Fast-gradient method

Fig. 12.19) that seemed to have the widest range of coefficients for the molecular descriptors in the solvation equation. Table 12.10 shows the solvation equations obtained and their normalised coefficients. The equations were obtained from the chromatographic hydrophobicity index data (CHI) of the compounds listed in Table 12.7 by using a 2.5 min linear gradient of the oi anic solvent from 0 to 1009F with 2 ml/min flow rate. The columns used were 4.6 x 50 mm short columns. The application of this fast gradient method makes it possible to obtain CHI values in a given HPLC system in 5 min. 

Based on the cpo concept, a fast gradient method RP-HPLC has been proposed to determine the chromatographic hydrophobicity index (CHI) as a high-throughput alternative to other lipophilicity measures.Eor this... 

The fast-gradient method, in contrast, retains analytes on-column until well after the solvent front has eluted. Overall sample throughput is increased with fast-gradient methods due to reduced analytical run time, decreased method development time, and fewer repeat analyses. Onorato et al. [90] used a multiprobe autosampler for parallel sample injection, short, small-bore columns, high flow rates, and elevated HPLC column temperatures to perform LC separations of idoxifene and its metabolite at 10 s/sample. Sample preparation employed liquid liquid extraction in the 96-well format. An average run time of 23 s/sample was achieved for human clinical plasma samples. 

Based on the (po concept, a fast gradient method RP-HPLC has been proposed to determine the chromatographic hydrophobicity index (CHI) as a high-throughput alternative to other lipophilicity measures.For this purpose, gradient retention times (tg) are measured and converted to CHI values by means of a calibration equation, derived by a set of standards with well-determined CHI (tpo) values ... 

The CHI can be obtained without preliminary method development direcdy from a single fast-gradient run with a cycle time less than 15 min with a 150-mm column [40] or 5 min with 50-mm column [42]. In this case, the obtained retention time, tr, is expressed within an organic phase concentration (< o) scale using a calibration set of compounds. CHI value can be obtained from ... 

Valko et al. [37] developed a fast-gradient RP-HPLC method for the determination of a chromatographic hydrophobicity index (CHI). An octadecylsilane (ODS) column and 50 mM aqueous ammonium acetate (pH 7.4) mobile phase with acetonitrile as an organic modifier (0-100%) were used. The system calibration and quality control were performed periodically by measuring retention for 10 standards unionized at pH 7.4. The CHI could then be used as an independent measure of hydrophobicity. In addition, its correlation with linear free-energy parameters explained some molecular descriptors, including H-bond basicity/ acidity and dipolarity/polarizability. It is noted [27] that there are significant differences between CHI values and octanol-water log D values. 

Others have examined the necessary parameters that should be optimized to make the two-dimensional separation operate within the context of the columns that are chosen for the unique separation applications that are being developed. This is true for most of the applications shown in this book. However, one of the common themes here is that it is often necessary to slow down the first-dimension separation system in a 2DLC system. If one does not slow down the first dimension, another approach is to speed up the second dimension so that the whole analysis is not gated by the time of the second dimension. Recently, this has been the motivation behind the very fast second-dimension systems, such as Carr and coworker s fast gradient reversed-phase liquid chromatography (RPLC) second dimension systems, which operate at elevated temperatures (Stoll et al., 2006, 2007). Having a fast second dimension makes CE an attractive technique, especially with fast gating methods, which are discussed in Chapter 5. However, these are specialized for specific applications and may require method development techniques specific to CE. 

The use of high flow and fast gradient HPLC has gained a lot of popularity because of the ability to reduce LC/MS/MS cycle times during bioanalysis. In the case of fast gradient HPLC, peak shapes were improved and method development times were minimized, especially when multiple analytes with diverse functionalities had to be separated. Flows as high as 1.5 to 2 mL/min were achieved on a 2.1 x 30 mm Xterra C18 column.7 Details are discussed in a recent review.8... 

As a generic method, the SPE cartridge was conditioned with 1.5 mL methanol (5.0 mL/min) and 1.5 mL water (5.0 mL/min), after which 100 /tL of spiked plasma was injected and washed with 3.0 mL water (2.0 mL/min). After switching online, the analytes were flushed and eluted with a fast gradient of mobile phase A (5 95 v/v acetonitrile water, 0.1% formic acid, and 10 mM ammonium acetate) and B (95 5 v/v acetonitrile water, 0.1% formic acid, and 10 mM ammonium acetate). The lower limit of quantitation was 0.2 to 2 ng/mL and linear range was 2 to 4 orders. Carry-over was 0.02 to 0.1 %. 

In addition, the use of fast gradients elution mode has become the bioanalytical mainstream as a possible way to improve peak parameters (shape and symmetry) and to minimize method development time, especially for the multi-analytes methods. 

Valko, K., Nunhuck, S., Bevan, C., Abraham, M.H. and Reynolds, D.P. (2003) Fast gradient HPLC method to determine compounds binding to human serum albumin Relationships with octanol/water and immobilized artificial membrane lipophilicity. Journal of Pharmaceutical Sciences, 92, 2236-2248. 

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