Reverse phase method development dimensions

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. 

Other applications that utilize different types of reversed-phase columns in both dimensions have been advocated by Carr (Stoll et al., 2006) for metabolomics work in small-molecule separations. These stationary phases include a pentafluorophenyl-propyl stationary phase in the first dimension and a carbon-coated zirconia material stationary phase in the second dimension. A common mistake in 2D method development is to mismatch the solvent system the two solvent systems must be miscible as discussed below. 

A sensitive reverse-phase HPLC method has been developed for the analysis of etodolac in tablet formulation [22]. The chromatographic separation was achieved using a reverse-phase Cu column, having dimensions of 3.3 cm x 0.46 cm i.d. (3 pm particles) and which was maintained at 30°C. The mobile phase consisted of pH 6.0 phosphate buffer / methanol (60 40 v/v), and was eluted at 1 mL/min. Analyte detection was effected on the basis of UV detection at 230 nm. Diazepam was used as an internal standard. The sample preparation entailed grinding the etodolac tablets, followed by extraction with methanol (using sonication). A retention time of 1.46 min was obtained for etodolac under these conditions, and the method was found to be linear, precise, and accurate over the concentration range of 0.01 to 0.1 mg/mL. 

Successful enantioseparation of individual N -protected amino acids stimulated the development of a rapid method of their simultaneous enantioseparation and quantification in a mixture. A feasibility study on this topic has been recently published by Welsch et al. [69]. The two-dimensional HPLC method involves online coupling of a narrow-bore C18 reverse phase (RP) column in the first dimension (separation of racemic amino acids) to a short enantioselective column based on nonporous 1.5 pm particles modified with t-BuCQD in the second dimension (determination of enantiomer composition). Using narrow-bore column resulted in fast analysis time for example, the mixture of nine racemic N-DNB-protected amino acids was completely analyzed within 16 min. 

In the last years following the nanotechnology trend, great efforts have been put towards miniaturization and the developments of nano-LC. This is actually mandated from the end needs of the MS detection in genome-pro-teome research Tiny amounts of the biomacromolecule are present in complex samples of limited volume and availability. Thus, miniaturization is a one-way road in proteomics, and microbore/capillary LC has become more popular. It is also believed that new bioanalytical LC/MS methods will be developed aiming at capillary or nano-LC dimensions. New, truly nano-LC pumps have now been commercialized to cover the niche. However, this has not yet passed into the bulk of the LC/MS reported works, where conventional LC columns still dominate the field. Reversed-phase (RP) LC is still by far the method of choice in LC/MS schemes, but more and more multidimensional schemes are reported employing size exclusion, ion exchange, or affinity modes and RP (Table 2). 

HPLC has been used in the extraction and purification of pollutants in environmental and biological samples. Its manifold applications are due to the availability of a number of stationary phases, particularly reversed phase columns of various dimensions. HPLC instruments used for this purpose are similar to the analytical machine but with the use of a different column, which depends on the nature of the pollutants to be extracted and purified. Many of the reports available in the literature deal with the use of HPLC as an extraction and purification technique for environmental and biological samples. For determinations of polar pesticides, HPLC appears to be the most appropriate technique for purification purposes [134, 135]. Smith etal. [136] used low pressure HPLC for the extraction of polychlorinated dibenzofurans and dioxins. Furthermore, Blanch era/. [137] used low pressure HPLC for the purification and extraction of PCBs from shark liver. Similarly, Ramos etal. [138] used the Smith etal. [136] method for the extraction of PCBs in dolphin liver. Bethan etal. [139, 140] used a LiChrosorb 100-Si HPLC column for the extraction of bromocyclen collected from river water and of ck-HCH in marine water. However, HPLC cannot achieve a reputation as the universal and highly applicable extraction technique, due to its low range with regard to preparative chromatography. Therefore, further advancement of the technique is required, especially with regard to the development of preparative columns. 

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