[RASMB] Buffers for CD

[Glen Ramsay]

Greetings David:

Acetate is commonly used. The acid and base forms for all buffers can have different absorbances, so expect some pH dependence. Also, if you are doing temperature melts, keep in mind that the enthalpy of protonization results in a pKa change that can cause the pH to drift with temperature. Acetate and phosphate are better, Tris and Tricine poor. Of course, keep in mind that there can be specific interactions between some samples and buffers, so some combinations will not work. At a low enough pHs (2?) self buffering kicks in, so the need for a buffer isn't as severe as at pH 7. At the other extreme, pH >10 will etch silica, so keep these experiments brief.

Keep in mind that that salts added for ionic strength can absorb too. If possible, keep these low, or substitute (when possible) non-absorbing salts for NaCl.

You can use the CD's dynode voltage to estimate the buffer absorbance. Most UV-Vis spectrometers don't go below 250 nm, so a CD spectrum of a new buffer is frequently the best test to see if it is going to work.

When possible, boost the sample concentration, use a shorter path length, and the CD signal will be the same but the buffer absorbance will be reduced. 1 mm may be the standard for CD, but it is not a requirement.

Glen

Glen Ramsay, Ph.D. 
Chief Scientist 
glen at avivbiomedical.com 
Aviv Biomedical, Inc. 
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Lakewood, New Jersey 08701 
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-----Original Message-----
From: David Scott [mailto:David.Scott at nottingham.ac.uk]
Sent: Thursday, February 9, 2012 08:00 AM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] Buffers for CD

st1\:*{behavior:url(#default#ieooui) }Hello All in RASMB-land,
I have a question on buffers for CD. I am performing CD experiments at different pH?s. Obviously for pH 7, a nice optically clear phosphate buffer means I can see down to 185 nm on a Jasco 815 spectrometer. However, I am having problems getting buffers that are equally optically clear at pH 5.5 and pH 3.0. Any suggestions? Advice and comments on suitable sources of chemicals to make the buffer that don?t have contaminating components in are also welcome.
Best wishes
Dave
>>>>>>>>>>>>>>>>>>>>>>>>>>
Dr. David J. Scott
Associate Professor and Reader in Physical Biochemistry
National Centre for Macromolecular Hydrodynamics
School of Biosciences
University of Nottingham
Sutton Bonington Campus
Leicestershire. LE12 5RD.
United Kingdom.
Phone: +44 (0) 115 951 6221
Fax: +44 (0) 115 951 6142
Email: dj.scott at nottingham.ac.uk
Web: www.nottingham.ac.uk/ncmh


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