Additives, mobile phase

The most important applications of chiral mobile phase additives in liquid ehromatogra-phy are ehiral ion-pair chromatography (section 4.3.3) and inclusion complex formation with cyclodextrins and similar chiral selectors. Other ehiral mobile phase additives have been used only oeeasionally, and with modest sueeess [ 1,3,32,153]. Multifunctional ion-pair reagents, such as 10-camphorsulfonic acid [154], derivatives of tartatie acid (e.g. di-n-butyltartrate) [155,156], peptides (e.g. N-carbobenzoxyglyeine-L-proline) [157-161], 

Chiral mobile phase additives provide a more versatile and cost-effective approach for enantiomer separations in thin-layer chromatography. Typically, chemically bonded layers with cyclodextrin and its derivatives, bovine serum albumin, or macrocyclic glycopeptides are used as chiral additives in the reversed-phase mode [59,60,172-178]. For [5- and y-cyclodextrins and their derivatives, a 0.1 to 0.5 M aqueous methanol or acetonitrile solution of the chiral selector is used as the mobile phase. Bovine serum albumin is generally used at concentrations of 1-8 % (w/v) in an aqueous acetate buffer of pH 5 to 7 or in a 0.5 M acetic acid solution, in either case with from 3-40 % (v/v) propan-2-ol (or another aliphatic alcohol), added to control retention. Enantioselectivity usually increases with an increase in concentration of the chiral selector, and may be non existent at low concentrations of the chiral selector. 

The low efficiency and short migration distances typical of thin-layer chromatography limit useful separations to those with relatively large enantioselectivity factors. Absorption by the chiral selector can cause baseline instability and reduced sample detectability for quantitative measurements by scanning densitometry. The chiral sepa- 

At equilibrium, the effective mobility of an enantiomer in the presence of a chiral selector in capillary electrophoresis is the sum of the electrophoretic mobility of each species containing the enantiomer, weighted by the mole fraction of each species. Assuming for a given separation the enantiomers exist as either the free enantiomer with a mobility, ixr, or the enantiomer-chiral selector complex, p,c,R, then the effective mobility for the enantiomer is given by 

Charged cyclodextrin chiral mobile phase additives for capillary electrophoresis 

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Two mechanisms for chiral separations using chiral mobile-phase additives, analogous to models developed for ion-pair chromatography, have been... 

An alternative model has been proposed in which the chiral mobile-phase additive is thought to modify the conventional, achiral stationary phase in situ thus, dynamically generating a chiral stationary phase. In this case, the enantioseparation is governed by the differences in the association between the enantiomers and the chiral selector in the stationary phase. 

High Performance Liquid Chromatography. Although chiral mobile phase additives have been used in high performance Hquid chromatography (hplc), the large amounts of solvent, thus chiral mobile phase additive, required to pre-equiUbrate the stationary phase renders this approach much less attractive than for dc and is not discussed here. 

Immobilization. The abiUty of cyclodextrins to form inclusion complexes selectively with a wide variety of guest molecules or ions is well known (1,2) (see INCLUSION COMPOUNDS). Cyclodextrins immobilized on appropriate supports are used in high performance Hquid chromatography (hplc) to separate optical isomers. Immobilization of cyclodextrin on a soHd support offers several advantages over use as a mobile-phase modifier. For example, as a mobile-phase additive, P-cyclodextrin has a relatively low solubiUty. The cost of y- or a-cyclodextrin is high. Furthermore, when employed in thin-layer chromatography (tic) and hplc, cyclodextrin mobile phases usually produce relatively poor efficiencies. 

The Effect of Mobile-Phase Additives and Cone-Voltage 147... 

Involatile inorganic buffers, when used as mobile-phase additives, are the prime canse of blocking of the pinhole. The situation can be alleviated either by replacing them by a more volatile alternative, such as ammonium acetate, or by nsing post-colnmn extraction to separate the analytes from the buffer, with the analytes, dissolved in an appropriate organic solvent, being introduced into the mass spectrometer. 

