Sodium Phosphate Buffer

WFID: 10001   Date: 10-20-2011  
Protocol Format: Public Draft.
Carlo Rago  
Molecular Biology, Buffers
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Introduction



Sodium phosphate buffer is used routinely in the life sciences. The protocol below is fairly straight forward and describes how to make buffered solutions at various pH's; I've used it successfully for PEGylation of proteins at pH 7.4 and a concentration of 50 mM.


The relevant pKa for sodium phosphate is 6.82 (the other pKa's are 2.15 and 12.38; these are not significant in the pH range used here).  The recommended buffering range is pH 5.8 to 7.8.

Materials

Catalog # Material Name Company Name Solute/Solvent
1S8282sodium phosphate monobasicSigmaSolute
2S7907sodium phosphate dibasicSigmaSolute
32613991pH indicator paperWhatman

Protocol



Stock solution A: 1 M monobasic sodium phosphate, anhydrous, MW 119.98 (thoroughly mix 120.0 g/liter of water)


Stock solution B: 1 M dibasic sodium phosphate, anhydrous, MW 141.96 (thoroughly mix 142 g/liter of water) note: I use 18 megaohm ddH20 water for critical applications.


Using the chart below, mix the appropriate volume of solutions A and B to achieve a 1M buffer solution at the desired pH:


Volume of Solution A (ml), Volume of Solution B (ml), pH
92.0, 8.0, 5.8
90.0, 10.0, 5.9
87.7, 12.3, 6.0
85.5, 15.0, 6.1
81.5, 19.5, 6.2
77.5, 22.5, 6.3
73.5, 26.5, 6.4
68.5, 31.5, 6.5
62.5, 37.5, 6.6
56.5, 43.5, 6.7
51.0, 49.0, 6.8
45.0, 55.0, 6.9
39.0, 61.0, 7.0
33.0, 67.0, 7.1
28.0, 72.0, 7.2
23.0, 77.0, 7.3
19.0, 81.0, 7.4
16.0, 84.0, 7.5
13.0, 87.0, 7.6
10.5, 89.5, 7.7
8.5, 91.5, 7.8


For example, 45 mls of Sol A + 55 mls of Sol B = 1M sodium phosphate solution at pH 6.9. Also, you may dilute this to a desired concentration with water and the pH should stay the same. However, use Whatman or EMD pH indicator strips to double check the pH after dilution. I use 18 megaohm distilled water for dilution. Also note that the buffering capacity depends on the concentration of the sodium phosphate, so dilution will decrease buffering capacity.

Attachments

References

1. www.openonward.org

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