I tried to make HEPES buffer solution by using C1V1 = C2V2 formula. But the prepared buffer isn't okay. could anyone please suggest me how to prepare 50mM 10mL HEPES buffer from 1M HEPES buffer solution?
Hi there,
Instead of using a formula you don't get, calculate the dilution factor as it doesn't require any volume data at first! Demonstration: You have a 1M stock and you want a 50mM solution (0.05M) so the dilution factor is (1M/0.05M)=20. So you need to dilute the stock solution 20 times. How to dilute 20x? It's simple: take one given volume of the stock and add 19 volumes of diluent! In your case, you need 10mL then these 10mL are representing the 20 volumes of the final dilution which means 1 volume is actually 10/20=0.5mL. So mix 9.5mL of diluent with 0.5mL of the stock and that's it!!!
In a more general context, including any value for stock and final solutions:
Dilution factor DF is the ratio stock/final=DF. The preparation of the dilution consists in mixing (DF-1)volumes of diluent with one volume of the stock.
Hi Afsana, go ahead with same C1V1=C2V2.
C1 - 1M or 1000mM
V1 - ?
C2 - 50mM
V2 - 10mL
V1 = (C2×V2)/C2
= (50mM×10mL)/1000mM
= 500/1000 mL
= 0.5 mL or 500uL
Mean you need to take 500uL of 1M HEPES stock + rest volume will be water (9.5 mL). Final volume will be 10mL.
Adam B Shapiro after adding buffer there should a color change of my nanoparticle solution. But in my case there is no color change. But when my senior make the same buffer, and add, the solution changes its color. I don't know what procedure he is following, that's the problem.
Adam B Shapiro Thanks as I've read your previous comments on another page. My simple question is can the diluent be just ddH2O, as I reduced HEPES conc from 1M with pH 7.4 to 25mM. I plan to use exact pH of 7.4 for cell treatment, so I'm wondering if the pH of ddH20 can be negligible since it is 7.0. Alternatively, do you recommend tipping up the pH with NaOH?
You can dilute the 1 M HEPES pH 7.4 to 25 mM with distilled water. The pH of distilled water is not necessarily neutral, but it has no buffering capacity, so its effect on the pH is minimal. However, dilution of the stock solution can have a measurable effect on the pH because the concentration can affect the pKa of the HEPES. Therefore, you may want to check the pH of the diluted solution and adjust the pH accordingly if it is too far from where you need it to be.
Adam B Shapiro Thank you, I checked the pH after dilution and itwas ok.
The molecular weight of HEPES is 238.3. So to make a 50 mM solution, add 0.12 g of HEPES to deionized water. Adjust the final volume of the solution to 10 mL with deionized water. Adjust the pH as needed, or take 0.5 ml from your 1M solution and add 9.5 ml of deionized water to make the final volume of 10ml. Your final concentration will be 50mM
- Badges
- Science method