Preparation of Solutions and making Dilutions

PRACTICAL 2: PREPARING SOLUTIONS AND MAKING DILUTIONS
INTRODUCTION
Preparing solutions is a very common activity in an analytical biochemistry lab. Proper solution
preparation requires basic math skills, accurate measurement using appropriate and suitable tools
or glassware, and the ability to follow instructions. A solution is a homogenous mixture of “solute”
dissolved in bulk liquid known as “solvent”. Solutions can be described by their solute
concentration, a measure of how much solute is present per unit of solution. A concentrated
solution is generally called a “stock solution,” and the diluted solution is called the “working
solution”. Preparing a concentrated stock solution saves a lot of time. It is easier to store stock
solution than a large volume of diluted working solutions. Making a working solution simply
requires diluting some volume of stock solution to the concentration needed. Every biochemistry
student must learn and know how to prepare a stock solution and making a working solution.
OBJECTIVES
1. To prepare a stock solution.
2. To prepare a working solution by diluting a stock solution using linear and serial dilution
methods.
3. To measure the absorbance of working solutions at various concentrations using a
spectrophotometer.
APPARATUS
1. 10-mL beaker
2. 100-mL beaker
3. Test tubes
4. Graduated cylinder
5. UV Spectrophotometer cuvette
6. Pipette
7. Spatula
8. Dispensing bottle with distilled water
9. Distilled water
10. Pipette bulb/Pipette pump
11. Test tube rack
12. Weighing paper
13. UV-Vis Spectrophotometer
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14. Analytical balance
Chemical: Bromophenol Blue
Lab Activity 1: Preparing solutions
Before preparing a solution, you must be able to identify or recognize the following questions; (i)
what is the substance or chemical to be dissolved?, (ii) what is the amount required?, (iii) what is
the most suitable solvent to be used?, (iv) how to calculate these amount, and, (v) what tools or
glassware should be used to obtain accurate measurements and right concentration. The amount
of substance is often measured as weight, i.e. in mg or g or kg. Mole is also used representing
the amount of substance, the mass of a mole of molecules if the formula weight is in grams. As an
example, the molecular mass of amino acid glycine is 75.07 g/mol or sometime in ‘Dalton’ unit, i.e.
75 Da.
Concentration is the amount divided by the volume it is dissolved or dispersed in. It is possible to
use different units to express concentration depending on the states of matter (i.e. solid, liquid or
gas) that the two substances are in. Typically, concentrations are measured as the mass of solute
per unit volume of solution, i.e.,
A. Molar solutions (Unit = M= moles/L)
A 1.0 molar (1.0 M) solution is equivalent to 1.0 mole (formula weight [FW] [g/mole]) of solute
dissolved in 1.0 L of solvent.Example 1: To prepare a litre of 0.15 M solution from a dry reagent with FW of 194.0 g/mole,
amount of solute required is 194.0 g/mole x 0.15 mole/L = 29.1 g/L
B. Percent solution (= parts per hundred)
1. In most cases, reagents are prepared as percent concentrations, a dry chemical is mixed as
dry mass (g) per volume (g/100 mL), [unit (w/v)].Example 2: To prepare a 10% (w/v) NaCl solution, 10 g NaCl is dissolved in 100 mL of solventExample 3: To prepare 150 mL of 6% (w/v) reagent A, 6 g/100 mL x 150 mL = 6 g x 1.5 = 9
g of A is dissolved in 150 mL solvent
2. If using liquid reagents, the percent concentration is based upon volume per volume (mL/100
mL), [unit (v/v)].
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Example 4: To prepare 75% (v/v) ethanol, mix 75 mL of ethanol with 25 mL water, making up
the volume to 100 mL
3. Concentration may also be expressed as percent of dry mass per mass of solvent (g/100 g),
[unit w/w)].Example 5: To prepare a 20% (w/w) of Triton X-100, 20 g of Triton X-100 is mixed with 80 g
solvent
Example
6: To prepare a 200 g of 20% (w/w) reagent B, 20 g/100 g x 200 g = 20 g x 2 = 40
g of B is mixed with 160 g solvent
C. Conversion from % to molarity and from molarity to %
To convert from percent concentration solution to molarity, use the formula below.
Example 7: To convert a 6% solution of C with FW of 325 g/mole to molarity value,
[6 g/100 mL) x 10]/325 g/mole = 0.2 M
To convert from molarity to percent concentration solution, use the formula below.
Example 8: To convert a 0.06 M solution of a chemical having FW of 178 g/mole to percent
solution,
[0.06 mole/L] x 178 g/mol /10 = 1.06% solution
Exercise 1: Preparation of a bromophenol blue stock solution
1. Prepare 50 mL bromophenol blue stock solution at concentration of 0.1 mg/mL. Use distilled
water as solvent. What is the amount of bromophenol blue powder required? Show your
calculation. Keep the solution for Exercise 2.
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