Chromatography microbore HPLC

Capillary column This term refers to a chromatographic column of small diameter and is used in both gas and high performance liquid chromatography. In HPLC, the term is usually apphed to columns with internal diameters of between 0.1 and 2 mm. The term microbore column is often used synonymously to describe these columns but is more correctly applied to columns with internal diameters of 1 or 2 mm. 

Recent advances in chromatography have made it possible to employ microbore HPLC for the determination of NOC. Its main advantage is that it uses a very low mobile-phase flow (20-100 /rl/min). This might make the TEA compatible with a reversed-phase system. Massey et al. (73), in fact, have successfully used reversed-phase chromatography for the HPLC-TEA determination of V-nitroso-V, 7V -di methylpiperazinium iodide. A 500-mm X 1-mm microbore ODS column and a mobile phase consisting of 0.1 M ammonium heptane-sulfonate in methanol water (70 30) (flow rate 20 /zl/min) was used for the HPLC separation. In another study, Riihl and Reusch (74) used a microbore Spherisorb 3 SW column for HPLC-TEA determination of volatile V-nitrosamines. The mobile phase was a mixture of 2-propanol and n-hexane (2.5 97.5). Further application of such techniques for the determination of various polar NOC, especially A-nitrosamides, in foods is desirable. 

Liquid chromatography-mass spectrometry (LC-MS) is an extremely power tool for the analysis of peptides, providing not only information on the purity of the product but also coMormation of structures. The s)rstem typically consists of a microbore HPLC system coupled to an electrospray mass spectrometer. Using such a system the composition of a crude peptide mixture can be quickly determined and by-products identified, enabling synthetic protocols to be rapidly optimized. 

During the initial years of HPLC, the most common internal diameter of conventional analytical columns was 2 mm, except for columns for size exclusion chromatography that often used to be 7-8 mm. Soon, the standard inner diameter changed to 4.6 mm, the standard length was reduced from 60 to 15-25 cm, and the particle size of the column packing materials was reduced from 40-50 to 5-10 pm. The dimensions of the columns were initially related to the commercial availability of stainless steel tubing. Today, the internal diameters of conventional HPLC columns and microbore HPLC columns are in the range of 2-5 and 0.5-1 mm, respectively. Capillary columns typically have 0.1-0.5 mm ID and nanoflow columns have <0.1 mm ID (Table 3.1). 

The development of microbore chromatography and nano-flow HPLC has greatly increased interest in detectors suitable for sub-microliter peak volumes. These include LIE, EC, and MS spectrometry detectors. Zare first described the application of lasers for detection in analytical chemistry in 1984, outlining the potential for the use of lasers in analytical science, especially their application to HPLC [20]. This was followed in 1988 by another review that focused more on HPLC applications and provided examples of the high sensitivity possible with LIE detection [21], Another good review was written by Rahavendran and Karnes outlining the usefulness of LIE in pharmaceutical analysis [22],... 

Although HPLC is not fundamentally different from low pressure chromatography, there are specific features of the equipment and column materials that should be noted. The columns are usually supplied ready-packed. For analytical use, column sizes of 4 mm - 125 mm or 4 mm - 250 mm are available, together with narrow bore (2 mm diameter) or microbore (1 mm diameter) columns preparative columns are usually 25 mm 250 mm, or 50 mm 250 mm, or even larger. They are expensive and should be used with care. They should not be subjected to major pressure variations which can lead to compaction of the column bed this can produce high back pressures and a large dead volume at the head of the column. Abrupt changes in solvent should also be avoided, and in particular it is not advisable to... 

The first exposure to spectroscopy for most scientists is ultraviolet/ visible absorbance. As virtually every HPLC chromatograph employed in the pharmaceutical industry uses UV absorbance as the detection method, it is no wonder that the most popular hyphenated technique is HPLC-DAD. DAD spectrographs have been coupled to all liquid-based chromatographic systems including HPLC (preparative, analytical, and microbore), capillary electrophoresis (CE) and supercritical fluid chromatography (SFC). There have been several successes with TLC plates,18 but it is more common for developed plates to be scraped and the sample analyzed offline. 

A standard technique for the HPLC analysis of amino penicillins (ampicillins) has been to use an aqueous acid phosphate buffer-acetonitrile mobile phase at a pH close to the isoelectric points of the amino penicillins (Margosis, 1982). Amino penicillins are most stable at their isoelectric point and despite their low solubility at this pH, it is optimal for their chromatography. The stationary phase used was bonded ODS on sihca and elution was achieved under isocratic conditions (Fig. 11.9.2). Recently, microbore columns have been used in the separation of cephalosporin antibiotics (Fig. 11.9.3) and provide good baseline separations (White and Laufer, 1984). 

Microbore columns (0.8 to 1 nun i.d.) offer a compromise between wide-bore and capillary columns. Chromatography separation with these colunms is carried out at flow rates of 20 to 100 p,Lmin HPLC hardware is available that can deliver a precise volume and solvent composition in this low flow range. These... 

In conventional high-performance liquid chromatography (HPLC), columns are usually of 10-25 cm length and 2—4 mm i.d. Recently, capillary HPLC columns have become available in the market. The terminology, abbreviations, and definitions have not yet been standardized. In the literature, we can find microbore, micro-LC, semimicro-LC,... 

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