Tubing, micro HPLC

Bile acids and their conjugates have been separated and mass analyzed by a direct combination of micro HPLC and FAB MS113, allowing the introduction of the total effluent into the mass spectrometer through a fused silica capillary tubing, ending with a stainless steel frit. The liquid mobile phase was vaporized on the surface of the frit, while the solute and the glycerol matrix were left on the surface and were subjected to FAB. 

This class is the simplest of all micro reactors and certainly the most convenient one to purchase, but not necessarily one with compromises or reduced fimction. HPLC or other tubing of small internal dimensions is used for performing reactions. There are many proofs in the literature for process intensification by this simple concept. As a micro mixer is missing, mixing either has to be carried out externally by conventional mini-equipment or may not be needed at all. The latter holds for reactions with one reactant only or with a pre-mixed reactant solution, which does not react before entering the tube. 

Micro-mixer Tees from Swagelock were used to connect the various feeds to a coil of 254gm stainless steel HPLC capillary tubing immersed in a Huber oil bath. 

Early in the development of capillary electrophoresis, it was noted that the successful detection of separated sample components present within the narrow confines of these capillary tubes posed a major challenge (2)- In response to this challenge, much research has been directed toward the development of sensitive and selective detectors for capillary electrophoresis. CE detector technology has been largely borrowed from the field of high-performance liquid chromatography (HPLC), especially from micro-column HPLC. 

Liquid Chromatograph. The liquid chromatograph was comprised of a Waters 660 Solvent Programmer, two Waters 6000A pumps, a Waters U6-K Injector and a Waters 440 absorbance detector (254 nm). Whatman micro-capillary tubing (0.007" ID) was used to transfer the HPLC column effluent from the 254 nm absorption detector to the fluorescence detector. 

HPLC/OMA System. In this study, the HPLC effluent stream was passed through three flow-cells connected in series with different lengths of Whatman micro-capillary (0.007 " I.D.) tubing. The first flow-cell monitored the absorption at 254 nm and was used to key the recording of absorption and fluorescence spectra of the effluent stream passing through the second and third flow-cells, respectively, at peak maxima. 

The micro-bore HPLC system is a modified form of chromatography. The utilization of micro-bore tubes with a stationary coating on the surface instead of conventional columns results in higher resolution, faster speed of separation, and minimal use of solvent. 

Low-dispersion HPLC systems are necessitated by the increasing trend of using shorter and narrower HPLC columns, which are more susceptible to the deleterious effects of extra-column band-broadening. HPLC manufacturers are designing newer analytical HPLC systems with improved instrumental bandwidths compatible with 2-mm i.d. columns by using micro injectors, smaller i.d. connection tubing, and detector flow cells. A new generation of ultra-low dispersion systems dedicated for micro and nano LC is also available. 

Determination of pyrogallol. Pyrogallol was assayed by HRP catalyzed imidazole chemiluminescence coupled to the micro-flow injection system at room temperature. Pyrogallol specimens (50 pL) were injected using an autosampler (AS-950, JASCO, Tokyo, Japan) every five min into a stream of water (100 uL/min) using a HPLC pump (PU-980, JASCO), and the other mobile phase (imidazole 100 mmol/L in the Tricine buffer 50 mmol/L, pH 9.3) was delivered at 100 pL/min. The light emitted from the reactor tube was detected with a... 

The fluorescence HPLC detector (HPLC FL) is similar to the UV-absorp-tion HPLC detector (HPLC UV) in that a source of UV radiation is made incident to a micro-flow cell in whieh the chromatographically resolved analyte passes through. However, a photomultiplier tube (PMT) and associated optics are positioned at right angles relative to the incident UV radiation. Lakowicz has articulated just what molecular luminescence is (93) ... 

Solid-phase extraction (SPE micro-columns) was efficient for the purification of alkaloidous solutions. Aliquots were passed through a pre-activated (by methanol than water) SPE cartridge (Supelclean LC-8 columns 500 mg, 3 mL, supplied by Supelco) which was then washed with water. Extraction was performed on a vacuum manifold processor (LiChrolut extraction unit Merck).The alkaloid (lobeline)-containing fraction was eluted from the tube with methanol. On the basis of the HPLC determination, the recovery of lobeline from the SPE step was total [40]. 

In HPLC, the sample is dissolved in a solvent (preferably same as the HPLC mobile phase) and injected onto the column. Attention must be paid to avoid precipitation of the injected sample and blockage of the column. The HPLC column is usually a 3-25 cm long metal tube of 1-5 mm diameter. Conventionally 4.6 mm columns are used in HPLC, with a flow rate of about 1 ml/min. Nowadays narrower columns (1 and 2 mm) are becoming very popular, especially combined with MS (using much less, 50-200 (xl/min solvent flow). Micro- and nano-HPLC is also gaining ground (e.g., using 75 pm diameter quartz tubes and —200 nl/min solvent rate), especially in the field of proteomics [34]. Note that narrow columns require very small amounts of sample (approximately proportional to the internal volume of the column), and thus require very sensitive detectors. 

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