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Researchers at the University of Illinois have found a simple solution to a problem that has plagued scientists for decades: the tendency of chemical buffers used to maintain the pH of laboratory samples to lose their efficacy as the samples are cooled. The research team, headed by chemistry professor Yi Lu, developed a method to formulate a buffer that maintains a desired pH at a range of low temperatures.

The study appears this month in Chemical Communications.

Scientists have known since the 1930s that the pH of chemical buffers that are used to maintain the pH of lab samples can change as those samples are cooled, with some buffers raising and others lowering pH in the cooling process.

Freezing is a standard method for extending the shelf life of biological specimens and pharmaceuticals, and biological samples are routinely cooled to slow chemical reactions in some experiments. Even tiny changes in the acidity or alkalinity of a sample can influence its properties, Lu said.

“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 said. “But when the pH changes at low temperature, the sample integrity can change.”

Graduate student Nathan Sieracki demonstrated this by repeatedly freezing and thawing oxacillin, a penicillin analog used to treat infections.

“After one freeze-thaw 50 percent of the drug was dead in several of the buffers investigated,” Sieracki said.

Sieracki was able to demonstrate that the loss of activity was due to changes in pH and not a result of the temperature changes.

To find a buffer that would maintain a stable pH at varying temperatures, Sieracki first evaluated the behavior of several commonly used buffers over a range of temperatures. He saw that some buffers became more alkaline at lower temperatures while others grew more acidic.

These observations led to an obvious methodology: “Why don’t we just mix them together?” Sieracki said.

Little by little, he varied the proportions of the combined buffers until he found a formula that exhibited minimal pH changes at a variety of temperatures. Instead of registering changes of 2 or more pH units while cooling, which was typical of some standard buffers, the new formula changed less than 0.2 pH units during cooling, he said.

“We’re canceling out 100-fold changes in proton concentration and bringing them down within an order of magnitude,” Sieracki said.

The creation of a temperature-independent pH (TIP) buffer could have broad implications for new – and previously published – research, Lu said.

“We’re not in the business of looking at the literature and correcting other 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 new buffer is immediately useful for biological research, and Sieracki said he is confident that a similar buffer could be made for use in many fields, such as biochemistry, biophysics, chemical biology and biomedical research.

Note for pH 
pH is a measure of the acidity or alkalinity of a solution. Aqueous solutions at 25°C with a pH less than seven are considered acidic, while those with a pH greater than seven are considered basic (alkaline). When a pH level is 7.0, it is defined as 'neutral' at 25°C because at this pH the concentration of H3O+ equals the concentration of OH− in pure water. pH is formally dependent upon the activity of hydronium ions (H3O+), but for very dilute solutions, the molarity of H3O+ may be used as a substitute with little loss of accuracy. (H+ is often used as a synonym for H3O+.) Because pH is dependent on ionic activity, a property which cannot be measured easily or fully predicted theoretically, it is difficult to determine an accurate value for the pH of a solution. The pH reading of a solution is usually obtained by comparing unknown solutions to those of known pH, and there are several ways to do so.

Note for X-ray Crystallography
X-ray crystallography is the science of determining the arrangement of atoms within a crystal from the manner in which a beam of X-rays is scattered from the electrons within the crystal. The method produces a three-dimensional picture of the density of electrons within the crystal, from which the mean atomic positions, their chemical bonds, their disorder and sundry other information can be derived. By definition, a crystal is a solid in which a particular arrangement of atoms (its unit cell) is repeated indefinitely along three principal directions known as the basis (or lattice) vectors. A wide variety of materials can form crystals — such as salts, metals, minerals, semiconductors, as well as various inorganic, organic and biological molecules — which has made X-ray crystallography fundamental to many scientific fields.
The oldest and most precise method of X-ray crystallography is single-crystal X-ray diffraction, in which a beam of X-rays is reflected from evenly spaced planes of a single crystal, producing a diffraction pattern of spots called reflections. Each reflection corresponds to one set of evenly spaced planes within the crystal. The density of electrons within the crystal is determined from the position and brightness of the various reflections observed as the crystal is gradually rotated in the X-ray beam; this density, together with supplementary data, allows the atomic positions to be inferred. For single crystals of sufficient purity and regularity, X-ray diffraction data can determine the mean chemical bond lengths and angles to within a few thousandths of an Ångström and to within a few tenths of a degree, respectively. The data also allow the static and dynamic disorder in the atomic positions to be estimated, which is usually less than a few tenths of an Ångström.

Note for Nucleic Acid
A nucleic acid is a macromolecule composed of nucleotide chains. In biochemistry these molecules carry genetic information or form structures within cells. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are universal in living things, as they are found in all cells. They are also found in viruses.
Artificial nucleic acids include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule.

In figure 1, Workflow for solving the structure of a molecule by X-ray crystallography
In figure 2, Chemistry professor Yi Lu, right, and graduate student Nathan Sieracki have developed a chemical buffer that maintains a desired pH at a range of low temperatures