Effect of Eluent

For the method development of the SEC separation, the main attention should be focused on the suppression of analyte interactions with the surface of packing material. This usually requires a careful selection of the SEC column. In GPC, commercially available synthetic organic polymer columns are usually packed with styrene-divinylbenzene copolymer particles, which are only capable of weak dispersive interactions. Any possible analyte interactions with the surface could be suppressed by using a strong solvent, which will be preferentially adsorbed on the packing material surface. Selection of such a solvent is limited since the polymer solubility in that particular solvent needs to be considered. Tetrahydrofuran is the most common solvent used for most GPC separations, although for polyimids and other high-temperature polymers the use of special solvents such as n-methylpyrrolidone may be necessary. 

The development of GFC methods generally has similar considerations, with much higher requirements for the packing material. GFC deals with water-soluble polymers thus the main solvent is water, with some additives. The suppression of possible surface interactions has to be done with these additives and with careful selection of the packing material. 

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Rauha JP, Vuorela H and Kostiainen R. 2001. Effect of eluent on the ionization efficiency of flavonoids by ion spray, atmospheric pressure photoionization mass spectrometry. J Mass Spectrom 36 1269-1280. 

Peyrin, E. et al., Dansyl amino acid enantiomer separation on a teicoplanin chiral stationary phase effect of eluent pH, J. Chromatogr. A, 923, 37, 2001. 

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. )...

Table XIII. Effect of Eluent Additives on -178 Yield from Bio-Rad AG1x8...

Fig. 24. Examples of the effect of eluent programme s>h(V) on retention volumes of macromolecules of different functionality (calculated data for D = 8,N = 10,Xq = 3.6,Xq, = 5.6, V0 = Vp = 40ml, verical lines in the figure correspond to retention volumes of macromolecules of different functionality)...

The effects of eluent pH upon the retention of various solute types in pure water as eluent are shewn in Fig. 5. The pH... 

The Effects of Eluent Temperature. m agreement with previous studies (67), both solute retention and separation selectivity were found to increase significantly as the eluent temperature was reduced from 30 to 0 C, as shown in Fig. 6 for the enantiomers of tryptophan. The change is much larger than what is observed on an octadecylsilica reversed-phase column and the improved separation selectivity is attributed to more pronounced inclusion complex formation. Solute retention decreases precipitously when the temperature of the eluent is raised to or above 60 C, an important fact that can be utilized advantageously for the rapid removed, of strongly adsorbed components (such as displacers) from the cyclodextrin-silica column. 

Each amine was dissolved in dilute (lxl0--3 M HC1). The working solution used in this study was 50 yg/ml amine. This is high concentration for the detector but assists in determining the effect of eluent concentration on retention time. The working solutions were protected from the light and fresh solutions were prepared weekly. 

A series of perchloric acid solutions were prepared as the eluent. Each eluent was flushed through the chromatograph until a stable baseline was obtained. Each individual amine was injected onto the column using the 0.5 ml sample loop. The amine was allowed to elute if possible. If after sixty minutes the amine had not eluted, the system was flushed with 1 M HCIO4 to remove the compound. The order of elution of the amine and the effect of eluent concentration on the retention time were determined. 

Another group of amines can be eluted in a reasonable time using higher concentrations of eluent. The effect of eluent concentration on retention volume is shown in Figure 5. Except for the 2-aminofluorene, these compounds are all diamines. 

Figure 4.37. Effect of eluent pH on selectivity in reverse-phase BPC. Column, 30 x 0.4cm, Bondapak-C, 10/jm mobile phase, 0.025M NaH2P04-Na2HP04 wiih 40%v methanol flowrate, 2ml/min temp., ambient detector, UV, 220nm, samples. 1—lOpg. (1) Salicylic acid (2) phenobarbitone (3) phcnacctin (4) nicotine ...

Figure 3. Effect of eluent pH on the retention and peak height of methionine enkephalin.

After the basic tests, were suitable solvents are selected and the desired solvent strength is established, the effect of eluent changes at a constant solvent strength are further investigated to find the optimum mobile phase composition. 

Fig. 6 Graphical representation of the van Deemter equation showing the effect of eluent velocity on the plate height of a chromatography column.

FIGURE 3. Gel permeation chromatography of humic acid on Sephadex G-lOO showing the effect of eluent on adsorption. 

Figure 10.1 Effect of eluent composition in reversed-phase chromatography (reproduced with permission from N.A. Parris, Instrumental Liquid Chromatography, Elsevier, 1st ed., 1976, p. 157).

Nivaud-Guemet, E. Guemet, M. Ivanovic, D. Medenica, M. Effect of eluent pH on the ionic and molecular forms of the non-steroidal anti-inflammatory agents in reversed-phase high-performance liquid chromatography. J.Liq.Chromatogr, 1994, 17, 2343-2357 [also aspirin, flufenamic acid, keta-zone, mefenamic acid, niflumic, niflumic acid, nixylic acid, oxyphenbutazone, phenacetin, salicylamide, salicylic acid, sulfinpyrazone]... 

Kostiainen R, Kauppila TJ, Effect of eluent on the ionization process in liquid chromatography-mass spectrometry, J. Chromatogr. A 2009 1216(4) 685-699. 

Figure 7.7 Effect of eluent ionic strength on the separation of anionic polymers (polyacrylic add sodium salt) in aqueous sodium nitrate solution. Sodium nitrate concentrations A, 0 B, O.OIM C, 0.025M Z), 0.05M E, 0.1M. Separations were performed on TSKgel GMPW (two 600 x 7.5 mm LD. columns) at a flow rate of ImLmin" and 25°C [15].

Law, B. The effect of eluent pH and compound acid-base character on the design of generic-gradient reversed-phase high-performance liquid chromatography (PR-HPLQ methods for use in drug discovery. J. Phaim. Biomed. 

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