I'm preparing tris buffers of molarity 1.5 and 0.5 M with respect to pH 8.5 and 6.5 respectively. The conductivity of molar concentration 1.5 is 26.0 ms/cm and for 0.5 M is 33.6 ms/cm. Is it a right calculation or is some thing is wrong with it. I was thinking that 1.5 M should have higher conductance.
The pK of Tris is 8.07 at 25 °C. Therefore, at pH 8.5, less than half of it is ionized, whereas at pH 6.5, it is fully ionized (incidentally, it looses its buffer capacity at such a low pH). Hence the higher conductivity at pH 6.5 in spite of the lower molarity
Use the Henderson-Hasselbalch equation.
Just remember that, unlike acetate or similar, the Tris base has no charge.
According to the conductivity data that you present, you carried out the preparation of the buffer by neutralizing Tris base with HCl. It is the only way for the Tris 0.5 M to give a conductivity higher than 1.5 M Tris. If you had used Tris`s conjugated acid to obtain the final solutions by partial neutralization with NaOH, the ionic load of 1.5 M Tris It would have been triple that of 0.5 M Tris.
The 1.5 M Tris (pH 8.5) is a buffer with only 26.6% of all Tris in the form of conjugated acid (R3NH+), that is, 0.4 mol/L and the solution also contains 0.4 mol/L of chloride approximately. The conductivity depends on this ionic load and a relatively small amount of OH- (10-5.5 mol/L).
On the other hand, 0.5 M Tris (pH 6.5) is not a buffer, 97.3% of Tris is in the form of R3NH+, that is, 0.487 mol/L and the solution also contains around 0.487 mol/L chloride. This ionic load is 22% higher than that of 1.5 M Tris and correlates more or less with the 29% increase in conductivity. The ionic load-conductivity relation is not necessarily linear but the difference may be partially due to the high ionic mobility of the H+ ions (10-6.5 mol/L) present in 0.5 M Tris.
Achim Recktenwald José Luis Duque-Rivera Pierre Béguin Is this mean that if by continuous adding HCl in Tris buffer pH would be ~2.0 and now by adding NaOH to maintain pH up to 8.5, there would be generation of NaCl which can ultimately interfere with its normal functioning such as in SDS-PAGE
Excuse me, instead of "ionic charge" I meant "ionic load". It was an idiomatic slip. Anyway, I already corrected my answer.
Achim Recktenwald, in effect, neutralizing R3NH+ with NaOH produces NaCl. This would make the conductivity of 1.5 M Tris really higher than 0.5 M Tris.
Dear Sandeep
If you plan to use the Tris-HCl buffer pH 8.5 for SDS-PAGE, you should titrate Tris base with HCl and bring the pH down to 8.5 and try not to overshoot. Bringing the pH back to 8.5 with important amounts of NaOH will be equivalent to adding NaCl and will jeopardize the stacking and migration of the samples.
Sandeep Kumar , the 1.5 M Tris is used in the preparation of the polyacrylamide gel, and its standard preparation is from Tris base. The initial alkaline pH is adjusted to 8.8 (8.5 in your case) with care, to avoid exceeding as it says Pierre Béguin.
The 0.5 M Tris (pH 6.8, in your case pH 6.5) is usually used in the preparation of the sample's 4X buffer. Its concentration in this buffer drops to 0.125 M. And finally, by reducing the sample buffer from 4X to 1X, this new dilution reduces the Tris to 0.03125 M. Overall, the dilution of the components of the 0.5 M Tris is 1/16 when the sample is injected into the gel, ie, any excess NaCl generated by mistake will also have a 1/16 dilution (there is an extra dilution when injecting well). Will the performance of the electrophoretic separation affect a lot? I do not know for sure.
If possible, and it would be an experience for you, use Tris 0.5 M with excess NaCl to run a sample in a single well of the gel, but work the rest of your samples with Tris 0.5 M prepared correctly (Tris base with HCl without exceeding pH 6.5). If you do not observe a resolution of different bands when comparing the two samples with different buffers (0.5 M Tris/NaCl and 0.5 M Tris correct), you could consider not discarding the 0.5 M Tris buffer (prepared with excess NaCl). It would be your decision. Then, if you decided to do this, tell us how it was.
José Luis Duque-Rivera I changed the buffers and now SDS-PAGE is working fine for me. This time I prepared 1.5M tris of pH 8.5 by careful adding HCl, then I prepared 0.5M by diluting 1.5M and again adjusted pH by carefully adding HCl for up to 6.5. Thanks for helping me José Luis Duque-Rivera Pierre Béguin Achim Recktenwald
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