After collecting supernatant which pore size filter is used to filter the supernatant? After filtration do we take its UV absorbance or to do anything in between? After taking its absorbance what to do next?
I agree with Rafiq Karaman, you should construct a callibration curve.
For this purpose, you will make a number of dilution of known concentration and take their absorbance in UV-Vis spectrophotometer. Plot a graph in MS Excel between concentrations (x-axis) and their absorbances (y-axis). Then, you can take trend line to see if calibration curve is predictable (R2 value is near to 1 i.e. 0.99) and show equation of graph.
In this equation, you will need to replace "y" with value of your absorbance of supernatant and you will find the the of "x", the concentration of drug in sample.
You can collect supernatant by centrifugation or by use of dialysis tube. Dialysis tube of differetn Molecular Weight Cut-Off are available that can retain nanoparticles but un-encapsulated molecules will come out of tube membrane due to smaller size. Usually we formulate drug loaded nanoparticle so that drug is released at desired rate. One disadvantage of using dialysis tube or similar membrane systems is that they need significant time for separation. During this time, a significant amount of drug is release which was actually encapsulated inside nanoparticles. Thus, you might find lower encapsulation than actual. Centrifugation may be less efficient in some cases but need relatively lower time.
Is this information helpful?
Regards, Mubashar Rehman
Dear Pharmacist,
You should use membranes with pore size of 0.2 micrometer. There are many types for such membranes: NIOSH-approved N95 and P100 and CE-marked FFP2 and FFP3 FFRs (Ann Occup Hyg (2009) 53 (2): 117-128. doi: 10.1093/annhyg/men086) and others such as 0.2um nylon membrane filter or even ultrafiltration membranes with smaller pore size (about 0.2um). After filtration, you can run UV-vis for the drug or other analysis techniques such LC-MS and HPLC.
Hoping this will be helpful,
Rafik
From UV-vis I will get absorbance but how to find drug concentration?
Thanks for the useful answers.
You have to make a calibration curve for a standard and then calculate the sample's concentration using the following equation:
Absorbance (sample)/absorbance (standard x standard concentration.
Good luck,
Rafik
Hi Pharmacist, one of the best and most sensitive methods to measure drug concentration in the supernatant is by ICP (inductively coupled plasma). In this way you don't have to concentrate your supernatant! Good luck!
I agree with Rafiq Karaman, you should construct a callibration curve.
For this purpose, you will make a number of dilution of known concentration and take their absorbance in UV-Vis spectrophotometer. Plot a graph in MS Excel between concentrations (x-axis) and their absorbances (y-axis). Then, you can take trend line to see if calibration curve is predictable (R2 value is near to 1 i.e. 0.99) and show equation of graph.
In this equation, you will need to replace "y" with value of your absorbance of supernatant and you will find the the of "x", the concentration of drug in sample.
You can collect supernatant by centrifugation or by use of dialysis tube. Dialysis tube of differetn Molecular Weight Cut-Off are available that can retain nanoparticles but un-encapsulated molecules will come out of tube membrane due to smaller size. Usually we formulate drug loaded nanoparticle so that drug is released at desired rate. One disadvantage of using dialysis tube or similar membrane systems is that they need significant time for separation. During this time, a significant amount of drug is release which was actually encapsulated inside nanoparticles. Thus, you might find lower encapsulation than actual. Centrifugation may be less efficient in some cases but need relatively lower time.
Is this information helpful?
Regards, Mubashar Rehman
By plotting a calibration graph you can find the concentration of unknown.
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