PREPARING SOLUTIONS AND REAGENTS PART 1

Understanding Concentration

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BASIC LABORATORY METHODS IN A REGULATED ENVIRONMENT

LECTURE OVERVIEW

• Where do solution recipes come from?
• Concentration vs amount
• Concentration of solute: calculations
• Preparing solutions, next lecture
• Making diluted solutions from concentrated ones
• Buffers
• Bringing solutions to proper pH
• Calculations for solutions with more than one solute, third solutions lecture

LECTURE OVERVIEW

• Where do solution recipes come from?
• Concentration vs amount
• Concentration of solute: calculations
• Preparing solutions
• Making diluted solutions from concentrated ones
• Buffers
• Bringing solutions to proper pH
• Calculations for solutions with more than one solute, next lecture

WHERE DO SOLUTION "RECIPES" COME FROM?

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• Original Scientific Literature

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• Lab manuals (instructional)

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• Lab Manuals (professional)

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• Handbooks

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• Manufacturers and suppliers

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INTERPRETING RECIPES

DEFINITIONS:

• SOLUTES -- substances that are dissolved

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• SOLVENTS -- substance in which solutes are dissolved (usually water)

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• AMOUNT -- how much

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LECTURE OVERVIEW

• Where do solution recipes come from?
• Concentration vs amount
• Concentration of solute: calculations
• Preparing solutions
• Making diluted solutions from concentrated ones
• Buffers
• Bringing solutions to proper pH
• Calculations for solutions with more than one solute, next lecture

CONCENTRATION VS AMOUNT

Each star represents 1 mg of NaCl.

What is the total amount of NaCl in the tube? _____

What is the concentration of NaCl in the tube (in mg/mL)? _____

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5 mL

Each star represents 1 mg of NaCl.

What is the total amount of NaCl in the tube? 4 mg

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What is the concentration of NaCl in the tube (in mg/mL)?

4 mg = ?_

5 mL 1 mL

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? = 0.8 mg, so the concentration is 0.8 mg/mL

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5 mL

LECTURE OVERVIEW

• Where do solution recipes come from?
• Concentration vs amount
• Concentration of solute: calculations
• Preparing solutions
• Making diluted solutions from concentrated ones
• Buffers
• Bringing solutions to proper pH
• Calculations for solutions with more than one solute, next lecture

WAYS TO EXPRESS CONCENTRATION OF SOLUTE

• Possible source of confusion: more than one way to express concentration of solute in a solution:

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CONCENTRATION EXPRESSIONS

1. WEIGHT PER VOLUME

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2. MOLARITY

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3. PERCENTS

a. Weight per Volume %

(w/v %)

b. Volume per Volume %

(v/v %)

c. Weight per Weight %

(w/w %)

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MORE CONCENTRATION EXPRESSIONS

4. PARTS

Amounts of solutes as "parts"

a. Parts per Million (ppm)

b. Parts per Billion (ppb)

c. Might see ppt

d. Percents are same category (pph %)

STILL MORE CONCENTRATION EXPRESSIONS

TYPES NOT COMMON IN BIOLOGY MANUALS:

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5. MOLALITY

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6. NORMALITY

• for NaOH and HCl, molarity = normality, however, this is not always true for all solutes

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WEIGHT / VOLUME

EXAMPLE:

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• 2 mg/mL proteinase K means there are 2 mg of proteinase K in each mL of solution.

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• How much proteinase K is required to make 50 mL of solution at a concentration of 2 mg/mL?

ANSWER:

MOLARITY

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• Molarity is number of moles of a solute that are dissolved per liter of total solution.

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• A 1 M solution contains 1 mole of solute per liter total volume

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MOLE

• How much is a mole?

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EXAMPLE: SULFURIC ACID

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For a particular compound, add the atomic weights of the atoms that compose the compound.

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H₂SO₄:

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2 hydrogen atoms 2 X 1.00 g = 2.00 g

1 sulfur atom 1 X 32.06 g = 32.06 g

4 oxygen atoms 4 X 16.00 g = 64.00 g

98.06 g

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EXAMPLE CONTINUED

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• A 1M solution of sulfuric acid contains 98.06 g of sulfuric acid in 1 liter of total solution.

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• "mole" is an expression of amount

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• "molarity" is an expression of concentration

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DEFINITIONS

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• "Millimolar", mM, millimole/L.
• A millimole is 1/1000 of a mole.

• "Micromolar", µM, µmole/L.
• A µmole is 1/1,000,000 of a mole.

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FORMULA

EXAMPLE

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How much solute is required to make 300 mL of 0.8 M CaCl₂?

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ANSWER

TO MAKE SOLUTION OF GIVEN MOLARITY AND VOLUME

1. Find the FW of the solute, usually from label.

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2. Determine the molarity desired.

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3. Determine the volume desired.

4. Determine how much solute is necessary by using the formula.

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PROCEDURE CONT.

5. Weigh out the amount of solute.

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6. Dissolve the solute in less than the desired final volume of solvent.

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7. Place the solution in a volumetric flask or graduated cylinder. Add solvent until exactly the required volume is reached, Bring To Volume, BTV.

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PERCENTS

X % is a fraction

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numerator is X

denominator is 100

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Three variations on this theme.

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CONCENTRATION EXPRESSED AS WEIGHT/VOLUME %

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Grams of solute

100 mL total solution

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Most common in biology.

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EXAMPLE

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20 g of NaCl in 100 mL of total solution

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= 20% (w/v) solution.

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EXAMPLE: BY PROPORTIONS

How would you prepare 500 mL of a 5 % (w/v) solution of NaCl?

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ANSWER

By definition: 5 % = 5 g

100 mL

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5 g = ?

100 mL 500 mL

? = 25 g = amount of solute

BTV 500 mL

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BY EQUATION

How would you prepare 500 mL of a 5 % (w/v) solution of NaCl?

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1. Total volume required is 500 mL.

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2. 5% = 0.05

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3. (0.05) (500 mL) = 25

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% EXAMPLE CONTINUED

4. 25 is the amount of solute required in grams.

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5. Weigh out 25 g of NaCl. Dissolve it in less than 500 mL of water.

6. In a graduated cylinder or volumetric flask, bring the solution to 500 mL.

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TWO OTHER FORMS OF % SOLUTIONS

v/v mL solute

100 mL solution

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w/w g solute

100 g solution

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WEIGHT/WEIGHT (W/W)

• How would you make 500 g of a 5% solution of NaCl by weight (w/w)?

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ANSWER

PARTS

Parts may have any units but must be the same for all components of the mixture.

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EXAMPLE:

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A solution is 3:2:1 ethylene: chloroform: isoamyl alcohol

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Might combine:

3 liters ethylene

2 liters chloroform

1 liter isoamyl alcohol

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PPM AND PPB

• Parts per Million: The number of parts of solute per 1 million parts of total solution.

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• Parts per Billion: The number of parts of solute per billion parts of solution.

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PPM EXAMPLE:

5 ppm chlorine = 5 g of chlorine in 1 million g of solution

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or 5 mg chlorine in 1 million mg of solution

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or 5 pounds of chlorine in 1 million pounds of solution

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Etc.

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CONVERSIONS

TO DELVE DEEPER INTO THE TOPICS IN THIS LECTURE

• Chapter 22 in Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference, 3^rd Edition has more information about concentration expressions, including an appendix on molarity calculations relating to DNA and RNA.

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