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The following guidelines should be followed while making reagents, buffers, etc.
1. Use highest grade of reagents wherever possible.
2. Prepare all solutions with double distilled or distilled water.
3. Autoclave solutions whenever possible. Solutions that cannot withstand autoclaving should be sterilized using 0.2 mm filter.
4. pH meter should carefully be checked using freshly prepared standard solutions of pH before using it for preparing pH buffers.
5. Label all containers with name of solution, percentage or concentration, date, etc.
6. Use plastic containers for highly basic solutions like 1(M) NaOH, since glass will be corroded by bases.
7. Store all solutions in cold or at 4°C, wherever possible.
8. Prepare concentrated stock solutions to make up a range of dilutions, e.g. 1(M) Trio. This saves time.
Percentage Solution:
The exact concentration of the solute in 100 ml of solvent is known as percentage solution. It can be weighted when it is expressed as W/V (g/ml) or millilitres, when it is expressed as V/V (ml/ml), e.g. for 1% (W/V) NaCl, 1 g of NaCl is dissolved in distilled water to make up the volume to 100 ml. For 1% (V/V) of acetic acid, 1 ml of glacial acetic acid plus distilled water to make up 100 ml. Another example where commercial strength is recorded on the bottle as 36-40% (V/V). When a 10% (V/ V) solution of HCl is required, it is erroneous to take 1 ml of 36-40% (V/V) HCl and make the volume up to 100 ml. It has to be prepared as follows:
Starting concentration (%) multiplied by (X) unknown volume (X) (ml) is equal to (=) final concentration (%) X total volume (ml)
36 x X = 1 x 100
36 X = 100
X = 100/36 = 2.78 ml of 36% (V/V) HC1 in 97.22 ml of distilled water.
Dilutions of Solutions (% by Volume):
The most accurate formula for making dilutions of solutions is:
Percentage you have X unknown volume = percentage you need X volume wanted (ml).
To make up 1000 ml of 70% (V/V) ethanol from 95% (V/V) ethanol, substitute the known amounts in the formula.
Percentage you have (95%) X unknown volume (X) = percentage you want (70%) X volume wanted (1000 ml)
95 x X = 70 X 1000
or, 95 X = 70,000
or, X = 70,000/95
or, X = 736.8 ml of 95% (V/V) ethanol should be added to 263.2 ml of distilled water.
Or
Subtract the percentage required from the percentage strength of the solution that is to be diluted. The difference will give the amount of water that is to be used.
e.g. Percentage volume required=70%(V/V)
The stock ethanol = 95% (V/V)
95-70=25 parts of distilled water is to be added to obtain a 70% solution of ethanol.
or, 700 ml of 90% alcohol to be made up to 950 ml.
Molar Solutions:
A molar (mole or molecular) solution is one in which the molecular weight of the solute is dissolved in one litre (1000 ml) of the solvent (Molecular weight is the sum of atomic weights).
e.g. 1(M) solution of NaCl is
At. wt. Na (= 22.997) + CI (= 35.459) = mol. wt.= 58.456
58.456 g of NaCl made up to 1000 ml with distilled water.
For 100 ml 1(M) NaCl.
Weigh 58.46/10= 5.85 g of NaCl made up to 100 ml with distilled water.
Preparation of Buffers:
Phosphate Buffer:
Stock Solutions:
A 0.2(M) solution of monobasic sodium phosphate (27.8g in 1000 ml distilled water)
B: 0.2(M) solution of dibasic sodium phosphate (53.65 g of Na2HPO4. 7H2O or 71.7 g Na2HPO4. 12H2O in 1000 ml distilled water).
Mix A and B as shown in Table and dilute to 200 ml:
Acetate Buffer:
Stock Solutions:
A: 0.2(M) solution of acetic acid (11.55 ml in 1000 ml distilled water).
B: 0.2(M) solution of sodium acetate (16.4 g of C2H2Na or 27.2 g of C2H3O2 Na. 3H2O in 1000 ml distilled water).
Mix A and B as shown below and dilute to 100 ml:
Citrate Buffer:
Stock Solutions:
A: 0.1 (M) solution of citric acid (21.01 g in 1000 ml distilled water).
B: 0.1 (M) solution of sodium citrate (29.41g C6H5O7.Na3. 2H2O in 1000 ml distilled water).
Mix A and B as follows and dilute to 100 ml:
TRIS (Hydroxy Methyl) Aminomethane Buffer (Tris HCl):
Stock Solutions:
A: 0.2 (M) solution of Tris (hydroxy methyl aminomethane (24.2 g in 1000 ml distilled water).
B: 0.2 (N) HCl (16.1 ml made upto 1000 ml with distilled water).
Mix A and B as shown below and dilute to 200 ml:
Glycine – NaOH Buffer:
Stock Solutions:
A: 0.2M solution of glycine (15.01 g in 1000 ml distilled water),
B: 0.2 NaOH.
Mix A and B and dilute to 200 ml.
Carbonate-Bicarbonate Buffer:
Stock Solutions:
A: 0.2 M anhydrous sodium carbonate solution (21.2 g in 1000 ml distilled water).
B: 0.2 M Sodium bicarbonate (16.8 g in 1000 ml of distilled water).
Add A and B and dilute to 200 ml.
Glycine – HCl Buffer:
Stock Solutions:
A: 0.2 (M) glycine (15.01 g in 1000 ml distilled water).
B: 0.2 (N) HCl.
Add A and B and dilute to 100 ml.
Boric Acid – Borax Buffer:
Stock Solution:
A: 0.2 (M) solution of boric acid (12.4 g in 1000 ml distilled water).
B: 0.05 (M) solution of borax (19.05 g in 1000 ml of distilled water; 0.2 (M) in terms of sodium borate).
Mix A and B and dilute to 200 ml.
