I prepared DPBS (pH 7.4) and filter sterilized it using a 0.2 micron membrane filter. When I checked the pH of filtered buffer, it raised to 7.79.
What could be the reason behind this?
Yes it does, even for preparing media for tissue culture, a decimal digit difference is often kept as a margin when steralization is done using filter emmbranes.
Water comes first from the membrane as it has the minimal molecular size thus, initially the solution becomes near neutrility first and then shifts to respective pH but it never reaches to the same as unfiltered one as some of teh left over also trapps some hydronium ions with it as for its own aqueous ionization.
Yes it does, even for preparing media for tissue culture, a decimal digit difference is often kept as a margin when steralization is done using filter emmbranes.
Water comes first from the membrane as it has the minimal molecular size thus, initially the solution becomes near neutrility first and then shifts to respective pH but it never reaches to the same as unfiltered one as some of teh left over also trapps some hydronium ions with it as for its own aqueous ionization.
The only thing I can think of is that during the filtration the sample is degassed, changing the level of carbonic acid. Does the pH return to the original 7.4 after exposure to air after a few hours? I've only observed this with unbuffered solutions though...
Thanks all for your explanations
@ Paul
I checked the pH after 12 hr storage, it is stable at 7.7.
DPBS is not a very strong buffer, i.e. the ionic strength is rather small. Therefore, it could be that leachables from the membrane interfere with the buffer and thus, change the pH. My recommendation would be to wash the filter with WFI prior to pass the buffer through. Alternatively, the filter could be washed with a part of the buffer (50-100 ml) first which is discarded. Then the rest of the buffer is filtered. This can easily be checked the next time when you prepare a buffer.
The Donnan effect is not applicable I think because the filter is not a semi-permeable membrane.
Thank you for your input Alexander Huwig. I'll take care of these suggestions in next preparation.
Recipe of buffer:
KCl : 200 mg/L
KH2PO4 : 200 mg/L
NaCl : 8000 mg/L
Na2HPO4.7H2O : 2160 mg/L
Dissolved all the ingredients in MQ water and adjusted the pH at 7.4
@ Ramon
I did following changes in preparation
1. Initially adjusted the pH to 0.2-0.3 units below the desired pH
2. As Alexander suggested, washed the filter with initial 50 ml of buffer and discarded
Thanks Namita,just to clarify: you did 1 AND 2? adding initial 50 ml of buffer wasn't enough?
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques
- Analytical Chemistry
- pH