How pure is ultrapure?

Defining standards

While industry standards for pure water have not changed dramatically over the past few decades, water purification technologies are undergoing continuous refinement, with point-of-use benchtop systems now making ‘ultrapure’ water routinely accessible.

Increasingly stringent quality control measures enforced within many laboratories make methods to reduce contamination critical at every step of the drug discovery and development process. Since water is so abundant in the lab, and in everyday life, it can often be overlooked as the potential source of a problem. As awareness has improved, the demand for ultrapure water systems has increased. And, so too, as analytical techniques have become more sensitive, the requirement to significantly reduce, if not eliminate, contaminating substances has grown.

Because water purity is so important, several professional organisations have established water quality standards. These include the American Society of Testing and Materials (ASTM) and the Clinical and Laboratory Standards Institute-Clinical Laboratory Reagent Water (CLSI-CLRW). These organisations have similar, but not identical, definitions for highly purified water, which are determined on the basis of parameters such as conductance, resistance, the presence of colloids, bacterial count, organic content and pH. The most commonly used standard, ASTM D1193-6 is summarised in Table 1. Using ASTM nomenclature, type IV is the lowest grade of purity, while type I is the highest grade, suitable for highly sensitive applications. These can then be further subdivided into A, B and C, where A is the most pure, as a measure of heterotrophic bacteria count (CFU/ml) and endotoxins (units/ml).

With a range of purification techniques available, it is important that all users are able to accurately match the purity level to their application. Ideally, all laboratory water would be purified to the highest possible level, but there is often an associated increase in cost. Six major technologies can be used to purify water: filtration; adsorption; ultraviolet radiation; distillation; reverse osmosis; and deionisation. Water purification is a sequential process, often requiring a combination of these technologies, each of which varies in its ability to remove specific contaminants. For example, distillation and deionisation are excellent at eliminating inorganic ions, whereas combined ultraviolet and ultrafiltration (UV/UF) is very effective for removing organics, particles, bacteria, pyrogens and nucleases.

Application critical

Highly critical analytical and life science applications, such as high performance liquid chromatography (HPLC), polymerase chain reaction (PCR) and electrophoresis, for example, will suffer the most from high impurity levels. In essence, the presence of such impurities will have a significant adverse effect on resulting data, rendering the experiment void. For example, if nucleases are present in a PCR reaction, they will effectively cleave any DNA, including the primers and probes. Amplification will therefore not occur.

As the techniques employed for these sensitive applications have advanced, users are able to detect increasingly minute quantities of various products. However, in detecting these small volumes, instrumentation is now also able to detect trace impurities as well, which will skew the obtained data. As a result, the water used must be as close as possible to theoretically pure.

Obtaining ultrapure water

As the most advanced level of purity, Type I water is produced free from nucleases and pyrogens, which have a negative impact on sensitive applications such as PCR. One of the most problematic impurities, nucleases are small enzymes that are present in all cell types, making them naturally present in all laboratories. They are heat-resistant and active over a broad pH range, thus they re-nature readily and are easily transferrable via an analysts fingers, for example. Consequently, nucleases are present on most laboratory glassware and solutions: contamination therefore occurs on a daily basis and appropriate steps need to be taken to minimise the detrimental effects that will occur as a result.

Traditional methods for ensuring that water is nuclease-free include the use of a 0.1% solution of diethyl pyrocarbonate (DEPC), which effectively inactivates ribonuclease enzymes (RNases) in buffers and water. However, since DEPC is a non-specific inhibitor, it must be removed from treated solutions before being used with nucleic acids. This method is therefore time-consuming and can also be toxic. More recent developments have included efforts to increase efficiency through improvements in filtration and separation technologies, as well as decreasing operational costs.

A point-of-use ultrapure water system that can produce nuclease-free water without DEPC treatment would therefore be a valuable piece of laboratory equipment, eliminating a lengthy and potentially toxic method. Such water systems use both UV and UF technology, successfully combining the advantages of each to provide ultrapure water.

The benefits of UV: Ultraviolet light can effectively eliminate trace organic compounds and inactivates microorganisms through its photochemical oxidative action, which essentially oxidises organic compounds within the sample to produce carbon dioxide. Through the use of UV light at both 185 and 254 nm, very low bacteria and total organic carbon (TOC) levels can be obtained. The light produced at 254nm has optimal antibacterial properties, causing DNA mutations, whereas light at 185nm has the ability to break organic bonds to generate free radicals, which subsequently causes cell death. This combination of light therefore allows for total oxidisable carbon levels of less than 5 ppb.

The benefits of UF: Ultrafiltration uses size exclusion processes to remove particles and macromolecules such as pyrogens and nucleases. They are captured on the hollow polysulfone fibers of the filter membrane and flushed out of the system via a reject stream. These filters are positioned at the end of the system to ensure the elimination of all nucleases and pyrogens.

One such system, which combines the advantages of the UV and UF technologies, is the Thermo Scientific Barnstead Nanopure Life Science (UV/UF) system, which produces ultrapure water with RNase and DNase levels below the detectable levels, at 0.003 ng/ml and 0.4 pg/μl, respectively.