Liquid chromatography flow rates

Elution volume, exclusion chromatography Flow rate, column Gas/liquid volume ratio Inner column volume Interstitial (outer) volume Kovats retention indices Matrix volume Net retention volume Obstruction factor Packing uniformity factor Particle diameter Partition coefficient Partition ratio Peak asymmetry factor Peak resolution Plate height Plate number Porosity, column Pressure, column inlet Presure, column outlet Pressure drop... 

The cracking of diphenylmethane (DPM) was carried out in a continuous-flow tubular reactor. The liquid feed contained 29.5 wt.% of DPM (Fluka, >99%), 70% of n-dodecane (Aldrich, >99% solvent) and 0.5% of benzothiophene (Aldrich, 95% source of H2S, to keep the catalyst sulfided during the reaction). The temperature was 673 K and the total pressure 50 bar. The liquid feed flow rate was 16.5 ml.h and the H2 flow rate 24 l.h (STP). The catalytic bed consisted of 1.0 g of catalyst diluted with enough carborundum (Prolabo, 0.34 mm) to reach a final volume of 4 cm. The effluent of the reactor was condensed at high pressure. Liquid samples were taken at regular intervals and analyzed by gas chromatography, using an Intersmat IGC 120 FL, equipped with a flame ionization detector and a capillary column (Alltech CP-Sil-SCB). 

It is apparent that diffusion is approximetely 10,000 times as fast in air as in water. Since the rate at which a chromatogram can be developed is dependent on the rate at which diffusional equilibrium can be established between the mobile phase and the stationary phase, the rate at which gas chromatograms can be run is much greater than the rate at which liquid chromatograms can be run. Development rates for liquid chromatograms vary from 0.01 to 1.0 ml per min per sq.cm. In gas chromatography, flow rates range from 50 to 1,000 ml per min per sq cm. Because of these rapid flow rates, it is possible to obtain rapid elution of... 

These factors make it necessary to reduce the amount of solvent vapor entering the flame to as low a level as possible and to make any droplets or particulates entering the flame as small and of as uniform a droplet size as possible. Desolvation chambers are designed to optimize these factors so as to maintain a near-constant efficiency of ionization and to flatten out fluctuations in droplet size from the nebulizer. Droplets of less than 10 pm in diameter are preferred. For flow rates of less than about 10 pl/min issuing from micro- or nanobore liquid chromatography columns, a desolvation chamber is unlikely to be needed. 

Liquid chromatography was performed on symmetry 5 p.m (100 X 4.6 mm i.d) column at 40°C. The mobile phase consisted of acetronitrile 0.043 M H PO (36 63, v/v) adjusted to pH 6.7 with 5 M NaOH and pumped at a flow rate of 1.2 ml/min. Detection of clarithromycin and azithromycin as an internal standard (I.S) was monitored on an electrochemical detector operated at a potential of 0.85 Volt. Each analysis required no longer than 14 min. Quantitation over the range of 0.05 - 5.0 p.g/ml was made by correlating peak area ratio of the dmg to that of the I.S versus concentration. A linear relationship was verified as indicated by a correlation coefficient, r, better than 0.999. 

Figure 12.8 Mia ocolumn size exclusion chromatogram of a styrene-aaylonitrile copolymer sample fractions ti ansfeired to the pyrolysis system are indicated 1-6. Conditions fused-silica column (50 cm X 250 p.m i.d.) packed with Zorbax PSM-1000 (7p.m 4f) eluent, THF flow rate, 2.0 p.L/min detector, Jasco Uvidec V at 220 nm injection size, 20 nL. Reprinted from Analytical Chemistry, 61, H. J. Cortes et al, Multidimensional chromatography using on-line microcolumn liquid chromatography and pyrolysis gas chromatography for polymer characterization , pp. 961 -965, copyright 1989, with peimission from the American Chemical Society.

Figure 13.9 Coupled-column RPLC-UV (215 nm) analysis of 100 p.1 of an extract of a spiked soil sample (fenpropimoiph, 0.052 mg Kg ). LC conditions C-1, 5 p.m Hypersil SAS (60 m X 4.6 mm i.d.) C-2, 5 p.m Hypersil ODS (150 m X 4.6 mm i.d.) M-1, acetonitrile-0.5 % ammonia in water (50 50, v/v) M-2, acetonitrile-0.5 % ammonia in water (90 10, v/v) flow-rate, 1 ml min clean-up volume, 5.9 ml transfer volume, 0.45 ml. The dashed line represents the cliromatogram obtained when using the two columns connected in series without column switcliing. Reprinted from Journal of Chromatography A, 703, E. A. Hogendoom and R van Zoonen, Coupled-column reversed-phase liquid cliromatography in envir onmental analysis , pp. 149-166, copyright 1995, with permission from Elsevier Science.

