PREPARING SOLUTIONS AND REAGENTS PART 3

Solutions with Multiple Components

BASIC LABORATORY METHODS IN A REGULATED ENVIRONMENT

SOLUTIONS WITH MORE THAN ONE SOLUTE

RECIPE I

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Na₂HPO₄ 6 g

KH₂PO₄ 3 g

0.4% glycerol 10 mL

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Dissolve in water.

Bring to a volume of 1 liter

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Easy to interpret, recipe lists amounts of solutes.

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MANY RECIPES DO NOT LIST AMOUNTS OF SOLUTES

RECIPE II

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1 M MgCl₂

0.1 M Tris

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Gives the final concentration of each solute.

You must calculate the amount of each solute to use.

A RECIPE WITH MULTIPLE SOLUTES, SM BUFFER

SM BUFFER

0.1 M NaCl

1 mM MgSO₄

0.2 M Tris, pH 7.5

0.01% gelatin

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STRATEGY 1:

PREPARING SM BUFFER WITHOUT STOCK SOLUTIONS, OVERVIEW:

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• Prepare a solution of 0.2 M Tris, pH 7.5.

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• Calculate grams of each of the other solutes required.

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• Weigh out solutes and dissolve directly in Tris buffer.

STRATEGY 1

1. Decide how much buffer to make, for example, 1 liter.

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2. 1 liter of 0.2 M Tris requires 24.2 g of Tris base. (MW = 121.1 g/mole.)

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3. Dissolve Tris in about 700 mL of water and bring pH to 7.5.

Do not bring Tris to volume.

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STRATEGY 1 CONT.

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4. 1 liter of 0.1 M NaCl requires 5.84 g of NaCl. Add this to Tris buffer.

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5. 1 liter of 1 mM MgSO₄ requires 1/1000 of its MW.

MgSO₄ comes in more than one hydrated form; read container to determine MW.

Weigh out correct amount.

Add it to Tris buffer.

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STRATEGY 1 CONT.

6. 0.01% gelatin is 0.1 g in 1 L. Weigh out and add to Tris buffer.

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7. Stir to dissolve.

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8. BTV 1 L

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9. Record pH.

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STRATEGY 2:

PREPARING SM BUFFER WITH STOCK SOLUTIONS:�

• In overview:

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• Four solutes are each prepared separately as concentrated stock solutions.

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STRATEGY 2 CONT.

• When the four stocks are combined, they dilute one another to the proper final concentrations

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.

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STRATEGY 2 CONT.

2. Prepare a stock solution of magnesium sulfate, for example, 1 M.

• To make 100 mL, dissolve 0.1 MW of MgSO₄ in water, bring to a volume of 100 mL.

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STRATEGY 2 CONT.

3. Prepare a stock solution of NaCl, for example,1 M.

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• To make 100 mL of stock, dissolve 5.84 grams in water and BTV 100 mL.

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4. Prepare a stock solution of gelatin, for example, 1%.

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• Dissolve 1 g in a final volume of 100 mL of water.

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STRATEGY 2 CONT.

• 5. To make the final solution, combine the right amounts of each stock.
• Since this is a situation where stocks are diluted, use the C₁V₁ = C₂V₂ equation
• Four times, once for each solute.

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For example, to calculate how much Tris stock is required:

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C₁ V₁ = C₂ V₂

(1 M) (?) (0.2 M) (1000 mL)

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? = 200 mL

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SUMMARY

To make 1000 mL of SM buffer, combine:

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200 mL Tris stock

1 mL MgSO₄ stock

100 mL NaCl stock

10 mL Gelatin stock

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7. BTV 1L; record pH.

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WHICH STRATEGY?

• Both strategy 1 and 2 are correct.

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• Generally efficient to make stock solutions of frequently used solutes because weighing out chemicals is time-consuming.

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