Running up against the buffers

Chemists have known since at least as early as the 1930s that the pH of a buffer solution, which is commonly used to maintain the acidity or alkalinity of a laboratory sample can change if the temperature of the sample drops. The cooling process leads to a fall in buffer pH towards higher acidity for some systems and a rise towards alkali in others when the buffered sample is cooled.

Now, researchers at the University of Illinois at Urbana-Champaign in the USA have developed a simple solution to this problem which has plagued researchers in laboratories across academia and industry for decades. Their new approach will hopefully avoid some of the frustrations of buffer failure. Put simply, the new buffer, developed by chemistry professor Yi Lu and colleagues Nathan Sieracki, Hee-Jung Hwang, Michelle Lee, and Dewain Garner maintains the desired pH across a range of low temperatures.

The team used ratiometric absorption spectroscopy and other techniques to monitor their temperature-independent pH buffer and explain that it works through a combination of buffers of opposite-sign temperature coefficients. They have already demonstrated proof of principle for the use of this buffer in low temperature spectroscopy and the storage of pH-sensitive compounds.

Writing in the journal Chemical Communications, Lu and colleagues point out that freezing is the standard physical method of extending the shelf life of biological specimens and pharmaceuticals. Biological samples, for instance, are routinely cooled to reduce the rate of chemical reactions in some experiments.

"We like to freeze proteins, nucleic acids, pharmaceutical drugs and other biomolecules to keep them a long time and to study them more readily under very low temperatures using different spectroscopic techniques and X-ray crystallography," Lu explains, "But when the pH changes at low temperature, the sample integrity can change." Even tiny changes in the acidity or alkalinity of a sample can influence its properties, Lu adds.

Graduate student Nathan Sieracki demonstrated this effect by repeatedly freeze-thawing oxacillin, a penicillin analogue. After just one freeze-thaw cycle, 50 percent of the drug was dead in several of the buffers investigated, Sieracki found. He was then able to demonstrate that this loss of activity was due simply to changes in pH as opposed to chemical change caused by the rising and falling temperature.

To find a buffer that would maintain a stable pH at different temperatures, Sieracki first tested the way several commonly used buffers behave over a range of temperatures. He showed that while some buffers became more alkaline on cooling others became more acidic with the same temperature change. This observation led to an almost obvious eureka moment. "Why don't we just mix them together," Sieracki thought.

By trial and error, he varied the proportions of the combined buffers until he found a formulation that showed a negligible change in pH at a variety of temperatures. Instead of registering changes of 2 or more pH units while cooling, which is typical of many standard buffer solutions, his optimal formulation changed by less than 0.2 pH units during cooling. "We're effectively cancelling out 100-fold changes in proton concentration and bringing them down within an order of magnitude," Sieracki explains.

Lu adds that the creation of such a temperature-independent pH, or TIP, buffer could have broad implications for new - and previously published - research. "We're not in the business of looking at the literature and correcting other peoples' mistakes," he said, "But some of the conclusions from previous studies could be on shaky ground if a buffer was used that changed pH dramatically at low temperatures."

The researchers say that their new TIP buffer will be of immediate use in biological research. It was about 75 years ago, that Finn and co-workers first reported the denaturation of proteins contained in muscle juice, which they explained as being due to variation in hydrogen ion and salt concentrations upon freezing. Subsequent studies showed similar pH cooling effects on enzymes, including aldolase, phosphofructokinase and several dehydrogenases in sodium and potassium phosphate buffers at lower temperatures.

Sieracki is confident that a similar buffer could be made for use in many fields, such as biochemistry, biophysics, chemical biology and biomedical research and in low-temperature spectroscopy. "It is surprising that no one has thought to try this in 75 years," Lu told SpectroscopyNOW, "We searched the literature but could not find a publication for such a method. We could not believe it, either."