Maintaining Linear Velocity (HPLC)

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Calculating the Appropriate Flow Rates for Columns of Differing Diameters or "Maintaining Linear Velocity"

An IonSource.Com mini-tutorial by Andrew Guzzetta
Introduction (flow rate, pressure and chromatography) The standard size HPLC column (4.6 X 250 mm) is run at a flow rate of 1 mL/min. 4.6 refers to the internal diameter and 250 refers to the length of the HPLC column in millimeters. The limiting factors in choosing a flow rate are, instrument pressure limitations, the effect on the quality of the chromatography, and time. Most HPLC's operate in the pressure range between 30 and 200 bar. Maintaining linear velocity is the single most important factor when trying to reproduce a chromatographic separation on columns of differing diameters. Since flow rate directly impacts HPLC system pressure a discussion of parameters that affect system pressure are listed below. Factors that affect system pressure: Column Dimension Shorter, fatter columns produce lower system pressures, which allows them to be run at higher flow rates. Often the chromatographer will exploit this and run short fat columns at higher flow rates to speed up analysis times. HPLC peaks do not always get narrower with increased flow rate. At faster flow rates the analyte may have insufficient time to interact with the stationary phase. Stationary Phase Particle Size Stationary phase particles range in size from 2 to 25 microns. The most common particle size is 5 micron. In general the larger the particle the lower the system pressure for a given flow rate. Smaller particles generally provide greater surface area and better separation but produce higher system pressures. Temperature System pressure is affected by temperature. The viscosity of the mobile phase will decrease with increasing temperature. For example if the HPLC system pressure is too high for a given solvent system the chromatographer may choose to raise the temperature of the column compartment to 40°C or even 60°C. In terms of trouble shooting high system pressure, adjusting temperature in not usually the first factor to consider. Often an increase in back pressure will be a sign of column plugging or fouling. Flow Rate It is not difficult to imagine that system pressure is affected by flow rate. Flow rate impacts HPLC system pressure, chromatographic quality, and analysis time. One must choose a flow rate that is appropriate for the HPLC system and column. A higher than usual flow rate may adversely affect the quality of the chromatography not giving the analyte sufficient time to interact with the stationary phase. Faster isn't always better. A lower than usual flow rate may leave the analyst waiting for the peak to appear at the detector.
"Maintaining Linear Velocity"
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Maintaining mobile phase linear velocity is important when attempting to reproduce chromatography obtained on columns of differing internal diameters. For example to maintain mobile phase linear velocity from the standard column mentioned above, 4.6 X 250 mm, to a small bore column with the dimensions 2.1 X 250 mm one must adjust the flow rate downward by a factor. When going from a 4.6 to a 2.1mm ID or to any smaller ID column decrease the flow rate by the square of the ratios of the column diameters. As mentioned above the standard flow rate for a 4.6 mm column is 1 ml/min.

In our example the new flow rate determined for the 2.1 mm column is 0.208 ml/min. Commonly 2.1 mm columns are run at a flow rate of 200 µl/min. It is important to note that sometimes adjustments in mobile phase flow rates from the standard flow rate can have minor beneficial effects in chromatography. Sometimes flowing a column at one half the normal flow rate will have a beneficial effect on a chromatographic separation.
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Good Luck. Happy Chromatography.
Sincerely,

Andrew Guzzetta
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