Chromatographic conditions optimization

The different optimization equations derived in chapter 12 will then be used with these realistic chromatographic conditions in a simple optimization procedure. The conditions chosen are typical and might represent the average LC analysis. The values for (X) and (yp) are those estimated by Giddings [1] for a well-packed... 

Method development remains the most challenging aspect of chiral chromatographic analysis, and the need for rapid method development is particularly acute in the pharmaceutical industry. To complicate matters, even structurally similar compounds may not be resolved under the same chromatographic conditions, or even on the same CSP. Rapid column equilibration in SFC speeds the column screening process, and automated systems accommodating multiple CSPs and modifiers now permit unattended method optimization in SFC [36]. Because more compounds are likely to be resolved with a single set of parameters in SFC than in LC, the analyst stands a greater chance of success on the first try in SFC [37]. The increased resolution obtained in SFC may also reduce the number of columns that must be evaluated to achieve the desired separation. 

This method requires about 40 g of tobacco which are extracted with ethyl acetate in the presence of ascorbic acid. A trace amount of C-NDELA is added as an internal standard for quantitative analytical work. The filtered extract is concentrated and NDELA is enriched by column chromatography of the concentrate on silica gel. The residues of fractions with p-activity are pooled and redissolved in acetonitrile. Initially, we attempted to separate NDELA on a 3% OV-225 Chromosorb W HP column at 210 C using a GC-TEA system with direct interface similar to the technique developed by Edwards a. for the analysis of NDELA in urine (18). We found this method satisfactory for reference compounds however, it was not useful for an optimal separation of NDELA from the crude concentrate of the tobacco extract (Figure 4). Therefore, we silylated the crude concentrate with BSTFA and an aliquot was analyzed by GC-TEA with direct interface. The chromatographic conditions were 6 ft glass column filled with 3% OV-... 

Snyder s classification of solvent properties is important in the selection of the chromatographic conditions and the optimization of the chromatographic processes. 

Chromatographic conditions should be optimized wherever possible to achieve baseline separation of the analyte peak from other peaks produced by co-extracted compounds. The retention time of the analyte should be at least twice the column... 

Also in this case the calculated (predicted) retention values showed good agreement with the experimental results. It has been concluded that pH gradient elution may enhance the separation efficacy of RP-HPLC systems when one or more analyses contain dissociable molecular parts [81]. As numerous natural pigments and synthetic dyes contain ioniz-able groups, the calculations and theories presented in [80] and [81] and discussed above may facilitate the prediction of the effect of mobile phase pH on their retention, and consequently may promote the rapid selection of optimal chromatographic conditions for their separation. 

Fig. 2.61. Separation of grape seed extract (a) and grape seed extract spiked with mixture of eight compounds (b) using optimized conditions. Chromatographic conditions are discussed in the text. Peak identification 1 = 2-phenylethanol 2 = vanillin 3 = ferulic acid 4 = protocatechoic acid 5 = caffeic acid 6 = gallic acid 7 = catechin 8 = epicatechin. Reprinted with permission from A. Kamangerpour et al. [167].

Underivatized a-amino acids have successfully been separated on almost all the gly-copeptides CSPs teicoplanin [30, 84, 87], ristocetin A [33], TAG [45], A-40,926 [41], CDP-1 [55], and eremomycin [22, 23] in different chromatographic conditions. Vancomycin and avoparcin CSPs are not optimal for such substrates. A commercially available teicoplanin CSP was successfully employed for the determination of the enantiomeric purity of a sample of L-arginine [129]. For the lack of chromophore groups in the majority of them (i.e., aliphatic a-amino acids), UV detection at 205-210 nm usually yielded to loss of detection sensitivity (see Section 2.3.1.4). This problem was circumvented by the recent interfacing to the MS detection [116,117]. 

Hoke et al. [47] recently did a detailed comparison of SFC-MS-MS, EFLC-MS-MS, and HPLC-MS-MS (hexane/2-propanol/trifluoroacetic acid) conditions for thebioanalytical determination ofR and S ketoprofen in human plasma. The optimum chromatographic conditions included 55% methanol/45% CO2 (EFL conditions) with a Chiralpak AD column. The performance parameters (specificity, linearity, sensitivity, accuracy, precision, and ruggedness) for SEC, EELC, and HPLC were found to be comparable. However, the optimized EELC conditions provided the analysis in one-third the amount of time for the LC-MS-MS conditions, which is 10-fold faster than an LC-UV method [48,49],... 

Analytical SFC units are perfectly suited for optimizing the chromatographic conditions to maximize the process throughput. The separation of TSO racemate developed on an analytical system was therefore successfully extrapolated to a pilot unit equipped with a 50-mm id DAC column (System Supersep 50, Novasep) and integrating a CO2 recycling loop. 

Confirmation of FLU in catfish muscle by electrospray LC/MS was done (199). Residues of CIPRO, ENRO, SARA, and DIFLX were positively identified at 10-80 /ug/kg. The extraction procedure was based on LLE with acidic ethanol. Extracts were cleaned up on a PRS SPE cartridge. Analytes were eluted with 30% ammonium hydroxide-MeOH (1 4). Chromatographic conditions were optimized to be compatible with the electrospray interface. To obtain maximum sensitivity, separate MS acquisition programs were developed for CIPRO/ENRO and SARA/ DIFLX pairs. This method was used to confirm residues in tissues fortified in the 10-80-ppb range. All relative abundances were within 10% of the value calculated for standard compounds. 

Figure 3. Four parameter, simplex-optimized SFC separation of a 12-component mixture. Chromatographic conditions as in Vertex 13 of Table II. Sample components isoquinoline, n-octadecane (n-CigH3g), naphthalene, quinoline, acetophenone, undecylbenzene, benzophenone, 2 -acetonaphthone, diphenylamine, o-dioctylphthalate, unidentified impurity, N-phenyl-1-naphthylamine, phenanthrene quinone. Other conditions as described in the experimental section.

Figure 10. Density and temperature-optimized SFC separation of the eight component mixture of Table III. Result corresponds to the global optimum in Figures 8 and 9. Chromatographic conditions 0.19 g/mL, 104°C.

Optimization of High-Performance Liquid Chromatographic Conditions... 

OPTIMIZATION OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC CONDITIONS... 

Medic-Saric M, Jasprica I, Smolcic-Bubalo A and Momar A, Optimization of chromatographic conditions in thin layer chromatography of flavonoids and phenolic acids. Croatica ChemicaActa 77 361-366 (2004). 

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