Isocratic conditions

A method offering the possibility for the separation, identification, and determination of alkyl- and alkylphenol ether carboxylates, even in mixtures with other nonionic and amphoteric substances, is carried out by HPLC using a reverse phase RP18 column and a mixture of methanol, water, and acetonitrile with the addition of an ion-pairing reagent as mobile phase working under isocratic conditions [242]. 

Figure 1.17 Separation of large ring polycyclic aroaatic hydrocarbons extracted from carbon black on a 1.8 x 0.2 n I.D. fused silica capillary column packed with 3 micrometer spherical octadecylsllanized silica gel eluted with a stepwise solvent gradient at a flow rate of 1.1 mlcroliters/min with an inlet pressure of about 360 atmospheres. Under isocratic conditions this column yielded ca. 225,000 theoretical plates. (Reproduced with permission from ref. 238. Copyright Friedr. Vieweg t Sohn).

NMR spectroscopy is essential for the structure determination of carotenoid isomers because the TI-NMR signals of the olefinic range are characteristic for the arrangement of the isomers. The stereoisomers of astaxanthin, as shown in Figure 4.16, can be separated on a shape-selective C30 capillary column with methanol under isocratic conditions. 

A typical HPLC separation using a 15-cm column of 15,000 theoretical plates produces peak capacity (Giddings, 1991) of about 80-100 under isocratic conditions and up to 150 under gradient conditions in 1 h(Eq. 7.3, n peak capacity, A number of theoretical plates of a column, and fR and t retention time of the last and the first peak of the chromatogram, respectively). An increase in the number of separated peaks per unit time can be achieved by increased separation speed made possible by monolithic silica columns (Deng et al., 2002 Volmer et al., 2002). This has also been shown for peptides and proteins (Minakuchi et al., 1998 Leinweber et al., 2003). 

Note 1 Best examined with a less aqueous, more organic mobile phase gradient, or isocratic condition. 

One drawback of this approach is the relatively low sample throughput of traditional HPLC systems. The primary reason for this low throughput is that each standard and sample must be assayed under a minimum of three different isocratic conditions. Assuming a run time (injection to injection) of 10 min, it would take 50 hr to analyze 4 standards and 96 unknowns. /./PLC is ideally suited for determination of log P since it facilitates parallel analysis of a large number of compounds under identical chromatographic conditions (Table 6.4). 

Natural extracts generally contain molecules with highly different retention characteristics which cannot be separated under isocratic conditions. The application of gradient elution is a necessity for these types of natural samples. However, the optimization of gradient elution on the base of isocratic data is cumbersome and the prediction of retention in gradient elution from isocratic data is difficult. Retention in an isocratic system can be described by a polynomial function ... 

Fig. 2.19. Chromatogram of carotenoid solution in THF. 1 lutein, 2 canthaxanthin, 3 /Tcryptoxan-thin, 4 lycopene, 5 /1-carotene. ODS column, 5/an, 150mm X 4.6mm. Mobile phase eluent A (methanol-acetonitrile 6 11), eluent B (THF) gradient profile 0-5min, isocratic conditions 95 per cent A 5-20min, gradient to 20 per cent A. Flow rate lml/min. Detection 450 nm. Reprinted with permission from C. Tricard el al. [45].

SEC offers several advantages that make it a desirable technique for both preparative and analytical applications. First, separations are rapid with an 8 x 300 mm analytical column operated at 1 ml/min, all analytes elute in about 10 min. Second, because the stationary phase is designed to eliminate interactions with the sample, SEC columns exhibit excellent recovery of mass and biological activity. Third, because all separations are performed under isocratic conditions, peak area and retention time precision are high. 

FIGURE 18 HPLC separation of a stabiiity sampie under isocratic conditions showing iow ieveis of various reiated substances (API active pharmaceutical ingredient). Reprinted with permission from Reference 19. 

Percentage organic modifier in the mobile phase at the start of the gradient or isocratic conditions. % 1 % absolute... 

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