If you are using the same quantities as always maybe you have to check water pH?

I don't prepare my Tris Buffer as you do, because I always adjust pH with HCl solution.

Sounds like whoever made your pre-mixed salts may have had a problem with their process.  You should use the equation of Henderson and Hasselbalch.  We have a calculator on our site that does it for you.  It tells you exactly how much tris acid and tris base you need to weigh in, to get a given molarity and pH.  That way you have control over the process and aren't blindly dependent on some company's fallible quality control procedures. 

You can contact Sigma customer support and confront them with the problem. Tell them the lot number of your package and they can check whether there were discrepancies during manufacture.

Regards, Christian

Thanks for all the comments. I did contact Sigma and it looks like the pouched premixed salts have been withdrawn, probably already some years ago. I assume the products passed QC and do not share the view that they did have dodgy QC or that they did not know what they were doing as they correctly instruct people to weigh in the acid and base salt in fixed ratios to get the desired pH.

"If Trizma components are thoroughly desiccated, it is possible to weigh the correct ratio and achieve a predicted pH with an accuracy of ± 0.05. Thus, under careful conditions, the use of a pH meter for confirming the pH is unnecessary. "

I am more worried about interactions with the foil pouches or something like that as they still sell the premixed salts, but in an plastic container instead. I assume that the mix ratio of the Supelco document is correct

https://www.sigmaaldrich.com/Graphics/Supelco/objects/4800/4709.pdf

Trizma Mixing Table (for 0.05M Solutions)

pH                           TrizmaHCl        TrizmaBase

5°C    25°C    37°C     (Grams/Liter) + (Grams/Liter)

8.07    7.50    7.22      6.35               1.18

is correct.

I have never been fond of titrating buffers as it should not be necessary if you weigh in the correct salts. At the time I was sceptical of the pH meter used thus chose the premixed salts to avoid issues with non dessicated salts and having experienced people adjusting the pH of CO2 dependent buffers told me that the world has gone mad. In fact thermo fisher gives the following protocol

Procedure A: Prepare Autoclavable MEM Powdered Medium (1X) (Cat. no. 11700*)

To a mixing container that is as close to the final volume as possible, add 950 mL of distilled water.

Add powdered medium to room temperature (15°C to 30°C) water with gentle stirring.

Rinse inside of package to remove all traces of powder.

When completely dissolved, adjust the pH to 4.1 to 4.2 with 1 N HCl.

Dispense 95 mL of the liquid into 100-mL bottles. Do not cap tightly.

Autoclave the medium for at least 15 minutes at 121°C on a slow exhaust cycle. However, due to equipment variability, we recommend that you qualify your autoclave sterilization cycle.

Allow the medium to cool to room temperature.

Aseptically add 3 mL of 7.5% NaHCO3 solution to each bottle. Mix well.

Aseptically add L-glutamine and antibiotic solutions, serum, or other supplements, as desired. Mix well.

Aseptically adjust the medium to final pH 7.2 to 7.4 by slowly adding, with stirring, 1 N NaOH or 1 N HCl, if necessary.

This last step told me that the people at Thermo Fisher struggle with the concept of a CO2 dependent medium as DMEM is designed to work at 5% or 10% CO2 depending on the amount of sodium bicarbonate used and that you destroy the buffer by adjusting the pH in ambient atmosphere.

If someone else uses the premixed salts in pouches I would be interested to find out what pH the product gives them, as it would invalidate a lot of science

If the pH meter is OK, this might be from wrong protocol you follow. Try the protocols in the Lab FAQ book of Roche

+Mostafa

If you buy pre-set salts for 1 litre there is no protocol to follow apart from dissolving the salts in 1 litre of water

Sorry, I might get it wrong, but I think Tris-Base should be in pH about 8.0 this is why we should add HCL if we are supposed to make a Tris-HCl buffer. right? 

I have used this protocol but for regular tris-base powder, not the product you are using. 

*Dissolve 121.14 g tris(hydroxymethyl)aminomethane

(Tris, MW = 121.14) in 800 ml H2O.

**Adjust pH to the desired value by adding concentrated HCl:

l pH 7.4: ~70 ml

l pH 7.6: ~60 ml

l pH 8.0: ~42 ml

** Adjust volume to 1 liter with H2O.

** Sterilize by autoclaving and store at room temperature (+15 to +25°C).

+Mostafa

Thanks for that. In order to cut out all the problem of pH adjustment and the handling of concentrated HCL I bought the salt premix of Tris-base and Tris-HCL which is how you make a pH standard as we can weigh in the components most accurately. I find it hard to believe that they had a weighing issue at the manufacturers end and a QC-issue so I wonder if there is a reaction with the pouch material which might have wider implications with regards to materials compatibility that would need looking into

indeed. I had the same experience with an RNA extraction kit in the past!

+Mostafa

what do you mean? interference of the pouch packing?

I know that for our pregnancy tests we insisted on secondary packing to protect the foil pouches from puncture as the tests have a desiccant to keep them dry but I would not think hygroscopic interactions would foul the premixed salts. They could however effect the weight of single salts depending on exposure.

I never got a satisfactory answer from Sigma explaining the discrepancy, the best explanation for the pH discrepancy could have been that someone prepared the buffer for use in the hot, but the pouch clearly promised pH7.5 at 25 degrees. So just be aware that your pH 7.5 buffer will turn pH8 in the fridge and at 8.4 you would have to be in a freezer at-16. But then PBS turns from pH7.4 to pH 3 upon freezing, so whatever is worse for our samples :-)