1. PREPARING LABORATORY SOLUTIONS AND REAGENTS II: Chapter 22

2. SOLUTIONS WITH MORE THAN ONE SOLUTE RECIPE I Na 2 HPO 4 6 g KH 2 PO 4 3 g 0.4% glycerol 10 mL Dissolve in water. Bring to a volume of 1 liter. Recipe lists amounts of solutes.

3. RECIPE II 1 M MgCl 2 0.1 M Tris Gives the final concentration of each solute. You must calculate the amount of each to use.

4. A RECIPE WITH MULTIPLE SOLUTES, SM BUFFER SM BUFFER 0.1 M NaCl 1 mM MgSO 4 0.2 M Tris, pH 7.5 0.01% gelatin

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 1. Decide how much buffer to make, for example, 1 liter. 2. 1 liter of 0.2 M Tris requires 24.2 g of Tris base. (MW = 121.1 g/mole.) 3. 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. Add this to Tris buffer. 5. 1 liter of 1 mM MgSO 4 requires 1/1000 of its MW. MgSO 4 comes in more than one hydrated form; read container to determine MW. Weigh out correct amount. Add it to Tris buffer.

8. 6. 0.01% gelatin is 0.1 g in 1 L. Weigh out and add to Tris buffer. 7. Stir to dissolve. 8. BTV 1 L 9. 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: 1. Prepare a stock solution of Tris buffer at pH 7.5. a. No set rule as to what should be concentration of stock. b. To make 1 L of 1 M stock, dissolve 121.1 g of Tris base in about 900 mL of water. c. Bring pH to 7.5. d. BTV 1L.

12. 2. Prepare a stock solution of magnesium sulfate, for example, 1 M. To make 100 mL, dissolve 0.1 MW of MgSO 4 in water, bring to a volume of 100 mL.

13. 3. Prepare a stock solution of NaCl, for example,1 M. To make 100 mL of stock, dissolve 5.84 grams in water and BTV 100 mL.

14. 4. Prepare a stock solution of gelatin, for example, 1%. 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. Since this is a situation where stocks are diluted, use the C 1 V 1 = C 2 V 2 equation Four times, once for each solute.

16. For example, to calculate how much Tris stock is required: C 1 V 1 = C 2 V 2 (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 MgSO 4 stock 100 mLNaCl stock 10 mL Gelatin stock 7. BTV 1L; 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.