HPLC methods silica-based

With the development of HPLC, a new dimension was added to the tools available for the study of natural products. HPLC is ideally suited to the analysis of non-volatile, sensitive compounds frequently found in biological systems. Unlike other available separation techniques such as TLC and electrophoresis, HPLC methods provide both qualitative and quantitative data and can be easily automated. The basis for the HPLC method for the PSP toxins was established in the late 1970 s when Buckley et al. (2) reported the post-column derivatization of the PSP toxins based on an alkaline oxidation reaction described by Bates and Rapoport (3). Based on this foundation, a series of investigations were conducted to develop a rapid, efficient HPLC method to detect the multiple toxins involved in PSP. Originally, a variety of silica-based, bonded stationary phases were utilized with a low-pressure post-column reaction system (PCRS) (4,5), Later, with improvements in toxin separation mechanisms and the utilization of a high efficiency PCRS, a... 

Silica stationary phases display some ion exchange properties, which may also influence the separation characteristics of silica. One of the main disadvantages of the use of silica and silica-based stationary phases is their instability even at slightly alkaline pH, such as 8.0. HPLC stationary phases can be characterized with the average particle diameter and the distribution of particle size. Smaller average diameter and narrow particle size distribution generally enhances the efficacy of separation. The average particle diameter can be calculated with different methods ... 

HPLC methods can be ntilized for the pre-concentration of aromatic amines from polluted waters on silica gel or octadecyl silica (ODS) colnmns [55], The determination is then performed by RP HPLC using ODS packings as the stationary phases and a mixture of methanol, isopropanol, and water as the mobile phase [55], RP HPLC with diode array detector (DAD) methods coupled on-line with a continnons seqnential anaerobic/aerobic reactor system have been employed in wastewaters treatments [56], A continnons monitoring of the possible presence of aromatic amines in azo dyes wastes is based on indncing in the waste, the reaction of a reduction of the dye, followed by HPLC/ UV or HPLC/MS analysis [57-59], The redncing agent solutions are sodium dithionite or tin(II) chloride in an aqneons acidic medinm at 70°C, followed by SPE [58,59], LLE [60,61], or SEE [60-62],... 

Since the 1970s numerous HPLC methods using lEC, RP and ion-pair chromatography have been proposed. In the last years, RP chromatography has become the most used method, thanks to its simplicity, sensitivity, and compatibility with different detection techniques. The stationary phases usually used are C18 or phenyl-bonded silica-based phases. More recently, alternative stationary phases, such as polar-embedded, polar endcapped, and perfluorinated phases, have been successfully tested for folate analysis [577]. The mobile phase is usually a mixture of phosphate or acetate buffer and acetonitrile or methanol. 

In normal-phase HPLC on a silica column, separation is based on the number and position of methyl substituents on the chromanol ring. In reversed-phase HPLC on a Cl8 column, separation is based on the structure of the side chain and the number of methyl substituents. It is therefore difficult to completely separate [3-and y-tocopherol and (3- and y-tocotrienols by reversed-phase HPLC, because both have the same side-chain structure and number of methyl substituents on the chromanol ring. Only six peaks are usually found in the reversed-phase HPLC method. Thus, reversed-phase HPLC is recommended for samples from animal tissues, which contain little or no P and y vitamers. 

Current chromatographic methods for these alkaloids are based on TLC or electrophoresis on silica gel plates and RP-HPLC (362-368). The most significative HPLC methods presented in the literature are summarized in Table 9. An example is shown in Fig. 12. 

Applications of HPLC for Hg speciation studies have been reviewed by Harrington.83 Practically all HPLC methods for Hg speciation reported in the literature are based on reversed-phase separations, involving the use of a silica-bonded phase column and a mobile phase containing an organic modifier, a chelating or an ion pair reagent, and in some cases, a pH buffer. 

The silica-based TSK-2000 size exclusion HPLC column has excellent separation properties for proteins in MW range 2000-20,000, when 0.1% aqueous TFA is used as eluent (3,10). We have not seen this property when solvents at neutral pH were used or with other size-exclusion HPLC columns (resin-based as well as zirkoniumoxide-based). The use of 0.1% aqueous TFA solution also dramatically increases the recovery of chemokines. Nevertheless, the TSK-2000 column should be used only when other methods of purification cannot be applied. 

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