In HPLC, the mobile phase is a liquid delivered under high pressure (up to 400 bar (4 x 10 Pa)) to ensure a constant flow rate, and thus reproducible chromatography, while the stationary phase is packed into a column capable of withstanding the high pressures which are necessary. 

Figure 5.1 Pesticides included in the systematic investigations on APCI-MS signal response dependence on eluent flow rate the parameter IsTow represents the distribution coefficient of the pesticide between n-octanol and water. Reprinted from J. Chromatogr, A, 937, Asperger, A., Efer, 1., Koal, T. and Engewald, W., On the signal response of various pesticides in electrospray and atmospheric pressure chemical ionization depending on the flow rate of eluent applied in liquid chromatography-mass spectrometry , 65-72, Copyright (2001), with permission from Elsevier Science.

Figure 5.6 Positive-ion electrospray spectrum obtained from the major component in the LC-MS analysis of a purified recombinant 62 kDa protein using a Cig microbore 50 X 1 mm column and a flow rate of 50 p.lmin . The starting buffer (buffer A ) was 0.1% TEA in water, while the gradient buffer (buffer B ) consisted of 0.1% TEA in acetonitrile-water (9 1 vol/vol). The running conditions consisted of 0% B for 5 min, followed by a linear gradient of 100% B for 55 min. Reprinted from J. Chromatogr., B, 685, McAtee, C. P., Zhang, Y., Yarbough, P. O., Fuerst, T. R., Stone, K. L., Samander, S. and Williams, K. R., Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry , 91-104, Copyright (1996), with permission from Elsevier Science.

The stationary phase matrices used in classic column chromatography are spongy materials whose compress-ibihty hmits flow of the mobile phase. High-pressure liquid chromatography (HPLC) employs incompressible silica or alumina microbeads as the stationary phase and pressures of up to a few thousand psi. Incompressible matrices permit both high flow rates and enhanced resolution. HPLC can resolve complex mixtures of Upids or peptides whose properties differ only slightly. Reversed-phase HPLC exploits a hydrophobic stationary phase of... 

The concentration of biodiesel (fetty acid methyl esters) and glycerides were analyzed by liquid chromatography (Shimadzu-lOA HPLC). An ODS-2 column (250x4.6mm) was used for the separation. The flow rate of the mobile phase (acetone acetonitrile=l l) was set to 1 ml/min. Peaks were identified by comparison with reference standards. Standards of methyl esters, monoglycerides, digjycerides and triglycerides were bought from Fluka. 

Analyses were performed by gel permeation chromatography (GPC) and by high performance liquid chromatography (HPLC). Gel filtration of oligosaccharides was effected in a thermostated (65 <>C) column (210 X 1.5 i.d.) filled with polycrylamide gel (Bio-gel P2, 200-400 mesh Bio Rad-USA), using stilled water as eluent (flow rate 30 ml.h-1). 

Heat and reflux a 5-g portion of soil sample with 50 mL of methanol-phosphate buffer (pH 7)-water (15 7 28, v/v/v) solvent mixture in a round-bottom flask for 1 h. After cooling, transfer a 10-mL portion of the supernatant to a test-tube and mix with 11 mL of 0.02M H3PO4 solution. Load this solution on to a silica-based SPE cartridge (Analytichem International Clin-Elut 1020) at a flow rate of 1-2 drops per second. Discard this fraction. Elute the analytes with 30 mL of dichloromethane. Concentrate the eluate to dryness with air in a water-bath at a temperature of 40 °C (do not use vacuum). Dissolve the residues in 5mL of HPLC injection solution [900 mL of water - - 50 mL of phosphate buffer (pH 7) 4-50 mL of ACN 4-4 g of TBABr]. Pinal analysis is performed using liquid chromatography/ultraviolet detection (LC/UV) with a three-column switching system. 

The viscosities of supercritical fluids are intermediate between those of a gas and a liquid, but this tine, are much closer to those of a gas than a liquid. For a fixed column pressure drop much longer columns or higher flow rates are possible in SFC compared to liquid chromatography. Liquids, however, are virtually incompressible while gases and... 

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