Mobile-phase composition

ED imposes certain restrictions on the type of chromatography that is employed and the mobile phase that is used. The detecting system requires a conducting mobile phase and thus must contain water. Therefore, the majority of normal phase systems are not usable. Reversed-phase high performance liquid chromatography (RP-HPLC), however, is ideally suited to ED. Nevertheless, certain precautions must be taken for its effective use. 

1 Purity Solvents and salt buffers developed for HPLC analysis are normally 

Taking into account this advice, it is necessary to observe some tips in the election of the 

The presence of antioxidants in eluents and extraction solvents Antioxidants can be readily oxidized electrochemically and generate high background currents or interfering broad peaks. Thus, eluents and extraction solvents containing such compounds should be either avoided or purified before use. For example, ethers, such as diethyl ether, diisopropyl ether, and tetrahydrofuran are likely to contain up to 0.1% (w/v) pyrogallol or quinol (hydroquinone) as stabilizer. If the stabilizer is removed, peroxides will form and their concentration will increase with time unless the solvent is stored under nitrogen. Not only do peroxides present a hazard from explosion, but they may also oxidize susceptible analytes. Methyl f-butyl ether (MTBE), on the other hand, is stable to oxidation. 

Presence of metals ED is sometimes particularly susceptible to increases in background noise caused by metal ions present in the eluent, the samples, or introduced from components of the HPLC system. Some analysts add EDTA to their eluents, typically at concentrations of 30-150 mg 1 (0.1 -0.5 mmol 1 ), with the aim of chelating iron and possibly other metal ions. EDTA may also enhance the stability of analytes, such as catecholamines, during sample storage and analysis. 

The previously discussed considerations cover very fundamental aspects of an LC/MS method setup they are discussed typically once, at the beginning of your work, as they deal with generic questions such as which instrument should I use or how should my LC/MS method look like. Once decided, these things do not change significantly across the lifetime of a method. But there are many minor 

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Organic Solvent/H20 Mobile-Phase Compositions Having Approximately Equal Solvent Strength... 

A mixture of methyl paraben, ethyl paraben, propyl paraben, diethyl phthalate, and butyl paraben is separated by HPLC. This experiment emphasizes the development of a mobile-phase composition capable of separating the mixture. A photodiode array detector demonstrates the coelution of the two compounds. 

Two variations of the technique exists isocratic elution, when the mobile phase composition is kept constant, and gradient elution, when the mobile phase composition is varied during the separation. Isocratic elution is often the method of choice for analysis and in process apphcations when the retention characteristics of the solutes to be separated are similar and not dramaticallv sensitive to vei y small changes in operating conditions. Isocratic elution is also generally practical for systems where the equilibrium isotherm is linear or nearly hnear. In all cases, isocratic elution results in a dilution of the separated produces. 

The stationary phase is selected to provide the maximum selectivity. Where possible, the retention factor is adjusted (by varying the mobile phase composition, temperature, or pressure) to an optimum value that generally falls between 2 and 10. Resolution is adversely affected when k 2, while product dilution and separation time... 

The development of micellar liquid chromatography and accumulation of numerous experimental data have given rise to the theory of chromatographic retention and optimization methods of mobile phase composition. This task has had some problems because the presence of micelles in mobile phase and its modification by organic solvent provides a great variety of solutes interactions. 

Usually goodness of fit is provided by adding new parameters in the model, but it decreases the prediction capability of the retention model and influences on the optimization results of mobile phase composition. 

A computer program was compiled to work out the ray-tracing of UV detector of high performance capillary electrophoresis at the investigation of 5 and 6 (98MI59). The capacity factor of 5 at different temperature and at different mobile phase compositions was experimentally determined in bonded-phase chromatography with ion suppression (98MI15). 

Figure 8.6 The basic possibilities of "D in one-dimensional TLC the shading illusti ates variation in the mobile phase composition.

MD method Abbreviation Development distance Mobile phase composition... 

Figure 8.12 The universal elution gradient during the stages of AMD separation (a) change in mobile phase composition (b) change of solvent strength (c) change of selectivity value.

The enantioselectivity of the macrocyclic CSPs are different in each of the operating modes, probably because of different separation mechanisms functioning in the different solvent modes. The possible chiral recognition mechanisms for three mobile phase compositions on glycopeptide phases are listed in Table 2-3 in descending order of strength. 

Fig. 2-2. The enantiomeric sepai ation of P-blockers on teicoplanin CSP (250 x 4.6 mm) with the same mobile phase composition methanol with 0.1 % acetic acid and 0.1 % triethylamine (v/v). The flow rate was 1.0 mL min at ambient temperature (23 C).

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. 

This an excellent example of the value of the diode array detector. If the chromatogram shown in figure 3 was monitored at two different wavelengths, then a peak ratio curve would immediately disclose the presence of the second peak (see page 175) and it would no longer be necessary to resort to changes in mobile phase composition to establish the presence of the impurity. 

It is seen that the profile of the combined peaks is perfectly symmetrical and displays no hint that there are two solutes present. Obviously an absorption ratio curve from a diode array detector would quickly disclose the presence of the two components, as would an appropriate changes in mobile phase composition. However, there would be a further clue for the analyst to follow that would give warning of the "duplicity" of the peak. The double peak would be very broad and be inconsistent with the change in peak width of the other solute peaks with retention time. The peak width of a solute increases regularly with retention time but, unfortunately, the relationship is not smooth. There are good reasons for this, but they... 

The effect of the mobile-phase composition on the operation of the different interfaces is an important consideration which will be discussed in the appropriate chapter of this book but mobile-phase parameters which affect the operation of the interface include its boiling point, surface tension and conductivity. The importance of degassing solvents to prevent the formation of bubbles within the LC-MS interface must be stressed. 

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