Conversion Tables, Tools & Useful Links
Our aim at Rubicon Science is to help you with all aspects of liquid chromatography. This page includes useful tables and links to help you in your work.
Scaling up in chromatography using Linear Flow Rate
Scale-up is often an issue with protein purification. Preliminary runs are performed on small columns and then often larger amounts of product is purified on larger columns.
Because protein purification is usually performed in volume mode and not time, when scaling up it is easier to calculate the extra flow rate required for the larger column using Linear Flow Rate, at cm/hour. This takes into account the increase in the cross-sectional area of the larger column so that it will have identical flow characteristics of the small column. Attached are 2 conversion tables for the finding of Linear Flow Rate from Volumetric Flow Rate and the other way around.
Use these two tables to help you convert Volumetric Flow Rate to Linear Flow Rate and vice versa.
Scaling up from Analytical to Preparative HPLC columns
When scaling up HPLC columns from analytical to preparative, multiple variables need to be taken into account. These might include changes in column length, column internal diameter, particle size increase, flow rate increase etc.
The Knauer ScaleUp Converter is a free software tool that automatically calculates the scale-up parameters when moving an analytical-scale separation method up to a preparative one.
Go to the Knauer page (and scroll down) to download this free software tool. : https://www.knauer.net/en/knauer-scaleup-converter/p14082
Example scale-up of an analytical HPLC method for steviol glycosides to a preparative method
Calculating the linear flow rate for CIM® monoliths
Linear flow rate calculations are commonly used with traditional chromatography resins to calculate the residence times of analytes prior to scaling up. Monolithic columns are not affected by diffusion limitations, hence residence time calculations become redundant when scaling up.
With CIMmultus™ and CIM® Tube monolith columns the linear flow rate can be calculated with the following equation:
Where F is the flow rate in mL/min, L is the length of the monolith, Do and Di are the outer and inner diameter of the column.
For more details on calculating linear flow rates for CIM monoliths take a look at the BIA Separations web site at https://www.biaseparations.com/en/technology/calculating-linear-flow-rate-for-cimr-monoliths
Table of capillary tube diameters for calculating delay volumes
In order to make sure that chromatography peaks are collected accurately, it is important to know the delay volume of the capillary tube between the monitor and the fraction collector. This is known as the time or volume delay. This can be calculated by knowing the internal diameter of the tubing and its length.
The table below shows the internal diameters of typical capillary tubing in HPLC and FPLC. Select which is the one you are using, and then note the tube volume μl/cm that applies to it. Then multiply by the length of the tubing in cm to get the total volume of the tubing in μl. For example a capillary tube with an internal diameter of 0.5 mm, that is 60 cm long, will have a volume of 117.6 μl.