I was looking through quantification of Vancomycin using UV absorbance spectrum and I came across multiple papers that uses wavelength around 215-240 nm for UV detector in HPLC whereas the most common one used for UV-vis is at 280 nm.
When I checked Vanco's UV at 215-240, I found it to be absurdly high compared to the peak at 280 nm, which can make quantification a lot different.
Is it because HPLC allows for a clearer separation? I also notice that many organic products also have peaks around 200-250 which can interferes with measurement at 215-240 nm using UV-Vis so I can see why people uses 280 nm. If that is the case, are quantity reported using UV-vis at 280 nm viable?
As long as you know the correct extinction coefficient at 280 nm in the solvent you are using, you can use 280 nm absorbance to calculate the concentration. However, that only tells you the concentration of the UV chromophore plus any contaminants that might also absorb in the UV. Employing a separation method such as HPLC, and using a standard comparator, will increase your confidence in the result.
There are many factors which must be considered:
1. Solvent
2. Brand of spectrophotometer used
3. Instruments are caliberted or not
4. Good laboratory practices are followed or not.
5. Glass wares are caliberted or not.
6. Date of manufacture and expiry and grade of chemical used etc.
I see. From what I understand - ignoring other possible factors - with a proper standard curve, the usage of 280 nm is acceptable. It is not the best option but I must proceed with it as HPLC isn't available right now.
Thank you for your answer.
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