Starting with solid Tris base (FW 121.1) and 1 M HCl, describe how you would make 2 liters of 200 mM Tris-HCl buffer pH 7.5. pKa of Tris 8.14
Let's call TRIS base, TNH2.
pH = pKa + log (base)/(acid)
7.5 = 8.14 + log b/a
b/a = 0.23 approximately. You do it more accurately.
So base = 0.23*acid
You want 2000 mL x 0.200 M = 400 mmols.
Therefore you want 0.23*400 = 92 mmols base.
.........TNH2 + HCl ==> TNH3^+ + Cl^-
I.........x......0.........0
add..............400.........
C........-400...-400......400
E........x-400 ....0......400
If x-400 = 92, then x must be 492.
So you take 492 mmols TNH2, add 400 mmols HCl, add H2O to the 2L mark, and you have it. You calculate grams TNH2 from this and mL HCl from this (depending upon how strong the HCl is).
I recommend you start with the grams and redo those calculations on paper to make sure the pH will be 7.5 when you finish.
To prepare 2 liters of a 200 mM Tris-HCl buffer at pH 7.5, you will need to follow a series of steps. First, let's break it down into the required components:
1. Tris base (FW 121.1): This is in solid form and will be used to make the Tris-HCl buffer.
2. 1 M HCl: This is a concentrated hydrochloric acid solution used to adjust the pH of the buffer.
3. Water: You will need distilled or deionized water to make up the total volume of the buffer.
Now, let's move on to the step-by-step process:
Step 1: Calculate the amount of Tris base needed:
To make a 200 mM solution, you need to determine the amount of Tris base (in grams) required to achieve this concentration. Use the formula:
mM = (grams / volume in liters) / Molecular weight
Rearrange the formula to solve for grams:
grams = (mM * volume in liters * Molecular weight) / 1000
Plugging in the values:
mM = 200
Volume in liters = 2
The molecular weight of Tris base (FW) is given as 121.1.
grams = (200 * 2 * 121.1) / 1000
Calculate the result:
grams = 48.44 grams
So, you will need 48.44 grams of Tris base.
Step 2: Dissolve Tris base:
Start by weighing out 48.44 grams of solid Tris base on a balance. Then, add the Tris base to a suitable container, such as a beaker.
Step 3: Add distilled or deionized water:
Add distilled or deionized water to the beaker containing the Tris base. The volume should be enough to make up the final 2 liters of buffer solution.
Step 4: Adjust pH with HCl:
To achieve a pH of 7.5, you need to add a calculated amount of 1 M HCl dropwise while monitoring the pH. Since the pKa of Tris is 8.14, the buffer will be most effective as a pH 7.5 buffer.
To calculate the exact amount of HCl required, you will need to use the Henderson-Hasselbalch equation:
pH = pKa + log ([A-]/[HA])
In this case, [A-] = concentration of Tris in the buffer (200 mM) and [HA] = concentration of HCl added. Rearrange the equation to calculate [HA]:
[HA] = [A-] * 10^(pH - pKa)
Plugging in the values:
[A-] = 200 mM = 0.2 M
pH = 7.5
pKa = 8.14
[HA] = 0.2 M * 10^(7.5 - 8.14)
Calculate the result:
[HA] ≈ 0.0188 M
So, you will need to add approximately 0.0188 M HCl dropwise to the Tris base solution until the desired pH of 7.5 is reached. It's important to measure the pH as you go to avoid overshooting the target pH.
Step 5: Adjust the final volume:
Once the desired pH is attained, you can carefully adjust the final volume of the buffer to exactly 2 liters using distilled or deionized water.
That's it! You have successfully prepared 2 liters of a 200 mM Tris-HCl buffer at pH 7.5 using Tris base and 1 M HCl.