Fast gradients

Valko, K., Bevan, C., Reynolds, D. Chromatographic hydrophobicity index by fast-gradient RP-HPLC a high-throughput alternative to log P/log D. Anal. Chem. 1997, 69, 2022-2029. 

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 ... 

In case of fast gradient (below 15 min), S could be considered constant for all the investigated molecules and wiU only have a small influence on the retention time of the compounds. Thus, the gradient retention times, of a calibration set of compounds are linearly related to the ( )o values [39]. Moreover, Valko et al. also demonstrated that the faster the gradient was, the better the correlation between t, and < )o [40]. Once the regression model was established for the calibration standards, Eq. 8 allowed the conversion of gradient retention times to CHI values for any compound in the same gradient system. Results are then suitable for interlaboratory comparison and database construction. The CH I scale (between 0 and 100) can be used as an independent measure of lipophilicity or also easily converted to a log P scale. 

In order to speed up the log P estimate by gradient RPLC, several solutions were investigated, i.e. fast gradient elution obtained with the help of monolithic supports or short columns and the use of mass spectrometry (MS). 

Donovan and Pescatore described another fast-gradient approach with very short columns (20x4.6 mm internal diameter) packed with a porous polymer (known as ODP columns) [38]. This chromatographic support presents a high chemical stability and can be used at pH 2, 10 or 13 to analyze neutral analytes. This procedure allowed a relatively high flow rate (2mLmin ) and a gradient from 10 to 100% methanol in only 7 min. The mathematical treatment was simplified and based on the direct transformation of retention time to log P. For this purpose, two standards (toluene and triphenylene) were used to minimize retention time variations from run-to-run and instrument-to-instrument, and to facilitate the... 

Stationary phase. Supelcosil Cig ABZ (Supelco Scientific, Bellefonte, PA, USA) was the most often employed support and gave the best correlations. This stationary phase should be selected in a first instance with a geometry adapted to the application for conventional gradient experiments, supports of 150X4.6 mm, 5 pm represent a good choice while a shorter column (i.e. 50 mm or lower) with smaller particle size (i.e. 3-3.5 pm) must be preferentially selected for fast gradient analysis. 

In this approach, the mathematical treatment is simplified. It is recommended to perform a fast gradient with short columns and high flow rate. Only a single gradient is necessary to determine CHI. Retention time of the compounds is obtained and the mathematical treatment is achieved as described above with the help of several standards. 

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. 

Stoll, D.R., Cohen, J.D., Carr, P.W. (2006). Fast, comprehensive online two-dimensional high performance liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-phase hquid chromatography. J. Chromatogr. A 1122 (1-2), 123-137. 

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 two major approaches for HPLC purification are fast gradient separation and parallel purification. Yan et al.178 utilized the former (Figure 1.51). The purification lab received a 96-well plate containing synthesized products at 0.1 to 0.2 mmol/well. A Hydra 96-probe liquid handler prepared QC plates for all samples that were analyzed with a MUX-LCT eight-channel parallel LCMS instrument at a throughput of 2000 samples/day. Only samples with purities above 10% were purified on a... 

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... 

Most modern HPLC instruments include a column oven that can thermostat the column to at least 100°C. A typical HPLC analysis can be done in half the time by elevating the column temperature from ambient to 50 or 60°C. At temperatures above 100°C, it is not uncommon to decrease analysis time by a factor of 5.26 Also, re-equilibration time for the column is much shorter, so it is possible to achieve ultra-fast gradient analysis with HTLC. 

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 %. 

Hsieh Y. et al., 2001. Quantitative screening and matrix effect studies of drug discovery compounds in monkey plasma using fast-gradient liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 15 2481. 

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. 

Romanyshyn and Tiller [12] with ultra-short columns (20 mm x 2 mm, 10, 5 mm) and fast gradients were able to increase sample throughput and maintain the reliability of chemically different analytes during the chromatographic separations. Using this approach, the authors avoided matrix... 

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