HEPES Stock Solution (0.1 M, pH 7.4)

Youssef Farhat, Written 12/21/13, Last updated 12/21/13

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The purpose of this protocol is to prepare a 0.1 M HEPES stock solution at pH 7.4.  HEPES is a buffer that can be used to control the pH of many solutions, and this particular buffer is used in our lab to make assay buffers for fluorogenic substrate assays to measure enzyme activity in the presence of various inhibitory substances.

In this protocol, we will use a solid powder of pure HEPES (MW 238.3 g/mol) to make 100 mL of 0.1 M HEPES, pH 7.4.


  • HEPES (Sigma-Aldrich, #H3375)
  • NaOH pellets (J.T. Baker, #3722)
  • Deionized water
  • Normal lab equipment, such as a graduated cylinder, small chemical spatula, beakers, stir plate, stir rod, a pH meter, a scale


  1. Add 2.38 g of HEPES to an appropriate beaker (100-200 mL beaker in this case).
  2. Add about 80 mL of deionized water to the beaker.
  3. Add a stir bar to the beaker and leave it on a stir plate until completely dissolved (~1 min).
  4. Begin monitoring pH of the solution.  It should be acidic (pH ~5).
  5. Add one NaOH pellet to raise the pH towards 7.4.  It should reach about pH of 7
  6. Once the first pellet is fully dissolved, add a second NaOH pellet if necessary to raise the pH to 7.4.  Monitor carefully, and if the pH approaches 7.3/7.4 before the pellet is fully dissolved, stop the stir plate from spinning the rod and carefully remove the NaOH pellet with a clean spatula.
    1. Note: In my experience, about 1.5 pellets are just the right amount to raise the pH to 7.4, so retrieving the second pellet is necessary for achieving the right pH
  7. If the pH goes too high, lower it back to a pH of 7.4 by carefully adding a little HCl, while monitoring the pH (Caution: wear gloves, eye protection and exercise extreme caution with this acid solution)
  8. Once the pH of the solution is 7.4, add enough deionized water to raise the volume to 100 mL.
  9. Sterile-filter, if possible, and store in the refrigerator for up to 4 months or aliquot and freeze at -20 C for future use.

Citing this Protocol

Farhat, Y. “Reagent Preparation: HEPES Stock Solution (0.1 M, pH 7.4.” Protocol Place. Dec 2013. Accessed on mm/dd/yyyy. <http://protocol-place.com>

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