I have been struggling trying to create a standard curve with cisplatin. I have been using the method attached in the article below. Article Development and Validation of a Simple and Sensitive Spectro...
Here is the method I have been following...
1. 10 mg cisplatin HCl was dissolved in 5 mM phosphate buffer pH=6.8 in a 100 mL standard volumetric flask and diluted using phosphate buffer to make 100 ug/ml cisplatin stock solution.
2. 10 ug/ml cisplatin solution was made by diluting 10 mL of stock solution in 100 mL volumetric flask using phosphate buffer.
3. from 10 ug/ml cisplatin solution, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 mL aliquots were withdrawn and placed into 10 mL standard volumetric flask.
4. 1 mL of 1.4 mg/ml OPDA solution (made in DMF pH=6.4 adjusted with 0.1 N HCl) was added into each solution.
5. 2 mL of phosphate buffer was added to each solution
6. the solutions were heated on a hot plate at 100 C for 10 minutes
7. solutions were cooled to room temperature
8. solutions were diluted with DMF
9. absorbance was measured at 706 nm using DMF as blank (750 baseline correction).
I am really not sure what I am doing wrong. I believe I am following the protocol correctly but when I measure the absorbances they are all reading low and relatively the same value. What might I be doing wrong?
My possible guesses....
1. cisplatin stock solution is not fully dissolved and should be heated?
2. the standards should be heated longer than 10 mins.
3. using a hot plate is incorrect?
4. temperature is actually lower than expected because I am heating all the standards together at one time?
thank you in advance for your help and suggestions
Please look at the following below attached file which may help you in your analysis.
Thanks
If you are using my method (as I understand, you are using some other method) and will draw a graph via standard addition, you should dissolve cisplatin in 0.15M KCl. Phosphate buffer might decrease the epsilon of the complex in the spectrum, because of increase of the ionic strength. I was experiencing problems in the complexation at high ionic strength. The optimum conditions found in my study for complexation was 90oC and 30 min and pH between 6.1-6.2. pH is critical , you should stir for a while and control 2-3 times after stirring, each urine obtained at different times, from different people will have different pH varying between 4-8. Don't use phosphate buffer try with acetic acid. It will take more time to adjust pH but you should know that there are ions in urine that you can utilize their buffer feature of course in a certain buffer capacity limits.
And one more thing you should use waterbath not hotplate, and be very quick because the complex decomposes after a while. You should keep all solutions while preparing standard curve in the dark don't let them stay too much in the day light.
Here is my work: I believe that it will help you.
If you try 0.15 M KCl, it might help your solution to be stable for a longer time.
Why adjust the pH of OPDA before mixing? Why not after mixing?
The solutions were diluted with DMF or acidified DMF?
Why adjust pH with HCl? Why not use weak acid not to miss the pH value?
Do you have a pH meter for organic solvents? Why not make easier with addition of some water to the organic solvent as I have performed in my study?
I would very much like to hear your response if you could increase the absorbances.
Antonia Marashio Hello, I am facing the same issue , could you please tell me how did you solve the problem ?
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