Eluent composition

Sodium, potassium or ammonium orthophosphate, acetate, perchlorate or citrate salts are commonly used for buffering the eluent and should be of the highest 

ED is sometimes particularly susceptible to increases in background noise caused by metal ions present in the eluent, introduced from the samples, eluent or from components of the HPLC system. Some analysts add EDTA to their eluents, typically at concentrations of 30-150 mg L (0.1-0.5 mmol L ), with the aim of chelating iron and possibly other metal ions, EDTA may also enhance the stability of analytes, such as catecholamines, during sample storage and analysis. 

Air in the eluent can cause bubbles to form in the detector cell, but degassing prior to use will reduce this, as will recycling the eluent. Back pressurisation of ED cells to minimise the risk of bubble formation is usually not advisable since many of them readily leak. 

One aspect of eluent compatibility with EC detection is that there should be no effect on the components of the detector. Detector cell bodies are now routinely constructed of PTFE, other fluoroplastics, glass or stainless steel, and seem stable to most eluents. Nevertheless electrodes are vulnerable to chemical attack. Problems with the longer term use of some eluents at potentials around +1 V vs Ag/AgCl have been experienced. For example, ammonium acetate buffers have caused flaking of the surface of glassy carbon electrodes held at as little as +0.1 V for one batch of electrodes. Noble metal electrodes are easily contaminated by a number of eluents unless the electrode is cleaned by pulsing the applied voltage as in carbohydrate analysis (Chapter 3, Section 6). 

Aliphatic alcohols, on the other hand, require alkaline pH values to oxidise them -this is, in part, the basis of the pulsed amperometric detection of carbohydrates. Because most silica-based columns are incompatible with aqueous buffers exceeding ca pH 7.5, it may be necessary to use a high pH stability column or to employ post-column pH modification. 

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In an ideal SEC separation, the mechanism is purely sieving, with no chemical interaction between the column matrix and the sample molecules. In practice, however, a small number of weakly charged groups on the surface of all TSK-GEL PW type packings can cause changes in elution order from that of an ideal system. Fortunately, the eluent composition can be varied greatly with TSK-GEL PW columns to be compatible with a wide range of neutral, polar, anionic, and cationic samples. Table 4.8 lists appropriate eluents for GFC of all polymer types on TSK-GEL PW type columns (11). 

In most situations the eluent composition is chosen to minimize the effects of hydrophobic interaction, but these secondary effects can be used to advantage. By careful selection of a salt and its concentration, specific selectivities for analytes can be achieved without the use of organic solvents. Therefore, many separations usually run by solvent gradient reversed-phase methods can be completed with a purely aqueous isocratic eluent (13,14). 

PL aquagel-OH consists of macroporous, hydrophilic particles that exhibit a polyhydroxyl functionality. The columns exhibit extremely good stability during operation they will tolerate an operating pressure up to 140 bar and an eluent composition containing organic modifier up to 50% by volume and eluent pH in the range of 2-10. 

Stock of solvents were prepared so that their properties changed only little in the course of experiments. All mixed eluent compositions are given in weight %. 

Another important parameter is the eluent composition. Binary mixtures (and obviously pure solvents) should be preferred to complex mixtures, since new systems perform an on-line analysis of the composition of binary eluents. These eluent systems allow the automatic eluent recycling, with a reduced number of controls. 

As a typical application, the separation of an octylphenoxy-terminated PEO with respect to the terminal groups by LCCC is presented in Fig. 17.6. Similar to previous investigations on RP-18 stationary phases (Gorshkov et al., 1990 Pasch and Zammert, 1994), the critical eluent composition was achieved with methanol-water 86 14% by volume. Five well-separated peaks appeared in the chromatogram, which could be identified by MALDI-TOF mass spectrometry as being different functionality fractions. Accordingly, separation took place strictly with respect to the chemical structure of the end groups. 

FIGURE 13.2 Plots of viscosity versus eluent composition for binary mixtures of water and methanol (top curve) and water and acetonitrile (bottom curve). 

