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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).

5 56.5 43.3 6.3 b 55.9 8.3 7.1 6.0 77.8 7.1 7.5 8.2 7.0 87.0 23.0 7.5 31.0 90.0 10.0 84.5 62.0 81.0 81.5 26.0 67.5 7.9 6.0 28.0 19.1294 g in 100 ml of distilled water (c) 0.5 68.5 73.0 16.8 a 45.0 39.9 7.4 6.6 .0 72.7 pH 6.2.0 13.5 51.7 85.5 93.0 0.0 12.5 8.Table A.0 5. a 93. b pH 6.6 6.0 87.5 22. Phosphate buffer. pH 5.0 87.5 92.7 7.0413 g in 100 ml of distilled water 2.0 Schedule for preparation of phosphate buffers.4 7.0 10.0 61.5 37.7 6.0702 g in 100 ml of distilled water (b) 0.0 94.5 49.5 6.7 5.15 M Phosphate Buffer 1.15 M KH2PO4 Dissolve 2.15 M Na2HPO4 Dissolve 2.3 15.8 5. Preparation of stock solutions (a) 0.5 77.15 M NaH2PO4 Dissolve 2.2 6.6 7.0 6.0 18.0 91.0 33.0 5.5 7.

7. Phosphate buffer. pH 9.Mix 13 ml of (b) with 187 ml of (a). (Adjust pH if necessary) 3. Phosphate buffer.0 1.2 .0 (Used in the conjugation of FA) Same as above without the addition of hydrochloric acid (Used for FA conjugation) . Dissolve 10. Adjust the pH to 8. pH 8. (Adjust pH if necessary) 4. then dilute to 500 ml with distilled water 3.15 M Phosphate Buffer.2 ml of (b) with 67. pH 6.15 M Phosphate Buffer.8 ml of (c). Check pH occasionally 0. (Adjust pH if necessary) 0.4 Mix 32.0 by the dropwise addition of 1 N hydrochloric acid.6 g of anhydrous Na2HPO4 in about 450 ml of distilled water 2. pH 7.3 Mix 24 ml of (c) with 76 ml of (b).