2 replies to this topic

[biotef]

We prepare tris glycine SDS buffer with the following composition [when diluted to 1X] 25 mM Tris, 192 mM Glycine 0.1% SDS in MilliQ water. We generally prepare 5X of the above solution and when we check pH its about 8.9 - 9.0 and diluting it with MilliQ to 1X it reaches 8.6 - 8.7.
Most protocols mention the pH to be about 8.3 and do not adjust pH. So how do we go about it. We use high purity chemicals i.e electrophoresis grade and that too from Sigma. Do we adjust the pH of this buffer or use it as it is.

Also with reference to 1.5 M tris pH 8.8 Buffer preparation. We adjust its pH at 25º C to 8.8 but when we check next day the pH is different. Can anyone guide me with exact amount of HCL to be added to prepare 1.5 M tris pH 8.8 and 1.0 M Tris pH 6.8. I tried henderson calculator for tris buffer preparation but have to add more HCL then prescribed there.

Kindly guide me on this.

regards

[protolder]

biotef, on 19 July 2011 - 08:39 PM, said:

We prepare tris glycine SDS buffer with the following composition [when diluted to 1X] 25 mM Tris, 192 mM Glycine 0.1% SDS in MilliQ water. We generally prepare 5X of the above solution and when we check pH its about 8.9 - 9.0 and diluting it with MilliQ to 1X it reaches 8.6 - 8.7.
Most protocols mention the pH to be about 8.3 and do not adjust pH. So how do we go about it. We use high purity chemicals i.e electrophoresis grade and that too from Sigma. Do we adjust the pH of this buffer or use it as it is.

Also with reference to 1.5 M tris pH 8.8 Buffer preparation. We adjust its pH at 25º C to 8.8 but when we check next day the pH is different. Can anyone guide me with exact amount of HCL to be added to prepare 1.5 M tris pH 8.8 and 1.0 M Tris pH 6.8. I tried henderson calculator for tris buffer preparation but have to add more HCL then prescribed there.

Kindly guide me on this.

regards

Hola, this is normal about tris. In my lab and probably in the Sambrock manual tris buffers specially high concentration is reajusted of pH and volume the next day, after pH is already mantained. The differences about tris-glycine depends of many factors, the quality of reagents, the pHmeter adjust etc, for me little variations as 0.3 pH units aren´t important and I would use it as you have, except that any of the excepcional forers and moderators that we have disagree. Buena suerte

[mdfenko]

temperature has an effect on the apparent pH of many buffers, including tris. when adjusting with hcl the temperature of the solution probably rose. you should ensure that your final pH determination is at the temperature that you want.

also, sds will have an effect on the electrode. that's why you are told not to adjust. it can be done but you have to take special precautions and you have to ensure that conditions of the reading are exactly reproducible or results between batches can vary a lot.