Hello,
I have made a concentration calibration curve on HPLC for Paclitaxel in acetonitrile.
I have then done a dialysis release of paclitaxel from nanoparticles.
However, when I calculate the release amount via the calibration curve it is coming to amounts higher than what I should have. e.g. I have a pax concentration of 80ug/mL in the nanoparticles and am getting concentrations of 100ug/mL... (should be less than 80ug/mL)
Would anyone have any ideas why this is occuring?
I am using ACN90% H2O10% mobile phase, 1ml/min.
I have a 3min elution time, with a Restek C18 5um 250x4.6mm column.
Thankyou.
I havnt run a dialysis control without PAX so will try that.
I so far just did a concentration profile with increasing amounts of known PAX.
I havnt heard of void volume before, so I will also try dropping ACN as well.
Thank you!
Yes, your HPLC method is invalid, you have no retention and have no method yet (see below for links to free articles to help you). Training in liquid chromatography fundamentals is required to make use of this valuable analytical method/technique. Without training, you will handicap your project by using unscientific methods to support your work.
To solve the problem you will need to develop a proper HPLC method which retains, holds, then elutes the compound of interest on the column. The method should be selective for the compound of interest, show specificity for it and be able to resolve any relevant compounds away from it (needed for any quantitative work).
Two fundamental HPLC concepts you should become acquainted with are (links provided for more info):
- "Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero)"; https://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html
- "K Prime (also known as: Capacity Factor, Ratio or Retention Factor): One of the Single Most Important HPLC Parameters of All"; https://hplctips.blogspot.com/2015/06/k-prime-also-known-as-capacity-factor.html
The above two fundamental concepts are part of any good HPLC training course and needed to begin planning any use of or work with an HPLC system (if it does not include them, then the course may in fact just be a sales talk, not a training class). These key concepts are extremely simple and also extremely important. Once you learn them see if you can find someone at your school who has practical experience in using HPLC to solve problems who can help you. This is a technique best learned locally with one or more people to guide you.
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