Analyte retention determination

Molecular sieves (zeolites) are artificially prepared aluminosilicates of alXali metals. The most common types for gas chromatography are molecular sieve 5A, a calcium aluminosilicate with an effective pore diameter of 0.5 nm, and molecular sieve 13X, a sodium aluminosilicate with an effective pore diameter of 1 nm. The molecular sieves have a tunnel-liXe pore structure with the pore size being dependent on the geometrical structure of the zeolite and the size of the cation. The pores are essentially microporous as the cross-sectional diameter of the channels is of similar dimensions to those of small molecules. This also contrilsutes to the enormous surface area of these materials. Two features primarily govern retention on molecular sieves. The size of the analyte idiich determines whether it can enter the porous... 

Commercially available GC-MS systems present major differences in their detection and recording system. Many quadrupole instruments use SIM for the determination of analytes at trace levels. With this type of instrumentation, more than 1-10 ng of the analyte is required to record a full-scan mass spectrum. In contrast, instruments based on ion-trap technology can record a full-scan mass spectrum on an analyte at pg level. With SIM, a limited number of ions are monitored during a selected time interval of the chromatogram. The presence of the analyte is determined by the presence of these diagnostic ions at the correct retention time and in the correct abundance ratio (33). 

Samples are injected onto the turbulent-flow column similar to single-column methods. The analytes of interest are retained in the turbulent-flow column while the large macromolecules are eluted to waste. Once the analytes are separated from the matrix, the samples are then eluted into the analytical column. The characteristics of the analytical column determine the peak shape and separation seen at the MS detector. Flow rates which are compatible with the mass spectrometer can then be used and the chromatograms are based on conventional HPLC parameters. The key to dual-column methods is that the retentive properties of the analytical column must be sufficiently stronger than that of the turbulent-flow column the dual-column approach is performed in such a manner so that the mobile-phase composition needed to elute the analyte from the turbulent-flow column does not elute the analyte from the analytical column. The sample is then focused at the head of the analytical column until the mobile-phase conditions are changed to elute the analyte. The choice of columns is critical to the success of dual-column methods. Table 10.2 lists some of the applications of dual-column methods found in the literature. 

As in analytical liquid chromatography (LC), analyte retention depends on sample concentration, solvent strength, and sorbent characteristics. An empirical approach to methods development initially involves screening the available sorbents. The first step is to determine which sorbents best retain the analyte. The second consideration is to evaluate the solvents needed to elute the compound and the compatibility of those sorbents to the chromatographic testing procedure. The third step is to test the blank sample matrix to evaluate the presence of possible interferents. Finally, recoveries of known quantities of analyte added to the sample matrix must be determined. 

The model was recently tested to determine whether it was able to model analyte retention in the presence of novel and unusual IPRs (see Chapter 7) such as chaotro-pic salts and ionic liquids. Chaotropes that break the water structure around them and lipophilic ions (classical IPRs and also ionic liquids) that produce cages around their alkyl chains, thereby disturbing the ordinary water structure, are both inclined to hydrophobic ion-pairing since both are scarcely hydrated. This explains the success of the theory, that is predictive in its own right, when neoteric IPRs are used [64]. Recently a stoichiometric model (vide supra) was put forward to describe retention of analytes in the presence of chaotropic IPRs in eluents [18] but its description of the system is not adequate [64]. 

It should also be noted that nitrogen is not an unbiased probe adsorbate. Obviously the surface accessibility for irregular materials depends on the size of the probe molecule a large probe cannot foUow the irregularity of the surface. Analyte molecules are usually larger than nitrogen molecules, and may not be able to penetrate aU pores. Thus only a fraction of the surface area is involved in analyte retention. To fulfill lUPAC recommendations for surface characterization methods [9], the most suitable method depends on the specific application. Recently an approach that involves IPR ion adsorption proved effective. The best probe to determine the packing area... 

The nature of the counter ion is important because it establishes the exact value of the potential difference between the stationary phase and the bulk eluent, and also, if it exhibits suitable absorption characteristics, indirect photometric detection of UV-inactive analytes is feasible. Moreover, the choice of the counter ion of the potential determining ion allows a tailor-made separation of the analytes since adsorbophilic counter ions may compete with the analyte for interaction with the potential determining ion, thereby decreasing analyte retention. Different counter ions may alter the elution sequences of a series of analytes with potential advantage for resolution and identification purposes [143]. 

Ion chromatography (1C) allows the separation of substances in the form of ions, chiefly in aqueous solutions. Mobile phases used in the technique contain relatively large amounts of salts stabilizing the pH and determining the sequence of analyte retention. They enable the separation of only cations or only anions the ions that are not separated by a selected phase (cationic or anionic) are eluted in the column dead volume. As the next step, they can be loaded into appropriate ion-exchange columns in the second chromatographic dimension [148]. 

