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Favourite TA solutions

  • timer  8.5 Minutes to read
  • 09 October 2020
  • Written by Michèle Heidemeyer
  • Life Science

Do you have a favourite solution? In other words, any buffer or solution you genuinely prefer to make – or one you can potentially create with particular precision?

For me, the absolute number-one laboratory formulation is the 0.5 M EDTA pH 8.0 solution. That may not initially come across as a very noteworthy or creative achievement – especially as this solution is one of the most common laboratory mixtures. However, its production is nowhere near as simple as many at first expect.

For example, I fondly recall my colleague "Mrs. Perfect" and her attempt to produce 0.5 M EDTA pH 8.0: It took her all of eight hours! Even then, the solution was still not ready. And who knows how long the no-doubt absolutely exhausted magnetic stirrer would have had to keep stirring and stirring?

Throughout the whole procedure, it never occurred to Mrs. Perfect to ask a colleague why this dumb substance didn't want to dissolve. Perhaps the magnetic stirrer would have gone on strike at some point if I hadn't asked her how long she was planning to use it for her solution? She looked embarrassed because, even though she was now about to boil the liquid, this stupid salt just didn't want to dissolve!

I was astonished to find I had to explain something to Mrs. Perfect, our little laboratory mouse, that she obviously wasn't yet aware of. So I rather smugly mentioned using heat was not the right way to persuade EDTA to go into solution. This should actually be done by adding the correct base (NaOH).

EDTA just happens to be a very stable substance that can only be converted into a solution with a lot of patience and the right amount of NaOH. So here's the process again, for all Mrs. Perfects to reference:

0.5M EDTA pH 8.0

  • Weigh out 186.1 g EDTA (x2H2O)
  • Add 800 ml MQH2O
  • Add about 18-20 g of NaOH pellets to adjust the pH
  • Top up the solution to 1 l

 

Second place on my all-time list of popular laboratory liquids goes to the 4% PFA solution. Like EDTA, PFA is also very sensitive to changes in pH, but the PFA reaction is even slower. While EDTA can be prepared relatively quickly, PFA requires a whole lot more patience and dedication. Here's a little story about "Mr. Slap-Dash". One morning, we were due to start a big experiment which we had been planning for months. Unfortunately, Mr. Slap-Dash had left the laboratory in a hurry the evening before, and in his haste had forgotten to prepare the PFA for the following day. And now he was stood in front of our magnetic stirrer trying to speed up the procedure – so it was quite predictable something might go wrong. Mr. Slap-Dash had the great idea of simply bringing the solution right to the boil. So while the PFA was simmering and spluttering away, I saw my chance and offered Mr. Slap-Dash the use of my fresh PFA solution from the previous day – on condition that he listen to my explanation about why a PFA solution should never be heated above 70°C. I then explained:

"If you heat up the whole lot, the paraformaldehyde breaks down and produces formaldehyde as a gas. The formaldehyde dissolves into the PBS solution. In this form, it is known as a formalin solution. When the whole liquid is heated to above 70°C, the formaldehyde produced in the formalin solution is driven off. That's why the concentration of the solution is degraded if it's allowed to overheat or boil. Visual changes become apparent if you use a lower concentration, especially under a microscope."

Since this incident, the penny has dropped – Mr. Slap-Dash now prepares with more patience and warms solutions up more slowly.

4% PFA solution for tissue fixation

  • Heat 800 ml of 1X PBS to 60°C
  • Add 40g PFA (under a fume hood)
  • Slowly increase the pH by adding 1N NaOH dropwise from a pipette until the solution clears
  • Let the solution cool
  • Adjust the volume of the solution to 1 l with 1X PBS
  • Filter the solution
  • Check the pH value (target value = 6.9) and adjust it with small amounts of dilute HCl if necessary

My favourite liquids bronze medal goes to the 10X-concentrated phosphate-buffered saline solution, or PBS for short. In principle, this solution is very simple to manufacture. The more precisely you weigh it out, the more accurate your final solution will be. And if you can perfect the weighing out, there will be no need for any subsequent adjustment of the pH value.

Although there might not be any production problems, I still have a funny story from the laboratory to share with you. Our apprentice "Greenhorn" tried to produce 10X PBS several times, failing miserably each time because of the pH value. For some reason, he always ended up with a pH of 3.5! Yet with a physiological solution, an optimal value should be much more like 7.4. Since 3.5 is well off this target value, I decided to monitor Greenhorn and watched him very carefully, looking over his shoulder throughout the preparation. He was really thorough and weighed each substance with absolute precision. It was so accurate I thought I might not find an error before completion. Then I suddenly noticed Greenhorn's problem: the weighing in was quite correct, he had just chosen the completely wrong option! Where he should have used NaH2PO4, he had used Na2HPO4 instead. Then everything went wrong! As a result of this, we now not only mention reagents in all our recipes, but also include a note about their molar mass. Na2HPO4 x 2H2O at 177.99 g/mol clearly has a different molar mass to NaH2PO4 x 2 H2O at 156.01 g/mol.

10x PBS

  • 2 g KCl
  • 2 g KH2PO4
  • 80 g NaCl
  • 14 g Na2HPO4 x 2H2O
  • Add 800 ml MQH2O
  • Check the pH value (target value = 7.4)
  • Top up to 1 l

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