buffer - (Jan/29/2009 )

hello,
how should i prepare buffer with 150mM NaCl, 100mM TrisCl, 1mM EDTA at pH 8 in 1L all together. plzz im new at this...

msaifkhusaini on Jan 29 2009, 08:00 PM said:

I believe the best way to go about this is to prepare stock solution. Prepare a stock solution
1M Tris-HCl solution at pH8.
500mM EDTA at pH8

Basically weight out enough Tris to make 500ml solution at 1M. Add miliQ grade water until you reach a volume of 400ml. Then gradually add concentrated HCl. Use a pH meter to monitor the pH. Once pH 8.0 add sufficient water to reach a final volume of 500ml

The same thing is done for the 500mM EDTA solution. Weight out appropriate amount of EDTA to make 500ml solution of 500mM EDTA. Add the EDTA powder to bottle and add MiliQ grade water until a volume of 400ml is reach. Include a magnetic stir bar. Throw in a few (2-3) pearls of NaOH and stir. The acid form of EDTA is not very soluble. It more soluble in the salt form. So you need a fair bit of stirring and NaOH. Continue adding NaOH, until pH 8 is reached. You should use drops of a 1M NaOH solution once you start getting close to pH8. The EDTA powder is completely solubalised once pH8 is reached.

Once you have the stock solutions, add the appropriate amount of each stock solution, and NaCl to produced your buffer.

A 1M solution is defined by the molecular weight of the chemical being used. 1M =grams of molecular weight/ 1 Liter. For example, the molecular weight of NaCl is 58.44. Look on the side of the container of the chemical in question. It should always indicate the molecular weight. So, for a 1M solution of NaCl you would dissolve 58.44 grams into 1 liter. There are 1000 mM per M so a 150mM solution is 0.15M. Multiply 58.44 by 0.15 and you have the amount of NaCl you need to make one liter of a 150mM solution. You can do the same for Tris and EDTA. Tris base molecular weight: 121.14 or 157.56 depending on which your lab buys. You'll have to check which one you have. Multiply by 0.1 and this is grams per liter for 100mM (0.1M). EDTA molecular weight: 292.24. You want only 1mM which is 0.001M so you only want 0.29gr. You can measure all these, throw them in a container. Add about 800mL of water (NOT the whole liter!!) and then pH with concentrated HCl. It will take quite a bit of acid if you only have the Tris base and not Tris-HCl and, if you add all the water before pHing, your solution will be too dilute. Only once you get the pH to 8.0 do you add the rest of the water to 1L. Measure the volume of solution that has been made and not neccessarily the water you've added since the acid you use to pH counts into the final 1 liter!

Now, you should know that this is not typically how buffers are made. As mentioned earlier, usually high concentration stock solutions are made and then you just dilute an appropriate amount of these to make the buffer. This way you can throw together buffers easily and quickly. For example, the common stock solution for NaCl is 5M. You still use the molecular weight but multiply by 5. This is the grams you need to make one liter 5M NaCl. Same for Tris (usually 1M or 0.5M stock at varying pH) and EDTA at 1M. Just beware that EDTA will not go into solution unless you pH to 8.0.