Isocratic separation conditions

Fig. 2.12.14. (a-c) Selected mass traces and (D) TIC of ESI-LC-MS(+) analysis of polyglycol amine surfactant mixture R-N H((CH2-CH2-0),c yH)2X. Isocratic separation conditions applying a PLRP column using the ion-pairing reagent methane sulfonic... 

STRATEGIES FOR SELECTING AND OPTIMIZING ISOCRATIC SEPARATION CONDITIONS... 

After these calculations, which are performed immediately after entering the structures, the system automatically commences wthe experimental runs. The results of the experimental runs are used to fine-tune the first theoretically set up retention model. Thus, the retention model becomes accurate enough to finally predict the optimum isocratic separation conditions. 

Lewis, J. A., Snyder, L. R., and Dolan, J. W., Initial experiments in high-performance liquid chromatographic method development. II. Recommended approach and conditions for isocratic separation, ]. Chromatogr. A, 721,15,1996. 

Fig. 8. Separation of polyaromatic hydrocarbons using commercial stationary phases. (Reprinted with permission from [56]. Copyright 1997 VCH-Wiley). Conditions voltage 20 kV,capillary column 100 pm i. d., total length 33.5 cm, active length 25 cm, isocratic separation using 80 20 acetonitrile-50 mmol/1 TRIS buffer pH=8. Peaks thiourea (1), naphthalene (2), and flu-oranthrene (3)...

Recent chromatographic data indicate that the interactions between the hydrophobic surface of a molded poly(styrene-co-divinylbenzene) monolith and solutes such as alkylbenzenes do not differ from those observed with beads under similar chromatographic conditions [67]. The average retention increase, which reflects the contribution of one methylene group to the overall retention of a particular solute, has a value of 1.42. This value is close to that published in the literature for typical polystyrene-based beads [115]. However, the efficiency of the monolithic polymer column is only about 13,000 plates/m for the isocratic separation of three alkylbenzenes. This value is much lower than the efficiencies of typical columns packed with small beads. 

Figure 1 shows a schematic drawing of a CAC apparatus. The apparatus consists of two concentric cylinders standing one inside the other, forming an annulus into which the stationary phase is packed. This annular bed is slowly rotating about its vertical axis. Under isocratic elution conditions the feed mixture to be separated is introduced continuously at the top of the bed at a space that remains fixed in space while the rest of the annulus is flooded with elution buffer. As time progresses, helical component bands develop from the feed point, with... 

The method has been proposed for the prediction of retention data in isocratic systems from data measured in gradient elution and vice versa [84], Similar calculation methods may be very important in the analysis of natural extracts containing pigments with highly different chemical structure and retention characteristics. The calculations make possible the rational design of optimal separation conditions with a minimal number of experimental runs. 

Figure 2.2 Separation of aromatic compounds using isocratic elution. Conditions column, 5 pm Cis-bonded silica gel, 15 cm x 4.6 mm i.d. eluent, 0.001 M phosphoric acid in 55% aqueous acetonitrile flow rate, 1ml min-1 temperature, ambient, detection, UV 254 nm. Peaks 1, phenol, 2, 4-methylphenol 3, 2,4-dimethylphenol 4, 2,3,5-trimethylphenol 5, benzene, 6, toluene, 1, ethylbenzene, 8, propylbenzene and 9, butylbenzene.

White recently illustrated the use of fast supercritical fluid and EFLC for drug discovery and purification [46]. The optimized isocratic separations used to scale up to preparative-scale separations were often EFL mixtures. For example, Figure 9.13 shows the optimized conditions for the separation of a drug candidate included 30% methanol (with 0.2% isopropyl amine)/C02 on a Chiralcel OJ-H column at 5 mL/min [46]. His work also illustrates by using gradients that start in supercritical conditions and then move into EFL mixture conditions provides efficient and fast separations. 

Figure 7.5 Separation of a cis/trans isomer mixture by SFC using 6% methanol, isocratic elution, (a) Analytical SFC separation. Conditions column 250 X 4.6 (i.d.) mm Berger NH2 flow rate 2.5 mbmin oven temperature 35°C nozzle temperature 40°C outlet pressure 120 bar sample concentration 5 mg/ml in methanol injection volume 5 pi UV 220 nm. (b) Preparative SFC separation. Conditions column 150 X 21.2 (i.d.) mm Berger NH2 flow rate 50 mPmin oven temperature 35°C nozzle temperature 60°C outlet pressure 100 bar sample concentration 50 mg/ml in methanol injection volume 1 ml UV 220 nm.

When performing HPLC (see Basic Protocol 2) the time of analysis will depend on the conditions used—i.e., isocratic versus gradient. Isocratic separation of the individual betacyanins, as shown in Figure F3.1.2, requires 20 min, compared to 9 min using a gradient elution system. 

The optimum separation conditions for 11 CPs of DCPs and TCPs were examined in an isocratic elution. The separation column used was oct-adecyl silica, Inertsil ODS-3, which was provided from GL Sciences Inc. (Tokyo, Japan). Figure 11.11 indicates the relationship of capacity factor, k, and pH for different organic solvents in (A) 50% acetonitrile buffer and (B) 60% methanol buffer. The value of k represents alternatively the elution time. In the case of acetonitrile solvent shown in Fig. 11.11A, the separation of the selected 11 CPs was not observed completely in any pH region. On the contrary, the mobile phase of the 60% methanol buffer... 

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