Preparation of buffers

pH, the negative log of H⁺ ion concentration is an important factor in most of the biological reactions. The change in pH value can be resisted by adding suitable buffers to the reaction mixture.  

A buffer solution is one that resist pH changes caused by addition of acid or alkali. Such solutions are used in many biochemical experiments where pH needs to be accurately controlled. A buffer system consists of a weak acid and its conjugate base.

In a buffer system, when the concentration of proton donor and its conjugate proton acceptor is equal, the addition of even small amount of acid or alkali does not make any detectable change in the pH. This point at which there is no net charge is called as isoelectric point. The pH at isoelectric point is equal to pKa.

                   Henderson Haselbach equation

pH = pKa + log [A-]

                        [HA]            

         Acetate buffer consists of mixture of acetic acid and sodium acetate

                                 [CH3COO¯ ]
pH = pKa + log   _______________
                                 [CH3COOH]

At isoelectric point [CH3COO¯] = [CH3COOH] hence, pH = pKa

Buffer capacity is the ability of a buffer solution to resist the change in pH when adding acid or alkali. Buffers with higher concentrations show higher buffering capacity. 

Common laboratory buffers are

Phosphate buffer

Citrate Buffer

Acetate Buffer

Carbonate- Bicarbonate Buffer

Tris buffer  (tris (hydroxymethyl) aminomethane)

HEPEs buffer (4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid) 

Things to be taken care of while preparing Buffers

1. Use analytical grade of chemicals
2.  Dissolve the chemicals in higher quantity of distilled water
3. It is better to prepare concentrated stocks of buffer solutions in order to save time and space. These concentrated stocks will last for a long period of time and can be easily diluted for use. These stocks are commonly labeled as 10X, 5X, 100X etc. X-factor indicates that the solution is concentrated and must be diluted usually with water to 1X concentration for use. eg., A 100X concentrated solution should be diluted to 100 fold.
4. Sterilize the solution wherever possible by autoclaving. If autoclaving is not possible, filtration through 0.2um filter is to be done
5. Calibrate the pH meter using standard buffer solutions
6.  Always label the prepared buffer bottle with the name of the solution, percentage concentration and date of preparation
7. The highly basic solutions such as 1M sodium hydroxide should be taken in glass containers
8.  Store the buffer in refrigerator

 

1. PREPARATION OF ACETATE BUFFER

Aim

To prepare 0.1M acetate buffer of pH 4.

Procedure

Stock solution A : 0.2M solution of acetic acid

Stock solution B : 0.2M solution of sodium acetate

 Mixed 41 ml of A and 9ml of B and diluted to a total of 100ml using distilled water.

Measured pH using pH meter.

A (ml)	B (ml)	pH
46.3	3.7	3.6
44	6	3.8
41	9	4
36.8	13.2	4.2
30.5	19.5	4.4
25.5	24.5	4.6
20	30	4.8
14.8	35.2	5
10.5	39.2	5.2
8.8	41.2	5.4
4.8	45.2	5.6

 

Result

0.1 molar acetate buffer of pH 4 was prepared and confirmed by measuring pH using pH meter.

 

 2. Preparation of Carbonate-Bicarbonate Buffer

Aim: To prepare 0.1 M carbonate- bicarbonate buffer of pH 10.1 

Procedure

Carbonate-Bicarbonate buffer has a pH range of 9.1–10.6 at 37^oC. To obtain a buffer of respective pH, mix A (sodium carbonate) and B (sodium acetate) in the proportions indicated below and adjust the final volume to 200 ml using distilled water. The pH can be checked using a sensitive pH meter.

Stock solution A : 0.2M solution of anhydrous sodium carbonate

Stock solution B : 0.2M solution of sodium bicarbonate

Mix 27.5 ml of A and 22.5ml of B and diluted to a total of 100ml using distilled water to obtain 0.1 M carbonate and bicarbonate buffer of pH 10.1. Measured pH using pH meter.

A (ml)	B (ml)	pH
4	46	9.2
9.5	40.5	9.4
16	34	9.6
22	28	9.8
27.5	22.5	10
33	17	10.2
38.5	11	10.4
42.5	7.5	10.6

 Result

0.1 molar carbonate bicarbonate buffer of pH 10 was prepared and confirmed by measuring pH using pH meter.

 

3. Preparation of Citrate Buffer (pH 3.0–6.2)

Aim: To prepare 0.1 M Citrate buffer of pH 4 

Citrate buffer has a pH range of 3.0–6.2. To obtain a buffer of respective pH, mix A (citric acid) and B (trisodium citrate) in the proportions indicated below and adjusted the final volume to 100 ml using distilled water The pH can be checked using a sensitive pH meter.

Stock solution A - 0.2 M citric acid monohydrate

Stock solution B - 0.2 M trisodium citrate, dihydrate

Mixed 59 ml of A and 41 ml of B diluted to 200 ml using distilled water to obtain 0.1 M Citrate buffer of pH 4

 Result

The buffer prepared is 0.1 M Citrate buffer with pH 4. It is confirmed using a pH meter.

 

Solution A (ml) (citric acid)	Solution B (ml) (trisodium citrate)	pH
82.0	18.0	3.0
77.5	22.5	3.2
73.0	27.0	3.4
68.5	31.5	3.6
63.5	36.5	3.8
59.0	41.0	4.0
54.0	46.0	4.2
49.5	50.5	4.4
44.5	55.5	4.6
40.0	60.0	4.8
35.0	65.0	5.0
30.5	69.5	5.2
25.5	74.5	5.4
21.0	79.0	5.6
16.0	84.0	5.8
11.5	88.5	6.0
8.0	92.0	6.2

 

4. Preparation of Phosphate Buffer

Aim: To prepare 0.1 M Phosphate buffer of pH 7 

Phosphate buffer has a pH range of pH 5.8–8.0 at 25°C. To obtain a buffer of respective pH, mix A (sodium phosphate, dibasic dihydrate) and B (sodium phosphate, monobasic) in the proportions indicated below and adjusted the final volume to 100 ml using distilled water. The pH can be checked using a sensitive pH meter.

Stock solution A - 0.2M sodium phosphate, dibasic dihydrate

Stock solution B - 0.2M sodium phosphate, monobasic, monohydrate

Mixed 30.5 ml of A and 19.5 ml of B diluted to 100 ml using distilled water to obtain 0.1 M phosphate buffer of pH 7  

Solution A (ml) (sodium phosphate, dibasic dihydrate)	Solution B (ml) (sodium phosphate, monobasic)	pH
4.0	46.0	5.8
6.15	43.85	6.0
9.25	40.75	6.2
13.25	36.75	6.4
18.75	31.25	6.6
24.5	25.5	6.8
30.5	19.5	7.0
36	14	7.2
40.5	9.5	7.4
43.5	6.5	7.6
45.75	4.25	7.8
47.35	2.65	8.0

  Result

The buffer prepared is 0.1 M Phosphate buffer with pH 7. It is confirmed using a pH meter.