Unlike GC, HPLC can handle a variety of both aqueous phases (with various pHs and salt contents) and organic phases, and there are a variety of stationary phases that can be used depending on the analytes to be separated and the eluent being used. It is also possible to switch between columns and eluent composition during analysis so that a wide range of analytes can be separated and analyzed. 

In order to determine the applicability of retention indices, based on the alkyl arylketone scale, as the basis of a reproducible method of reporting retentions, the separation of 10 barbiturates and a set of column test compounds were examined on an octadecylsilyl bonded silica (ODS-Hypersil) column with methanol-buffer of pH 8.5 as eluent [100]. The effects on the capacity factors and retention indices, on changing the eluent composition, pH, ionic strengthened temperature, showed that the retention indices of the barbiturates were much less susceptible to minor changes in the eluent than the capacity factors. 

The adduct formation can be largely controlled and directed into the formation of a single selected species by adequate choice of the ionisation mode (possibly at the expense of sensitivity), the eluent composition (buffer addition, pH adjustment, type of organic modifier) and by optimisation of the ion source parameters influencing the stability of individual (adduct) ions. In contrast to the variations in adduct or cluster formation, which principally can be diagnosed by recording more than one (adduct) ion in SIM mode, the occurrence of ion suppression requires more careful diagnosis. 

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... 

This very large selectivity or relative retention, makes normal isocratic elution (constant eluent composition) inappropriate. Instead, gradient elution or stepwise elution are used. The eluent pH or ionic strength is changed either in a continuous gradient or in a series of steps to desorb and so elute one protein, or group of proteins, at a time. An example of stepwise elution is shown in Figure 19.9. 

Several methods are applied to reduce the separation time. The best way is the selection of a suitable column and an eluent using isocratic elution. However, much skill and knowledge are required to make such a system. A flow rate gradient, step-wise elution, or eluent composition gradients are commonly applied to reduce separation times. 

Pressure Drop Changes due to Changes in the Eluent Composition... 

To demonstrate these differences, the experimental relationship between log P and log k across a range of eluent compositions was determined for each group of analytes. The results were then used to calculate the predicted logk in each case for a theoretical model compound with log P = 5 as a hydrophobic model, for log P = 1 as a hydrophilic model, and for log P = 3 as an intermediate model. 

Fig. 6. Graph illusirating the effect of eluent composition on solute retention ( cihyl acetate from l.iChrosorh SI 100 with covalently botinti dinitinphenyl giniips at the stitface. (Courtesy of Springer-Verla. )...

As already discussed, chromatography on silica or alumina stationary phases nowadays should be restricted to the separations of organic soluble compounds. Of course, also ionic and other water-soluble substances have been and will be separated with such phases. But in order to obtain highly efficient separations (i.e., elution of solutes without tailing) the eluent composition has to be selected and adjusted carefully. Such polar components are better separated by applying tended phases or ion exchange or ion pair chromatography. 

Coupled columns packed with different stationary phases can be used to optimize the analysis time (71, 75). In this approach the different columns are connected in a series or in parallel. liie sample mixture is first fractioned on a relatively short column. Subsequently the fractions of the partially separated mixture are separated on other columns containing the same or other stationary phases in order to obtain the individual components. Columns differing in length (number of theoretical plates), adsorptive strength or phase ratio (magnitude of specific surface area), and selectivity (nature of the stationary phase) can be employed, whereas, the eluent composition remains unchanged. Identification of the individual sample components via coupled column technique requires a careful optimization of each column and precise control of each switching step. 

Gradient elution places special demands on solvent purity. Only carefully purified solvents should be used, and, it is recommended that prior to use they be passed over activated alumina or silica (7). The column acts as a collector of impurities which may elute as sharp peaks at a certain eluent composition and can be mistaken for sample components. It is therefore advisable to run the gradient first without injecting the sample in order to recognize the impurity peaks. 

The retention behavior of solutes in adsorption" chromatography can be described either by the "competition" model or by the "solute-solvent interaction" model depending on the eluent composition. It appears that both mechanisms are operative but their importance depends on the composition of the eluent mixture 84). 

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