Making a Tris Buffer for Click Chem, using SAOS-2 Cells

[pddxr]

Hi,

 

I'll soon be doing a click chemistry reaction which calls for 10% of the reaction solution to be a 1M Tris buffer of pH 8.5.

 

Assuming that I want 200ml of the buffer and use solid Tris with 1M HCl I would prepare in the following way:

(See my calculation below)

So I come up with:

24.23g Tris

68ml 1.0M HCl

BTV 200ml with H₂O

Then titrate to pH 8.5.

 

Questions:

1) Can I autoclave the buffer?

2) I really only need about 250ul of this Tris buffer per experiment run. Can I store the rest for future use and is room temp ok? or -4C, -20C, -80C?

 

 

Calculations:

200ml of 1M Tris @ pH 8.5. & 1M HCl stock

 

200ml(1/10³ml)(121.14g/mol) = 24.23g Tris

pH=pKa + log([A]/[HA])

[A]/[HA] = 10^ph-pKa = 10^8.5-8.21 = 10^0.26 = 1.95

[HA] = 1/2.95

[H⁺] = (1/2.95)(1M) = 0.34M

 

C₁V₁=C₂V₂

V_1(HCl) = C₂V₂/C₁ = (0.34M)(200ml)/(1.0M) = 68ml 1.0M HCl

 

So I come up with:

24.23g Tris

68ml 1.0M HCl

BTV 200ml with H₂O

Then titrate to pH 8.5.

 

[BabcockHall]

I am very short on time today, but this calculation does not look correct. 1 M Tris means the total concentration of Tris in all forms: [HA] + [A] = 1.0 M.

EDT

Now that I look at it again, it seems to me that you have correctly taken the point I made above into account.

Edited April 25, 2013 by BabcockHall

[CharonY]

With regards to autoclaving, you can do that. Just be aware that oftentimes autoclaves are not very clean and it may contaminate your solution (although it would be sterile contamination). Depends on the sensitivity of your application. If made cleanly Tris tends to be fairly stable at RT (6 months did not appear to be much of an issue), though I would check pH before use to be certain. Also note that the pH is temperature dependent. Should you put it in the fridge, let it warm up to RT (or whatever the reaction temp is) before use.

[BabcockHall]

Also, the apparent pKa of Tris is dependent on ionic strength. Therefore, your calculation of how much HCl to use, is just an approximation.

[CharonY]

Excellent point!