There are two notable features of the quantitative performance of this type of interface. It has been found that non-linear responses are often obtained at low analyte concentrations. This has been attributed to the formation of smaller particles than at higher concentrations and their more easy removal by the jet separator. Signal enhancement has been observed due to the presence of (a) coeluting compounds (including any isotopically labelled internal standard that may be used), and (b) mobile-phase additives such as ammonium acetate. It has been suggested that ion-molecule aggregates are formed and these cause larger particles to be produced in the desolvation chamber. Such particles are transferred to the mass spectrometer more efficiently. It was found, however, that the particle size distribution after addition of ammonium acetate, when enhancement was observed, was little different to that in the absence of ammonium acetate when no enhancement was observed. 

Factors may be classified as quantitative when they take particular values, e.g. concentration or temperature, or qualitative when their presence or absence is of interest. As mentioned previously, for an LC-MS experiment the factors could include the composition of the mobile phase employed, its pH and flow rate [3], the nature and concentration of any mobile-phase additive, e.g. buffer or ion-pair reagent, the make-up of the solution in which the sample is injected [4], the ionization technique, spray voltage for electrospray, nebulizer temperature for APCI, nebulizing gas pressure, mass spectrometer source temperature, cone voltage in the mass spectrometer source, and the nature and pressure of gas in the collision cell if MS-MS is employed. For quantification, the assessment of results is likely to be on the basis of the selectivity and sensitivity of the analysis, i.e. the chromatographic separation and the maximum production of molecular species or product ions if MS-MS is employed. 

Table 5.16 LC-MS-MS signal responses" obtained from wheat forage matrix samples using various mobile-phase additives (injection volumes of 50 p,l). From Choi, B. K., Hercules, D. M. and Gusev, A. I., LC-MS/MS signal suppression effects in the analysis of pesticides in complex environmental matrices , Fresenius J. Anal. Chem., 369, 370-377, Table 2, 2001. Springer-Verlag GmbH Co. KG. Reproduced with permission...

Cyclodextrins as chemically banded layers [102] or mobile phase additives [103-105] have been used successfully to resolve a wide variety of alkaloids, steroids and dansyl- and naphthylamide-amino acid derivatives. The low solubility in aqueous solution and f high cost of cyclodextrins restricted the use of these additives > initially. These limitations were overcome by the availability of ... 

There is a wide variety of commercially available chiral stationary phases and mobile phase additives.32 34 Preparative scale separations have been performed on the gram scale.32 Many stationary phases are based on chiral polymers such as cellulose or methacrylate, proteins such as human serum albumin or acid glycoprotein, Pirkle-type phases (often based on amino acids), or cyclodextrins. A typical application of a Pirkle phase column was the use of a N-(3,5-dinitrobenzyl)-a-amino phosphonate to synthesize several functionalized chiral stationary phases to separate enantiomers of... 

Ru(bpy)32+ to the mobile phase was investigated and compared with conventional postcolumn Ru(bpy)32+ addition. The detection limit using oxalate standards with Ru(bpy)32+ in the mobile phase was below 0.1 pM, which was significantly superior to the postcolumn technique. The mobile-phase addition method allowed the instrumentation to be simplified and reduced band broadening caused by postcolumn mixing. 

Therefore, the way to ensure reproducible adduct formation is to use mobile-phase additives (e.g. ammonium acetate or formate, formic, acetic or trifluoroacetic acid (in APCI), ammonium hydroxide, etc.). Their application in the mobile phase can be an effective way to improve the intensity of the MS signal and LC-MS signal correlation between matrix and standard samples. However, it is observed that some additives like trifluoroacetic acid or some ion-pairing agents (triethyl-amine) may play a role in ionisation suppression [3]. In addition, high concentrations of involatile buffers will cause precipitation on, and eventually blocking of, the MS entrance cone, leading to a fast decrease of sensitivity. For the in volatile NaAc buffer, it is advisable to maintain... 

The efficacy of CE separation depends considerably on the type of capillary. Fused-silica capillaries without pretreatment are used most frequently. Its outside is coated with a polymer layer to make it flexible and to lessen the occurrence of breakage. The polymer coating has to be dissolved with acid or burned away at the detection point. Capillaries with an optically transparent outer coating have also found application in CE. The objectives of the development of chemically modified capillary walls were the elimination of electro-osmotic flow and the prevention of adsorption on the inner wall of the capillary. Another method to prevent the adsorption of cationic analyses and proteins is the use of mobile phase additives. The modification of the pH of the buffer, the addition of salts, amines and polymers have all been successfully employed for the improvement of separation. 

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