I would like to know the must references (manuscripts or book chapters) for learning the fundamentals theory and practical calculation for determining flow rate for column during prepartative HPLC. I perform preparative HPLC for drug metabolites purification, and based on the reaction scale I use different length and diameter column e.g. 100*3, 250*4.6 and 250*10 mm etc. And I generally collect 200 to 1000 ul fractions per minute. Recently, I am planning to do a run with 250*4.6 mm column with a 350 uL/min flow rate as I am planning to collect the fractions in a 96 well plate for convenience of the following experiments. But it does not look quite right to me as the column is bigger and ideally I think it requires a faster flow rate. Then I have to switch to regular test tubes for collecting fractions which I did not want but can certainly do if needed. Considering all these, I would like to know about some must read references for learning the fundamentals better.
You will find several useful related articles at this link. Please note the differences between flow rate and linear velocity. Flow rate is adjusted to maintain it (ideally):
"Flow / Rate / Linear" (search):
https://hplctips.blogspot.com/search?q=linear+flow+rate
"Number of theoretical plates (N), Calculation Formulas"; https://hplctips.blogspot.com/2018/06/number-of-theoretical-plates-n.html
Quick comments (sorry, I do not have time to quote book chapters, but this is basic info which can be found in any of the classical books on LC):
(1) Column diameter has the largest effect on chosen flow rate (not length) as it plays a large role in peak dispersion and peak volume. Particle size becomes less and less important for efficiency as the particles get smaller in diameter.
(2) You can cheat with estimates. Flow rate is mainly effected by column diameter. For example, a 4.6 mm diameter column will have a linear flow rate just above 1 mL/min (so we use 1 mL/min). A 2.1 mm ID column will have a linear velocity/flow rate of around 208 ul/min. A 10 mm ID column ~ 5 ml/min. The optimum flow rate for any column can be calculated to determine an estimate, but then should be determined by running samples and analyzing the plate numbers and separation efficiency to find the flow rate which is applicable to your actual method.
(2) It does not make any sense to use a 4.6 x 250mm column at 350 ul/min. There is no advantage in using a column that large at 1/3 the optimum flow rate. Such a low flow rate will increase diffusion/dispersion and take 3 times longer than needed with no benefit of any kind. *Select a column for a project based on the actual application needs and parameters.
(3) Prep HPLC is all about FIRST learning how to run analytical scale columns and methods very well. Be sure to learn and understand all of the good fundamentals of liquid chromatography FIRST before moving to prep LC. It is critical that you understand the relationships of flow rate, column diameter, peak volume, separation efficiency, K prime and delay volume. Without command of these fundamentals, you will waste huge amounts of expensive solvents (and samples) trying to develop methods on Prep columns.
(4) We start Prep HPLC method development by first developing it on an analytical scale column to optimize the parameters. Scale up for high pressure LC columns with diameters up to 50 mm is straightforward. Ideally, the analytical and Prep columns would be packed with identical supports types. You must take into account differences in delay volumes, injection volumes, dispersion, particle size (if it is different) and even the heating or cooling effects present with large volumes of solvent vs a few mls. The analytical method should allow for losses in performance when converting. Once the analytical method is fine tuned, then it can be scaled up and tried.
Hi Navid,
maybe the following links are helpful to you:
https://www.sigmaaldrich.com/analytical-chromatography/hplc/method-transfer-calculator.html
https://www.agilent.com/en/products/liquid-chromatography/lccalcweb
https://www.phenomenex.com/Tools/preplc-scaleup
http://www.waters.com/waters/promotionDetail.htm?id=134713448&alias=Alias_prepcalculator_CHEMISTRY&locale=de_CH
https://epgl.unige.ch/labs/fanal/hplc_calculator:en
Regards
Markus
P.S.: Optimum flow rate depends on several factors. In our lab for example with use focussed or narrow gradients instead of a generic gradient. The desired peaks elute in a range of 3-5 (retention factor). For each compound the optimum narrow gradient is determined by a pre-analytic run. The retention time in this run is proportional to the respective narrow gradient. Used flow rate with 2 columns in a row (dimension 30 mm x 100 mm) is 50 mL at a column temperature of 60°C. Pump pressure under these conditions is in the range of 1500 -2000 psi. Max. injection volume is 6 mL. During the injection procedure we use at-column dilution.
You will find several useful related articles at this link. Please note the differences between flow rate and linear velocity. Flow rate is adjusted to maintain it (ideally):
"Flow / Rate / Linear" (search):
https://hplctips.blogspot.com/search?q=linear+flow+rate
"Number of theoretical plates (N), Calculation Formulas"; https://hplctips.blogspot.com/2018/06/number-of-theoretical-plates-n.html
Quick comments (sorry, I do not have time to quote book chapters, but this is basic info which can be found in any of the classical books on LC):
(1) Column diameter has the largest effect on chosen flow rate (not length) as it plays a large role in peak dispersion and peak volume. Particle size becomes less and less important for efficiency as the particles get smaller in diameter.
(2) You can cheat with estimates. Flow rate is mainly effected by column diameter. For example, a 4.6 mm diameter column will have a linear flow rate just above 1 mL/min (so we use 1 mL/min). A 2.1 mm ID column will have a linear velocity/flow rate of around 208 ul/min. A 10 mm ID column ~ 5 ml/min. The optimum flow rate for any column can be calculated to determine an estimate, but then should be determined by running samples and analyzing the plate numbers and separation efficiency to find the flow rate which is applicable to your actual method.
(2) It does not make any sense to use a 4.6 x 250mm column at 350 ul/min. There is no advantage in using a column that large at 1/3 the optimum flow rate. Such a low flow rate will increase diffusion/dispersion and take 3 times longer than needed with no benefit of any kind. *Select a column for a project based on the actual application needs and parameters.
(3) Prep HPLC is all about FIRST learning how to run analytical scale columns and methods very well. Be sure to learn and understand all of the good fundamentals of liquid chromatography FIRST before moving to prep LC. It is critical that you understand the relationships of flow rate, column diameter, peak volume, separation efficiency, K prime and delay volume. Without command of these fundamentals, you will waste huge amounts of expensive solvents (and samples) trying to develop methods on Prep columns.
(4) We start Prep HPLC method development by first developing it on an analytical scale column to optimize the parameters. Scale up for high pressure LC columns with diameters up to 50 mm is straightforward. Ideally, the analytical and Prep columns would be packed with identical supports types. You must take into account differences in delay volumes, injection volumes, dispersion, particle size (if it is different) and even the heating or cooling effects present with large volumes of solvent vs a few mls. The analytical method should allow for losses in performance when converting. Once the analytical method is fine tuned, then it can be scaled up and tried.
Thank you all for taking time out to share your valuable advises and important references. I am grateful to all of you for your valuable input. Thanks once again.
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