2 Solutions With More Than One Solute RECIPE INa2HPO gKH2PO g0.4% glycerol 10 mLDissolve in water.Bring to a volume of 1 liter.Recipe lists amounts of solutes.This is easy to follow
3 Solutions With More Than One Solute Cont… RECIPE II1 M MgCl20.1 M TrisGives the final concentration of each solute.You must calculate the amount of each to use.This is harder to interpret
4 A Recipe With Multiple Solutes, SM Buffer 0.1 M NaCl1 mM MgSO40.2 M Tris, pH 7.50.01% gelatinThis might be whole recipe as given in a manual
5 Strategy 1: PREPARING SM BUFFER WITHOUT STOCK SOLUTIONS, OVERVIEW: Prepare a solution of 0.2 M Tris, pH 7.5.Calculate grams of each of the other solutes required.Weigh out solutes and dissolve directly in Tris buffer.
6 Strategy 1 Decide how much buffer to make, for example, 1 liter. 1 liter of 0.2 M Tris requires 24.2 g of Tris base. (MW = g/mole.)Dissolve Tris in about 700 mL of water and bring pH to 7.5.Do not bring Tris to volume.
7 4. 1 liter of 0. 1 M NaCl requires 5. 84 g of NaCl 4. 1 liter of 0.1 M NaCl requires 5.84 g of NaCl. Add this to Tris buffer.5. 1 liter of 1 mM MgSO4 requires 1/1000 of its MW.MgSO4 comes in more than one hydrated form; readcontainer to determine MW.Weigh out correct amount.Add it to Tris buffer.
8 0.01% gelatin is 0.1 g in 1 L. Weigh out and add to Tris buffer. Stir to dissolveBTV 1 LCheck and record pH.
9 Strategy 2: PREPARING SM BUFFER WITH STOCK SOLUTIONS In overview: Four solutes are each prepared separately as concentrated stock solutions.
10 When the four stocks are combined, they dilute one another to the proper final concentrations
11 Steps Are: Prepare a stock solution of Tris buffer at pH 7.5. a. No set rule as to what should be concentration of stock.b. Example: To make 1 L of 1 M stock, dissolve g of Tris base in about 900 mL of water.
12 c. Bring pH to 7.5.d. BTV 1L.2. Prepare a stock solution of magnesium sulfate, for example, 1 M.a. To make 100 mL, dissolve 0.1 MW of MgSO4 in water, bring to a volume of 100 mL.
13 3. Prepare a stock solution of NaCl, for example,1 M. a. To make 100 mL of stock, dissolve 5.84 grams in water and BTV 100 mL.
14 Prepare a stock solution of gelatin, for example, 1%. a. Dissolve 1 g in a final volume of 100 mL of water.
15 5. To make the final solution, combine the right amounts of each stock. a. Since this is a situation where stocks are diluted, use the C1V1 = C2V2 equationfour times, once for each solute.
16 For example, to calculate how much Tris stock is required:C1 V1 = C V2(1 M) (?) (0.2 M) (1000 mL)? = 200 mL
17 Summary To make 1000 mL of SM buffer, combine: 200 mL Tris stock 1 mL MgSO4 stock100 mL NaCl stock10 mL Gelatin stockBTV 1L; check and record pH.
18 Which Strategy? Both strategy 1 and 2 are correct. Generally efficient to make stock solutions of frequently used solutes because weighing out chemicals is time-consuming.