Column chromatography flow rate

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... 

In most inorganic chromatography, resins of 100 to 200 mesh size are suitable difficult separations may require 200 to 400 mesh resins. A flow rate of 1 mL cm min is often satisfactory. With HPEC columns, the flow rate in long columns of fine adsorbent can be increased by applying pressure. 

Fig. 16. Separation of cytochrome c (1), myoglobin (2), and chicken egg albumin (3) by re-versed-phase chromatography on a monolithic poly(styrene-co-divinylbenzene) column at flow rates of a 5 ml/min b 25 ml/min. (Reprinted with permission from [53]. Copyright 1996 American Chemical Society). Conditions column 50 mmx8 mm i.d., mobile phase linear gradient from 20 to 60% acetonitrile in water... 

The amount of resin to pack in a column, column geometry, flow rates, pressure, column hardware, and wetted materials of construction should all be evaluated in development. Chromatography columns must be properly packed prior to validating the purification process. From a business perspective there should be some criteria other than purification of the product by which the quality of the packed column can be assessed prior to applying the feedstream, which by this time in the process is quite expensive. Height equivalent to a theoretical plate (HETP) and asymmetry determinations can be used to evaluate the quality of column packing, but may have limited value for some types of packed columns... 

FIGURE 3. Steric exclusion chromatography of water from waste trenches and inert atmosphere wells at Maxey Flats, sampled July, 1982. Water samples from waste trenches 27 and 19S (440 ml) and wells WIN (585 ml) and W2NA (238.5 ml) were concentrated to 10 ml, pH-adjusted to their original values and chromatographed on a Sephadex G-15 column at flow rates ranging from 28-33 ml/hr. Individual column fractions were collected every 10 min and analyzed for specific organic compounds and radionuclides. 

Optimize Chromatography (column, solvents, flow rate, gradient) using a solution of the analyte in mobile phase... 

FIGURE 8 Separation of rabbit polyclonal antibodies by ion-exchange chromatography on DEAE Trisacryl M. Column dimensions 16 mm i.d.X 100 mm initial buffer 50 mM Tris-HCI, 0.035 M sodium chloride, pH 8.8 load 5 mL of rabbit serum previously precipitated with ammonium sulfate at 50% saturation and redissolved in column buffer flow rate 50 mL/hr elution of adsorbed protein performed using I M sodium chloride solution in the initial Tris buffer. The first peak represents IgG the second peak is composed of all other serum proteins precipitated by ammonium sulfate. The straight line is absorbance at 280 nm, and the broken line represents the variation of ionic strength of the buffer. The purity of IgG estimated by gel electrophoresis was over 98% and the calculated yield was over 90%. 

Gas chromatography requires that samples remain stable when volatilized in a stream of helium, first in the injector used to introduce the analytes onto the GC column, and then during the time that the sample components traverse the column as it is heated progressively inside an oven. The requiranent for volatility means that only nonpolar or moderately polar compounds can be analyzed by GC-MS. For capillary GC columns the flow rate of the gas that moves compounds through the column (the carrier gas) is low, about 1 ml/min. Such a quantity of gas can be introduced into the ion source directly, without compromising the vacuum in the instrument. This simplifies the interfacing of GC with MS so that a heated interface tube can be used to link the two instruments and through which the GC column is run so that it abuts the ion source. 

Fig. 2. Gel exclusion chromatography of chro-matophore preparations on a Sepharose 2B column. Chromatography was performed essentially as described by Fraker and Kaplan (18). A 0.1 ml sample was loaded onto a 1.7 x 35 cm Sepharose 2B column the flow rate was 11 ml hr Sodium phosphate buffer (100 mM), pH 7.5, containing lOmM EDTA and 0.02% soium azide was used for both equilibration and elution. The effluent was passed through an LKB 2138 Uvicord S detector to...

The column must be equilibrated, re-equilibrated to the initial high aqueous solvent composition before another analysis can be performed. Normally this re-equilibration is included at the end of the gradient. How much equilibration time is enough In reality, it depends on the column length, flow rate, and the hydrophobicity of the sample peptides. Some chromatographers use 10 min as the standard equilibration time. Equilibration is all about fitness of purpose. One should determine the equilibration time experimentally the criteria should be, does the analyte really stick to the column and chromatograph appropriately and reproducibly for subsequent analyses. If this part of the method development is taken care of, it will undoubtedly be rewarded with improved chromatography and better cycle time. 

Fig. 4. Chromatogram of a mixture of retinyl ester standards. Column, Supelcosil LC-8 (5 jLm) mobile phase, acetonitrile water (88 12) from origin to arrow and acetonitrile water (98 2) for rest of chromatography flow rate, 3 ml/min. The esters of retinol are 1, acetate, 2 0 2, laurate, 12 0 3, 7-linolenate, 18 3 4, myristate, 14 0, 5, palmitoleate, 16 1 6, linoleate, 18 2 7, palmitate, 16 0 8, oleate, 18 1 9, stearate, 18 0 10, arachidonate, 20 4. (Reprinted with permission from Ross, 1981.)...

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. 

In general, the longer a chromatographic column, the better will be the separation of mixture components. In modem gas chromatography, columns are usually made from quartz and tend to be very long (coiled), often 10-50 m, and narrow (0.1-1.0 mm, internal diameter) — hence their common name of capillary columns. The stationary phase is coated very thinly on the whole length of the inside wall of the capillary column. Typically, the mobile gas phase flows over the stationary phase in the column at a rate of about 1-2 ml/min. 

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. 

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