Eluent optimization

In the UV-VIS range, personal computers are used in data evaluation, especially in multicomponent analysis. When connected to detection systems in chromatography, they are al.so used for peak detection, peak integration, and eluent optimization [105]. Although in UV-VIS spectrometry, libraries of spectra do not play an important role, standard spectra are used in chromatography for identification [106],... 

Structural formulae and data of one run Optimal eluent for separation of a mixture in isocratic RP HPLC on the column being used (first approximation). Starting conditions for RP HPLC on another column type with another eluent. Optimal gradient profile. 

Generally, optimizing the selectivity by choosing a gel medium of suitable pore size and pore size distribution is the single most important parameter. Examples of the effect of pore size on the separation of a protein mixture are given in Fig. 2.15. The gain in selectivity may then be traded for speed and/ or sample load. However, if the selectivity is limited, other parameters such as eluent velocity, column length, and sample load need to be optimized to yield the separation required. 

The latest trend is to smaller beads in smaller columns, as this saves eluent and shortens the time for a chromatographic analysis. This argument can be correct if only one suitable detector is used. However, these modern small columns are not optimal for a combination of detectors. So-called multiple detection is a combination of some detectors with different measurement principles (differential refractometer, spectral photometer, light-scattering detector, on-line viscometer) behind the last column, mostly in series, seldom in a branched ( parallel ) order. In this way, the tedious preparative fractionation of a polymer sample can often be avoided. 

K. Grob, Concurrent eluent evapor ation with co-solvent Capping for on-line reversed-phase liquid cliromatography-gas clir omatogr aphy. Optimization of conditions , J. Chromatogr. 477 73-86 (1989). 

Moore and Jorgenson eombined the rapid two-dimensional separation aehieved by LC-CZE with SEC to make the first eomprehensive three-dimensional separation involving an eleetrodriven eomponent in 1995. Size exelusion ehromatography separated the analytes over a period of several hours while the reverse phase HPLC-CZE eombination separated eomponents in only 7 min. A sehematie diagram of the three-dimensional SEC-reverse phase HPLC-CZE instrument is shown in Eigure 9.9 (18). A dilution tee was plaeed between the SEC eolumn and the reverse phase HPLC injeetion loop in order to dilute the eluent from the SEC eolumn, sinee it eon-tained more methanol than was optimal for the reverse phase HPLC eolumn. 

The vertex of a separation region points out the better operating conditions, since it is the point where the purity criteria are fulfilled with a higher feed flow rate (and so lower eluent flow rate). Hence, in the operating conditions specified by the vertex point, both solvent consumption and adsorbent productivity are optimized. Comparing the vertex points obtained for the two values of mass transfer coefficient, we conclude that the mass transfer resistance influences the better SMB operating conditions. Moreover, this influence is emphasized when a higher purity requirement is desired [28]. 

Modifier additives also play a role in method optimization and are typically added to the modifier at concentrations less than 1 % (v/v). Additives can provide increased efficiency by minimizing undesirable interactions between the analyte and the CSP, and may be necessary to elute certain types of compounds. The type of additive (acidic or basic) that will produce the best results depends upon the functionality of the analyte [72]. Certain additives are strongly retained on the stationary phase, and their effect may persist even after they are removed from the eluent [22]. The impact of both modifiers and additives can also be affected by the proximity of the operating conditions to the critical point of the eluent [73]. 

Use of 10 pm LiChrosorb RP18 column and binary eluent of methanol and aqueous 0.1 M phosphate buffer (pH 4.0) according to suitable gradient elution program in less than 20-min run time with satisfactory precision sensitivity of spectrophotometric detection optimized, achieving for all additives considered detection limits ranging from 0.1 to 3.0 mg/1, below maximum permitted levels Simultaneous separation (20 min) of 14 synthetic colors using uncoated fused silica capillary column operated at 25 kV and elution with 18% acetonitrile and 82% 0.05 M sodium deoxycholate in borate-phosphate buffer (pH 7.8), recovery of all colors better than 82%... 

The optimization of preparative and even micropreparative chromatography depends on the choice of an appropriate chromatographic system (adsorbent and eluent), sample application and development mode to ensure high purity, and yield of desirable compounds isolated from the layer. For the so-called difficult separations, it is necessary to perform rechromatography by using a system with a different selectivity. But it should be taken into account that achievement of satisfactory results frequently depends on a compromise between yield and the purity of the mixture component that is being isolated. 

Madden, J. E. and Haddad, P. R., Critical comparison of retention models for the optimization of the separation of anions in ion chromatography II. Suppressed anion chromatography using carbonate eluents, /. Chromatogr. A, 850, 29, 1999. 

System B is the most widely used for CL detection after an HPLC separation. In this system, two pumps are required for delivering the reagent solutions in the following cases (1) the solutions for CL reaction are first combined and then mixed with an eluent (2) CL reaction conditions (e.g., pH, water and organic solvent contents, and salt concentration) need to be optimized before mixing with the CL reagent. 

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