Efficient Buffer Preparation: 500ml MgCl2, Glucose, ATP, and Tris-Cl at pH 7.8

[vande060]

Homework Statement

Prepare 500ml of a reaction buffer containing .82% MgCl₂, 62.5 mM glucose, 950 ATP, and 100mM Tris -Cl, pH 7.8 Use the stock solutions you have prepared

we had to prepare in previous problems:

500ml 1.75M glucose
25mL 2.5% glucose
2L of 1.8 Tris, pH 8.5

Homework Equations

C1V1 = C2V2
Pka Tris = 8.1
glucose mol mass = 180.16
ATP mol mass = 507.8

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The Attempt at a Solution

my calculation for MgCl₂:

.82% = .82g/100ml = 8.2g/L
15% = 15g/100ml = 150g/L

C1V1=C2V2

150*x = 8.2*500

x = 28mL stck 15% MgCl₂

My calculation for glucose

1.75M glucose = 315g/L
62.5mM = 11.2 g/L

C1V1=C2V2

11.2*500 = 315*x

x = 17.7 ml 1.75M glucose stock

Here is my calculation for ATP

5mM ATP = 3g/L
950nM ATP = .00048g/L

C1V1=C2V2

3*x = .00048*500

x =.08ml of stocK 5mM ATP

My calculation for the Tris I am not sure about, can I use my original pH 8.5 solution the prof asked me to prepare, or do I have to start over with the Tris acid and base components and use henderson H equation? does this all look right so far?

 

[Borek]

C₁V₁ = C₂V₂ works for molar concentrations as well, you don't have to convert them to g/L.

My guess is you should prepare new buffer from HH equation.

 

[vande060]

C₁V₁ = C₂V₂ works for molar concentrations as well, you don't have to convert them to g/L.

My guess is you should prepare new buffer from HH equation.

is the rest of it right?

 

[Borek]

Looks OK, but I just skimmed.

 

[DeeNos]

First of all, great job on your calculations for MgCl2, glucose, and ATP! Your calculations seem to be correct and you have used the correct equations to determine the volumes of stock solutions needed.

For the Tris component, you can use the original pH 8.5 solution that you prepared in a previous problem. However, you will need to adjust the pH to 7.8 by using the Henderson-Hasselbalch equation. The pKa of Tris is 8.1, so you can use the equation pH = pKa + log([base]/[acid]) to determine the amount of Tris acid and base needed to achieve a pH of 7.8. Once you have adjusted the pH, you can use the same equation (C1V1 = C2V2) to determine the volume of stock solution needed.

Overall, your approach and calculations seem to be correct. Just make sure to double check your calculations and units before preparing the buffer. Good luck!