Eluent considerations

The use of formic acid, acetic acid and ammonium formate rather than triflu-oroacetic acid can substantially increase sensitivity because their proton affinities are lower than that of the TFA anion - though TFA is often used in the analysis of peptides. It is always advisable to keep the level of acid additives to less than 0.1% v/v, and preferably 0.03-0.05% v/v, in the final eluent. Triethylamine or ammonium hydroxide can be used successfully in negative mode because they promote deprotonation of acidic species. 

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Corrosive eluents such as diluted hydrochloric acid and sodium hydroxide solutions are often used in ion chromatography. Therefore, all parts of the chromatographic system being exposed to these liquids should be made of metal-free inert materials. Conventional HPLC systems with stainless steel tubings and pump heads are only partially suited for ion chromatography, since even stainless steel is eventually corroded by aggressive eluents. Considerable contamination problems would result, since metal ions... 

These three criteria—molar mass interval, eluent, and working temperature—are fixed by the group of samples to be analyzed and considerably restrict the number of suitable columns. The selection has to be done from current lists of the manufucturers. It is useless to collect these data here, as such tables would be antiquated before this book is printed. This chapter deals with the quality of the selected columns. At this stage, columns of the same application profile are compared. The most important properties are (1) the number of... 

The plate number for the same column also depends on the eluent, e.g., a permitted operation for some styrene-divinylbenzene columns is to change the eluent from tetrahydrofuran (THF) to dimethylacetamide (DMAC) and then return to THF. The plate number in DMAC is considerably lower than in THF. After the replacement of DMAC by THF the old N value is obtained again. 

As the vast majority of LC separations are carried out by means of gradient-elution RPLC, solvent-elimination RPLC-FUR interfaces suitable for the elimination of aqueous eluent contents are of considerable use. RPLC-FTTR systems based on TSP, PB and ultrasonic nebulisa-tion can handle relatively high flows of aqueous eluents (0.3-1 ml.min 1) and allow the use of conventional-size LC. However, due to diffuse spray characteristics and poor efficiency of analyte transfer to the substrate, their applicability is limited, with moderate (100 ng) to unfavourable (l-10pg) identification limits (mass injected). Better results (0.5-5 ng injected) are obtained with pneumatic and electrospray nebulisers, especially in combination with ZnSe substrates. Pneumatic LC-FI1R interfaces combine rapid solvent elimination with a relatively narrow spray. This allows deposition of analytes in narrow spots, so that FUR transmission microscopy achieves mass sensitivities in the low- or even sub-ng range. The flow-rates that can be handled directly by these systems are 2-50 pLmin-1, which means that micro- or narrow-bore LC (i.d. 0.2-1 mm) has to be applied. 

An additional consideration for sample preparation is to ensure that the final sample solution is miscible with the HPLC eluent and will not alter or degrade the column.62 The total time needed for sample preparation may be longer than that required to conduct chromatographic separation and therefore becomes is the rate-determining step for the analysis.63 A survey cited by several authors indicated that on average chromatography separation accounts for about 15% of the total analysis time, sample preparation, about 60%, and data analysis and reporting, 25%.64 66... 

This problem was resolved by Nakae et al. [7] using non-polar octadecylsilica as the stationary phase and a solution of 0.1 M of sodium perchlorate in methanol/water (80 20) as the mobile phase. The ternary system (water-alcohol-salt), previously used by Fudano and Konishi [8] as an eluent for the separation of ionic surfactants at higher concentrations, induced the so-called salting out effect . The addition of the organic solvent to the water modified the polarity of the eluent and produced a good separation within a short period of time [9]. It also has the function of dissociating the surfactant micelles in individual molecules that are dissolved in the eluent [8], The presence of the salt (NaC104) in the mobile phase has a considerable influence on... 

Global LSER calculations have also been applied to the study of the retention of ioniz-able analyses in RP-HPLC. While the retention of neutral analyses does not depend on the pH of the mobile phase the retention of analyses with one or more ionizable substructures considerably depends on the pH even at the same concentration of organic modifier in the eluent. The relationship between the retention and pH of the mobile phase and pK value of the analyte can be described by... 

The theoretical considerations discussed briefly above have been further enlarged and the enhanced calculation of optimal gradient programmes was achieved involving three factors gradient slope, initial eluent composition and gradient curvature. In the case of an ACN organic modifier the retention of an analyte can be described by... 

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