Dear all, i had made few times the krebs-HEPES solution. And i found that the solution tend to be cloudy with precipitate. expecially when i add in CaCl₂.2 H₂0, after the sodium HEPES. I tried turn over, which means that i add in CaCl₂.2H₂0 ,then i add in sodium HEPES. But it's still turn to cloudy solution. I would like to ask that is it very important that the order to put in the chemicals? and if i continue to use the cloudy solution, will it affect my assay? for you information, the solution is clear before i add in the calcium cloride ot the sodium HEPES, and i also add very slowly for these two chemicals....thanks!

Do you adjust the pH value? You can try to add the CaCl2 after you "finished" your buffer (which includes the pH). Or it may help to add a CaCl2 solution after sterilizing (autoclaving) the buffer.

Does the buffer contain a carbonate buffer?

yes....i did checked the pH values today. It's about pH9.0, and when i add in the HCl, the cloudy solution turn to clear already and the pH is about pH 7.48. thanks to remind me the pH values! ()

 

 

Do you adjust the pH value? You can try to add the CaCl2 after you "finished" your buffer (which includes the pH). Or it may help to add a CaCl2 solution after sterilizing (autoclaving) the buffer.

 

Thanks for reply, i solve the problem right now, as the sodium HEPES is a salt which will increase the pH values. It's go down when i add in HCl (and the cloudy solution turn to clear now..hehe...). However, I did add sodium bicarbonate, i would like to ask that will it affect the pH values also? Thanks!

 

Does the buffer contain a carbonate buffer?

 

pH and temperature can "clean" solutions, if they are in the correct values.

 

yes....i did checked the pH values today. It's about pH9.0, and when i add in the HCl, the cloudy solution turn to clear already and the pH is about pH 7.48. thanks to remind me the pH values! ()

 

 

 

 

Thanks for reply, i solve the problem right now, as the sodium HEPES is a salt which will increase the pH values. It's go down when i add in HCl (and the cloudy solution turn to clear now..hehe...). However, I did add sodium bicarbonate, i would like to ask that will it affect the pH values also? Thanks!

 

 

Bicarbonate is also a buffer, however heating it (e.g. during autoclaving) changes the equilibrium and it will get lost (in form of CO2). This can affect your final pH, depending on the concentration.

oic....Thank you for you all kindly explanation. Hopefully that i can do my assay well. Thanks!