APPENDIX 5

BUFFERS (FROM CONRATH 1972)

0.1 M Phosphate Buffer

1.

Prepare stock solutions.

(a)

0.2 M solution of monobasic sodium phosphate

(NaH2PO4.H2O)

Dissolve 27.8 g in 1000 ml of distilled

water.

(b)

0.2 M solution of diabasic sodium phosphate

Dissolve 52.65 g of Na2HPO4.7H2O or 71.7 g of
Na2HPO4.12H2O in 1000 ml of distilled water.

2.

Mix x ml of (a) with y ml of (b), according to the following

table, and dilute to a total of 200 ml with

distilled

water.

Phosphate buffered saline (0.01 M pH 7.1)

(Used in FA

purification)

Mix 330.0 ml of a with 670.0 ml of b, dilute with saline (8.5 g

of NaCl per liter of distilled water) to 20 liters.

Add

Merthiolate at a concentration of 0.01% (200 ml of a 1%

solution to 20 liters PBS).

Table A.2.

a
93.5
92.0
90.0
87.7
85.0
81.5
77.5
73.5
68.5
62.5
56.5
51.0

Schedule for preparation of phosphate buffers.

pH

6.5
8.0
10.0
12.3
15.0
18.5
22.5
26.5
31.5
37.5
43.5
49.0

5.7
5.8
5.9
6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8

a
45.0
39.0
33.0
28.0
23.0
19.0
16.0
13.0
10.5
8.5
7.0
5.3

b
55.0
61.0
67.0
72.0
77.0
81.0
84.0
87.0
87.0
91.5
93.0
94.7

pH
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0

0.15 M Phosphate Buffer

1.

Preparation of stock solutions

(a)

0.15 M NaH2PO4
Dissolve 2.0702 g in 100 ml of distilled water

(b)

0.15 M Na2HPO4
Dissolve 2.1294 g in 100 ml of distilled water

(c)

0.15 M KH2PO4
Dissolve 2.0413 g in 100 ml of distilled water

2.

Phosphate buffer, pH 5.6

Mix 13 ml of (b) with 187 ml of (a).

(Adjust pH if

necessary)

3.

Phosphate buffer, pH 6.4

Mix 32.2 ml of (b) with 67.8 ml of (c).

(Adjust pH if

necessary)

4.

Phosphate buffer, pH 7.2 - 7.3

Mix 24 ml of (c) with 76 ml of (b).

(Adjust pH if

necessary)

0.15 M Phosphate Buffer, pH 8.0

1.

Dissolve 10.6 g of anhydrous Na2HPO4 in about 450 ml of

distilled water

2.

Adjust the pH to 8.0 by the dropwise addition of 1 N

hydrochloric acid; then dilute to 500 ml with distilled
water

3.

Check pH occasionally

0.15 M Phosphate Buffer, pH 9.0 (Used in the conjugation of FA)

Same as above without the addition of hydrochloric acid (Used for

FA conjugation)