I use Tris-HCL as lysis buffer for thermophilic enzyme. Is it possible the PH of this buffer changes when I use high temperature for enzyme assay?
pH is based on the concentration of free H+ in solution. As the temperature increases the bonds holding the protons are broken and the pH increases. For example water is pH 7.47 at 0C, 7.00 at 25C and 6.14 at 100C. Different buffers have different temperature dependence, but Tris is know to be very poor for this property. See this link from Promega for some better details.
https://www.promega.co.uk/~/media/files/resources/technical%20references/temperature%20dependence%20of%20%20ph%20for%20common%20buffers.pdf
Hi,
It is a common phenomenon in biochemistry. Temperature will affect ionization capability in your solution then affect pH of buffer. This is why pHmeter devices have temperature electrode.
pH is based on the concentration of free H+ in solution. As the temperature increases the bonds holding the protons are broken and the pH increases. For example water is pH 7.47 at 0C, 7.00 at 25C and 6.14 at 100C. Different buffers have different temperature dependence, but Tris is know to be very poor for this property. See this link from Promega for some better details.
https://www.promega.co.uk/~/media/files/resources/technical%20references/temperature%20dependence%20of%20%20ph%20for%20common%20buffers.pdf
I completely agree with the above two answers. The pH of most of the buffers depends on the temperature at which it is being used. The pKa value of most of the amine buffers use to be temperature dependent, whereas the buffers containing carboxylic acid are comparatively less affected by temperature variation. The pH value of a Tris solution use to be 7.8, 8.4 and 7.4 at room temperature, 0°C and 37°C, respectively. Please refer to the link given bellow.
https://www.applichem.com/fileadmin/Broschueren/BioBuffer.pdf
The pH of your buffer will change with change in temperature, but extent will depend upon the variation in temperature. It is important that what temperature you are using for your thermostable enzyme, you can compare the changes by keeping a control. Otherwise, variations can be neglected.
You should prepare a Tris buffer in a such a way that at the temperature of the assay you`ll get the `desired` pH. For example, reading the deltapKa/deltaT for Tris at https://www.applichem.com/fileadmin/Broschueren/BioBuffer.pdf, pKa changes -0.028 pH units for each degree Celsius when temperature increases. If room temperature is 25 oC but you`ll use it at 50 oC, you will have a decrease of -0.028 x 25 = -0.7 pH units. Prepare your Tris buffer at room temperature adjusting the pH that you want plus 0.7. If you intend to have a pH 8.0 at 50 oC, adjust pH to 8.7.
Tris has a pKa of 8.07 at 25 °C, which implies that the buffer has an effective pH range between 7.07 and 9.07.The pKa declines approximately 0.03 units per degree Celsius rise in temperature. Source: http://en.wikipedia.org/wiki/Tris
USE one of GOOD's buffer have a lower deltapKA/degC
http://en.wikipedia.org/wiki/Good%27s_buffers
Good luck
If you apply the van't Hoff equation: dlnK/dT=dH/RT^2, to the proton dissociation of the buffer you get: dpKa/dT=-dHa/2.3RT^2, where pKa is the -log of the acid dissociation constant and dHa is the proton dissociation enthalpy (i.e. ionization enthalpy of the buffer). Thus, the ionization enthalpy of the buffer determines the extent of the pKa change with temperature, and, therefore, the change in solution pH with temperature.
The ionization enthalpy for Tris is quite high, a bit larger than 11 kcal/mol. Other buffers like phosphate or cacodylate have much smaller ionization enthalpies (close to or below 1 kcal/mol). That is the reason for the considerable temperature dependency of pH in Tris solutions. As already indicated, you may prepare the solutions for compensating such temperature-induced changes, or use other buffers with a smaller dpKa/dT (that is, smaller ionization enthalpy).
It is a question from a general chemistry/biochemistry course. Temperature do affect the equilibrium and, hence, the equilibrium constants (Ka or Kb). For sure you will get a more detailed explanation in a chemistry/organic chemistry text book or haveng a look at internet (search buffer pH and temperature just as an example).
Adding a reference for the ionization enthalpy correctly discussed above by Adrian, for further reading: http://www.nist.gov/data/PDFfiles/jpcrd615.pdf.
So you can prepare the buffer and adjust the pH at the temperature which you require for the enzyme assay to insure that you get the required pH value when it is at that temperature.
pH change is a problematic for lyophilization, especially if phosphate buffer is used. For example di basic phosphate will differentially precipitates than mono basic phosphate resulting a low pH, thus hurting the molecules. However, this pH change is relatively less for Tris. The best buffer in this case is Histidine or citrate buffer.
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