HPLC theory could be subdivided in two distinct aspects kinetic and thermodynamic. Kinetic aspect of chromatographic zone migration is responsible for the band broadening, and the thermodynamic aspect is responsible for the analyte retention in the column. From the analytical point of view, kinetic factors determine the width of chromatographic peak whereas the thermodynamic factors determine peak position on the chromatogram. Both aspects are equally important, and successful separation could be achieved either by optimization of band broadening (efficiency) or by variation of the peak positions on the chromatogram (selectivity). From the practical point of view, separation efficiency in HPLC is more related to instrument optimization, column... 

To derive the relationship of the analyte retention with the thermodynamic properties of chromatographic system, the mechanism of the analyte behavior in the column should be determined. The mechanism and the theoretical description of the analyte retention in HPLC has been the subject of many publications, and different research groups are still in disagreement on what is the most reahstic retention mechanism and what is the best theory to describe the analyte retention and if possible predict its behavior [8,9]. 

Base material provides mechanically stable rigid porous particles (mostly spherical) for reversed-phase HPLC adsorbents. Particle porosity on the mesoporous level (30 to 500-A diameter) is necessary to provide high specific surface area for the analyte retention. Surface of the base material should have specific chemical reactivity for further modification with selected ligands to form the reversed-phase bonded layer. Base material determines the mechanical and chemical stability—the most important parameters of future (modified) reversed-phase adsorbent. 

Knowledge of pKa for the target analyte and related impurities is particularly useful for commencement of method development of HPLC methods for key raw materials, reaction monitoring, and active pharmaceutical ingredients. This practice leads to faster method development, rugged methods, and an accurate description of the analyte retention as a function of pH at varying organic compositions. Relationship of the analyte retention as function of mobile-phase pH (spH) is very useful to determine the pK of the particular... 

When developing separation methods for analytes with known pKa values, determination of the starting mobile-phase pH is highly advisable. This estimation may help to avoid strange analyte retention behavior during further method optimization and variation of the mobile-phase composition. Below we include several examples where the methodology of the combined pH and pKa shift evaluation is outhned. 

The main process determining analyte retention is the hydrophobic interactions with the stationary phase and its competition with organic mobile-phase additive. This simplistic description allows for a rough estimation of the analyte retention and, in principle, is applicable only for very ideal systems, like the separation of alkylbenzenes in methanol/water mixtures. 

In step 3, for this study the upper pH for the mobile phase to be prepared was determined to be JpH 6.7 (at least two units greater than the highest spA a of the molecule). The lower pH for the mobile phase (containing 30v/v% MeCN) that should be prepared for this study should be 1.7, but this would mean that an aqueous mobile-phase wpH of 1.1 would have to be prepared to obtain a spH of 1.7 (see Chapter 4, Section 4.5 for pH shift). Remember that the pH shift of the mobile phase for a phosphate buffer is approximately 0.2 pH units in the upward direction for every 10v/v% acetonitrile. In this case, not to compromise the stability of the packing material (column chosen has recommended a lower pH limit of wpH 1.5), a pH of pH 1.6 was chosen to be prepared which correlates to a spH of 2.2 ( pH 1.6 -i- 0.6 units upward pH shift upon addition of 30v/v% acetonitrile). Most definitely the final method will not be set at this low pH, since the analyte would exist in multiple ionization states however, the experiment was performed at this low pH to elucidate the effect of the pH on the analyte retention in this low-pH region. 

HPLC is another convenient method for measurement of the NCE pKa values. As was shown by Melander and Horvath [13], the retention of any ionizable analyte closely resembles the curve shown in Figure 12-3. Chromatographic determination of the pKa could be accurately performed with very limited amount of sample. Fast HPLC method with optimum analyte retention is suitable for this purpose, but the influence of the organic mobile-phase modifier on the mobile phase pH and analyte Ka should be accounted for in order to provide the accurate calculation of the respective Ka value. Detailed discussion of the HPLC-based methods for the Ka determination is given in Chapter 4. 

Alternatively, the flow system can be exploited for in-line sample conditioning prior to chromatographic separation, as demonstrated by the determination of benzoic and sorbic acids in food products [243]. The flow system comprised an electro-osmotic pump, five solenoid valves and a homemade SPE unit, combined with capillary zone electrophoresis. Tetrabutylammonium bromide was used as an ion pair reagent to improve analyte retention on a Cs-bonded silica sorbent. 

This equation is one of the fundamental equations of analytical chromatography and offers the possibility to determine linear adsorption coefficients from analytical retention times. 

The distribution coefficient, fCo, is difficult to determine from the chromatogram. Thus, other chromatographic parameters are defined to characterize the analyte retention directly from the chromatogram. 

HPLC data may be influenced by the presence of background matrix components if we consider the presence of compounds in the matrix that have a high retention time then after several runs in which the analyte is determined a major baseline drift (= background) may occur, hindering further quantitation caused by the slowly eluting component. Applying a gradient as wash procedure to clear the column may substantially increase the analysis time. It may be better to develop another method for this analysis. 

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