Separation of ionizable compounds

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. 

IPC is a valuable addition to the toolbox of analytical chemistry for dealing with multi-faceted separations of ionizable compounds. The aim of this chapter is to perform a comparative evaluation of IPC and possible competitive analytical strategies (see Figure 13.1). No significant statistical differences were usually found among results obtained from IPC in comparison to other techniques. 

The separation of ionizable compounds by liquid-solid adsorption chromatography has long presented difficulties and the separation of un-... 

There were selectivity differences as a function of pH from 2.1 to 7.1 (pH of the aqueous phase), with concomitant changes in retention. This demonstrates that separations of ionizable compounds in RPLC should be performed where the molecules are in one predominate ionization state (s) to avoid sig-nihcant changes in retention and selectivity with minor changes in pH. 

We have discussed so far the general approach to method development for the separation of ionizable compounds, emphasizing modification of the mobile phase. Despite the fact that this approach was designed for conventional reversed-phase HPLC columns and a standard HPLC set-up, there are some important requirements for columns, analyte solutions, and reagents. 

Generally, a systematic search for the two-phase solvent systems for CCC is focused on the hydrophobicity of the solvent system for providing a proper range of partition coefficients of solutes. For the separation of ionizable compounds such as alkaloids, however, an additional adjustment is required with respect to the pH and ionic strength of the solvent system. 

The pH of the mobile phase is a major factor in the separation of ionizable compounds. As mentioned earlier, the most widely used model considers the retention factor as an average of and k according to the mole fraction of the neutral and ionic forms. The mole fraction depends on p Ta and pH of mobile phase. The pH of the mobile phase is taken to be the same as that of the aqueous fraction and this implies a false assumption. Even when pH is measured after mixing the buffer with the organic modifier, the potentiometric system, calibrated with aqueous standards, does not measure the true pH of the mobile phase. 

As we have seen, the pH value plays a major role with regard to the separation of ionizable compounds. This means that the pH is the most important factor in method development, and one should explore its effect at the beginning of the method development effort. We have devised a procedure that does exactly that it demonstrates relatively early on which combination of pH and mobile phase composition is most promising for a further fine-tuning of the method [10]. The foundations of this principle had already been developed earlier [11]. 

Under these drcumstances, it is absolutely necessary to have excellent control over the pH value. The consequence of this is that true buffers must be used in order to achieve and control the desired pH value. If this is not done, problems with the reproducibility of retention times are almost inevitable, and often problematic peak-shape phenomena such as fronting or tailing occur as well. In addition to the use of a true buffer, it is also essential to measure and control the pH with high precision to obtain reproducible separation patterns. On the one hand, a change in the pH value is the best tool to manipulate and optimize the separation of ionizable compounds. On the other hand, careful pH control is mandatory. 

An inductively coupled plasma formed by passing argon through a quartz torch is widely used for the mass spectroscopic analysis of metal compounds separated by online HPLC.6 Samples are nebulized on introduction into the interface. Plasma impact evaporates solvent, and atomizes and ionizes the analyte. Applications include separation of organoarsenic compounds on ion-pairing F4PLC and vanadium species on cation exchange. 

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 ... 

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

High-performance liquid chromatography (HPLC) techniques are widely used for separation of phenolic compounds. Both reverse- and normal-phase HPLC methods have been used to separate and quantify PAs but have enjoyed only limited success. In reverse-phase HPLC, PAs smaller than trimers are well separated, while higher oligomers and polymers are co-eluted as a broad unresolved peak [8,13,37]. For our reverse-phase analyses, HPLC separation was achieved using a reverse phase. Cl8, 5 (Jtm 4.6 X 250 mm column (J. T. Baker, http //www.mallbaker.com/). Samples were eluted with a water/acetonitrile gradient, 95 5 to 30 70 in 65 min, at a flow rate of 0.8 mL/min. The water was adjusted with acetic acid to a final concentration of 0.1%. All mass spectra were acquired using a Bruker Esquire LC-MS equipped with an electrospray ionization source in the positive mode. 

EKC is not restricted to the separation of neutral analytes, as it is widely employed for the simultaneous separations of charged and neutral analytes as well as of ionizable compounds having similar electrophoretic mobility. The separation of ionizable analytes by EKC is governed by differences in the partitioning between the pseudostationary phase and the surrounding electrolyte solution as well as electrophoretic mobility. For these analytes, the retention factor can be described by the following mathematical model ... 

In the last years, ILs have been applied as matrices for matrix-assisted laser desorption/ionization (MALDI) MS [42], thus expanding the use of MALDI. In Ref. 38 the suitability of alkylammonium- and alkylimidazolium salts of a-cyano-4-hydroxycinnamic acid was investigated as a MALDI matrix and at the same time as the additive of BGE. The alkylammonium salt produced better separation of phenolic compounds than the alkylimidazolium salt. The investigation suggests that it is possible to synthesize ILs suitable for electrophoretic analysis as well as for online MALDI-MS analysis. 

Helium is the most common carrier gas and is compatible with most detectors. For a flame ionization detector, N2 gives a lower detection limit than He. Figure 24-11 shows that H2, He, and N2 give essentially the same optimal plate height (0.3 mm) at significantly different flow rates. Optimal flow rate increases in the order N2 < He < H2. Fastest separations can be achieved with H, as carrier gas, and H2 can be run much faster than its optimal velocity with little penalty in resolution.11 Figure 24-12 shows the effect of carrier gas on the separation of two compounds on the same column with the same temperature program. 

A well-defined method development plan with clear aim of analysis is critical to the success for fast and effective method development. The general approach for the method development for the separation of pharmaceutical compounds was discussed, emphasizing that modifications in the mobile phase (organic and pH) play a dramatic role on the separation selectivity. The knowledge of the Ka of the primary compound is of utmost importance prior to the commencement of HPLC method development. Moreover, pH screening experiments can help to discern the ionizable nature of the other impurities (i.e., synthetic by-products, metabolites, degradation products, etc.) in the mixture. 

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