Zwitterionic analytes

The retention model by Cecchi and co-workers also quantitatively faced the prediction of the retention behavior of neutral and zwitterionic analytes in IPC. According to the electrostatic models, at odds with clear experimental data [1,50,52,53], the retention of a neutral solute is not dependent on the presence and concentration of a charged IPR in a chromatographic system. Equation 3.23 is very comprehensive if Ze is zero [50], it simplifies since ion-pairing does not occur (C2= C3 = 0). Adsorption competition models the retention patterns of neutral analytes in IPC and the slight retention decreases of neutral analytes with increasing HR concentration may be quantitatively explained [50,53]. 

Dinitrobenzoic acid proved capable of improving reversed phase retentions of chemical warfare agent derivatives [137] and [S-(R,R)]-(-)-bis(-a-methylbenzyl) amine hydrochloride was an effective IPR for zwitterionic analytes [138]. 

Figure 4-23. Effect of analyte pH on the retention dependencies of acidic, basic, and zwitterionic analytes.

Isoelectric focussing allows the separation of zwitterionic analytes such as proteins or peptides according to their isoelectric point, pi (section 1.1.1.1). lEF is applied to the separation and purification of proteins, peptides and amino acids on an analytical as well as preparative scale. The pi of a protein depends on the sum of all charges, as well as the 3D structure and post translational modiflcations such as phosphorylation, glycosilation and changes in oxidation state. The pi is, thus, a valuable parameter for studying post-translational modifications of proteins. 

Influence of IIR Concentration on the Retention Behavior of Charged, Neutral, and Zwitterionic Analytes... 

The simultaneous use of acidic and basic additives (triethylamine and acetic acid, for instance) is also possible. This practice allows for improving efficiency and resolution for zwitterionic analytes or for those that, even if neutral, establish strong polar interactions with the CSP. 

One of the few zwitterionic surfactant used in CL reactions is /V-dodccyl-MA-dimethyl-ammonium-3-propane-l-sulfonate (SB-12). Particularly, SB-12 has been assayed in the study of the CL reaction of lucigenin with various biological reductants [10]. The results show that SB-12 enhances CL intensity of the luci-genin-glucose and lucigenin-fructose systems by factors of 3.0 and 1.5, respectively, compared to the intensity obtained in aqueous medium. In these conditions detection limits were found for-both analytes of 0.7 X 10 4 and 2.5 X 10 5 M, respectively. 

Besides some rare experimental setups the mass analyzer of any mass spectrometer can only handle charged species, i.e., ions that have been created from atoms or molecules, more seldom from radicals, zwitterions or clusters. It is the task of the ion source to perform this crucial step and there is a wide range of ionization methods in use to achieve this goal for a wide variety of analytes. 

Since tetracyclines have several pKa values and can exist as zwitterions, one must always consider the control of the mobile-phase pH in the development of an analytical method. The property of tetracyclines to exist in the form of ions also makes the use of ion-pairing reagents attractive, and several methods have been developed that utilize this approach (289, 299, 303, 307). 

Capillary isoelectric focusing separates analytes based on differences in their isoelectric points (pi) using the same principles as in preparative solution IEF. After a focusing step, that builds up a linear pH gradient in the capillary (controlled with zwitterionic internal markers), the analytes move as a function of their respective charge until they reach a position of zero charge (isoelectric point). The solution is then mobilized in CIEF to the detector hydrodynamically. 

All solid amino acids exist as dipolar ions known as zwitterions (Figure 1.2(a)). In aqueous solution the structure of amino acids are dependent on the pH of the solution (Figure 1.2(b)). The pH at which an aqueous solution of an amino acid is electrically neutral is known as the isoelectric point (pi) of the amino acid (Table 1.1). Isoelectric point values vary with temperature. They are used in the design of electrophoretic and chromatographic analytical methods for amino acids. 

Capillary isoelectric focusing (cIEF) is used to separate zwitterionic compounds, such as peptides and proteins, on the basis of isoelectric point. The separation occurs as a result of a pH gradient that is established inside the capillary. It is important in cIEF to use coated capillaries to reduce or eliminate the EOF and to minimize solute adsorption if the EOF is too swift, it could sweep the analytes past the detector before the separation is complete. 

---