Dear all today I faced a problem while making mobile phase and the composition was A)potassium Dihydrogen phosphate(15g) + Di-sodium hydrogen phosphate(15g) in 1000ml water
B) Acetonitrile 300ml+methanol 300ml------
Final volume is buffer A is 400ml+solvent mix 600ml----...while mixing this composition the mobile phase forms precipitate.(turns turbid).i added buffer to ACN:MeOH mixtur and tried vice versa too..why this happen and what shall I do to make clear mobile phase.
the C of buffer should be 0,005M-0,05M always, if the peak is tailing u should modify pH then.
There is one more thing, maybe is not so important, but u need to use the HPLC or gradient grade (if it exist) of salts, to be sure that the salts are purified 100%
After sonnication pass the mobile phase through solvent filter 0.45u using Millipore assembly.
use the 0.45 micron filter for degassing and also add 0.1% sodium azide that will solve the problem of turbidity
Dear robin n rohit i had filtered mobile phase through 0.45u filter paper but salts ppt out.and @ rohit i will add 0.1% sodium azide and will let u know the final outcome
Dear jagadish, decrease the conc of buffer in term of mM neither it wil harm ur column and use proper ratio of buffer : ACN
Yes the problem is the concentrations of salts in buffer,
decrease the C to 0,05-0,025 and less, to 0,005M
or try to decrease the C of organic component
dear all i tried all whatever suggested me here but the fact is that today i added more buffer to my system that is
in previous mobile phase composition was buffer A is 400ml+solvent mix 600ml(ACN:MeOH::1:1) this system precipitates...but today i tried 600ml buffer and 400ml solvent mix(ACN:MeOH 1:1) this system didnt precipitate after increasing the buffer concentration but the peak tailing observed...if I increased buffer concentration then also mobile phase is clear now...wy this happend?
the C of buffer should be 0,005M-0,05M always, if the peak is tailing u should modify pH then.
There is one more thing, maybe is not so important, but u need to use the HPLC or gradient grade (if it exist) of salts, to be sure that the salts are purified 100%
Dear Munvar and Bruce thanks for guidelines...munvar I have not used that buffered mobile phase...I tried different that is ACN and water combination.and i have resolved the problem of peak tailing.
thanks for guidelines...but im not expert in chromatography,hope you all are here to resolve ma probles..heartly thanks to everyone.
dear osmani sir,
thanks for reply
sir i haven't used that mobile phase. after preparation of mobile phase i had sonicated as well as filtered...but was not getting filtered easily...so i left that system and developed new one.
but i have new question while developing the method for clioquinol, the correction of peak tailing was quite difficult.
and mobile phase is 98%ACN.,column C18-150x4.6mm,5um.
chemical structure shows prasence of Cl,I & N groups which may form complex with stationary phase..and give peak tailing..its ma opinion.
so what is the solution for correction of peak tailing?in this case???
hi, I would like to ask does the potassium phosphate buffer solution would affect the retention time of the standard peak?
I am doing standard folic acid. I have found the standard peak of folic acid (100ppm) wby using mobile phase of acetronitrile and potassium phosphate buffer (ph 2.3). The folic acid was dissolve in potassium phosphate buffer (ph 7.4).
I am using gradient elution. The first 5 min is 6% acetronitrile isocratic, then increase linearly to 25% acetronitrile within 20min. and the standard peak is come out at 22min.
But, the problem is when I doing serial dilution to calibrate standard curve. It was found that the retention time of the standard serial dilution is different from the stock solution (100ppm).
May I know the reason?
Hi, at first you can exert filtration after mixing the organic solvents with your buffer. secondly, your buffer is susceptible to precipitation due to the lower polarity of mixed solvents which results in turbidity. therefore you can ,and should, run your mobile phase through your column at the same day of preparation.
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