HEPES is a zwitterionic organic buffering agent. It is stable, water soluble and does not react with divalent cations. It has a pKa of 7.5 at 37ºC and offers an optimal buffering range of pH 6.8 - 8.2. It maintains the physiological pH despite changes in the carbondioxide concentrations which take place due to cellular metabolism. HEPES is widely used in cell culture media to maintain the pH levels of basal media. Unlike the bicarbonate buffers which require CO2, HEPES containing media do not need CO2 atmosphere for buffering. HEPES is better than bicarbonate buffer in controlling the pH of the media in cell culture.
HEPES is generally used at a concentration of 10mM to 25mM for cell culture application. After addition of HEPES the pH of the media is adjusted with NaOH or HCl. It is also necessary to maintain the appropriate osmolality in the medium. Concentrations of 50mM and above can be toxic to the cells and are not recommended.
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