Fast Gradient Separations

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... 

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... 

In order to talk about fast gradient separations, we need to review the principles involved in measuring the performance of a gradient separation. We can use the concept of peak capacity to measure the separation power of a particular gradient on a given column. The peak capacity (P) is defined as follows ... 

Fast gradient separations can be achieved, at a cost of decreased peak capacity, by using short packed columns and simultaneously decreasing the particle size, dp, of the packing material to keep a constant ratio of the column length to the mean particle diameter, lidp, e.g., with a 3 cm column packed with a 3 [im material, or even a 1 cm, 1 pm column for very fast separation instead of a conventional 5 cm, 5 pm or a 10 cm, 10 pm column. At constant Vq/V , and Hdp ratios, the... 

In a separate experiment, two regions of rat brain tissue sections that contained different levels of clozapine based on the MALDI-IMS results were isolated. The isolated tissue sections were transferred into plastic test tubes and mixed with 300 p,L of 95 5 methanol-water solution for protein precipitation. After centrifugation, a 10-p.L aliquot of the supernatant from each sample was injected for HPLC-MS/MS analysis. The HPLC was operated under a fast gradient condition using a cyano... 

In some cases, drug substance does not have chromophores with a molar absorbtivity sufficient for accurate quantitation using UV detection. If HPLC with UV detection is used as a basic quantitation technique, then MS detection as a complementary technique is desirable in most cases. LC-MS is essentially preferable in most preformulation assays. High selectivity of the MS detector allows the use of fast gradient HPLC separation methods, which does not require significant development time. Practically in all assays used in preformulation, the quantitation of only drug substance is required and MS detection provides an accurate quantitation. 

With the application of the fast gradient CHI values it takes approximately six times five minutes to determine the basic molecular properties of newly synthesised drug molecules. The method can be fully automated, and small impurities can be separated during the HPLC run so that they do not disturb the measurements. Very small quantities of compounds are used for these measurements, and we believe that this method will help design soluble drug molecules with the desired absorption, and brain penetration, etc., by using the general solvation equations already known for these systems. 

A multiresidue method for 21 sulfonamides in milk was described by Volmer [32]. Separation of all compounds was achieved in only 6 min on a 50x4.0-mm-ID ODS-AQ colunrn (3 pm) using a fast gradient program (10-45% acetonitrile in 0.1% aqueous formic acid in 7 min). The coluttm was ran at a flow-rate of 1 ml/min. A post-colunm split was used to deliver 90 pFmin to ESI. A three-step analytical strategy is adopted ... 

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. 

Metabolite ID 1.4 operates in both interactive and batch mode. In the interactive mode, the user reviews the full-scan data prior to MS/MS generation. In batch mode, the user submits a list of samples to be analyzed and starts automated acquisition. With such automated approaches, the metabolic profile of a single compound can be evaluated in approximately 1.5 hours, provided that adequate separation can be achieved with short, narrow-bore columns and fast-gradient chromatography. 

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