. It also is a complex sorbent with hydroxyl groups, partial positive and negative surface charges, onto which water is also attracted. Only TLC precoated... 

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