Fit for purpose?

Almost every laboratory experiment and diagnostic assay relies on using pure water to generate results that can be trusted. Jim Keary explains why selecting the right water supply is more important and complex than you might think If you’ve ever spent any length of time working in a lab, you’ll know that scientists make use of water in a myriad of processes throughout the day, from HPLC and cell culture through to buffer preparation and wiping down benches. As such, they get through a lot of it. A typical laboratory is estimated to use around five-times as much water as a comparably sized office block – that’s around 35 million litres per year^1,2,3. Despite it being used in such a massive volume, water isn’t given much – if any – consideration. Typically, you walk over to the lab’s water dispenser, grab whatever’s on offer and press on with your experiments. Perhaps it’s time we started to pay more attention: if you consider that approximately 70% of HPLC performance problems are likely to be directly attributable to water quality in our experience, or that water impurities can affect most enzymatic reactions, you quickly begin to realise that water purity is something we shouldn’t be overlooking. Accuracy and precision are the aims of the game in science and being able to achieve this is dependent upon the quality of the materials you use: poor reagents in, means poor results out. If we take something as sensitive as spectroscopy and spectrometry, you can quickly imagine that impurities in the water used could lead to erroneous results. For example, when detecting factors down into the parts per trillion (ppt) range using a technique such as inductively coupled plasma mass spectrometry (ICP-MS), additional elements, ions or particulate matter are going to result in more errors than you calibrate for! Another frequently used and highly sensitive technique is chromatography. If you’re conducting high performance liquid chromatography (HPLC), then you are going to need to make use of water free from impurities as not doing so can, and will, have an adverse effect on the data generated⁴. Dissolved gases in the water can affect the pH, while organic compounds compete with the analyte in the mobile phase to reduce the effective levels of analyte retained in the column. In addition, bacteria can form blockages and influence the process via organic by-products, and ionic contaminants can affect some chromatographic separations. In combination, these all contribute to wasting the precious time and resources of you and your fellow researchers. It isn’t just those working with high-grade analytical instruments that need to be aware of the effects of poor water quality. For example, consider something as routine as the polymerase chain reaction (PCR). Used on a daily basis in a wide range of labs across the world, PCR is wholly dependent upon the action of DNA polymerases, enzymes that amplify single stranded DNA ‘templates’, to produce a vast number of additional copies. The PCR process can be inhibited relatively easily by water contaminants. For example, nucleases are enzymes that cleave the phosphodiester linkages between nucleotides, severely disrupting the PCR reaction to essentially leave you without a stable product. Nucleases aren’t the only danger factor: bacteria in the water will inevitably lead to problems, and nobody wants to risk completing a series of experiments only to realise that they’ve been inadvertently amplifying lengths of contaminating bacterial DNA! Non-biological contaminants can also affect PCR, as DNA polymerases are highly sensitive to various common cations (e.g. Cu²⁺, Fe²⁺, Ni²⁺), which can disrupt substrate binding and inhibit enzyme activity, further disrupting your experiments. Water contamination can even affect procedures commonly perceived to be highly robust, such as histology and immunohistochemistry (IHC). In the case of these techniques, bacterial contamination can lead to the introduction of artefacts in mounted samples, primarily as a result of the bacteria adhering to tissue sections. Bacteria can also release alkaline phosphatase (AP), which can interfere with those IHC protocols making use of AP for chromogenic detection. At the molecular level, several metal ions can cause unwanted precipitation reactions when at high enough concentrations in staining solutions, or even interfere with antibody-antigen binding reactions when performing IHC. Given just these few examples, the next time you need to troubleshoot experiments, it would probably make a lot of sense to first investigate the quality of your water as the likely source of your problems. There are several categories of impurities that may be present in water, the most conspicuous of which are suspended particles. These can include anything from vegetation through to colloids and pathogens adsorbed onto other particles. Such factors are typically the first to be removed, since their presence can lead to blockages in filters, columns and membranes as well as affecting a wide range of experiments (spectrometry being an obvious example). Another important contaminant to consider is the presence of microorganisms. Bacteria and other microorganisms in lab water can spell disaster for those carrying out research or analysis where sterile conditions are essential. They can affect biochemical reactions by competing with substrates at enzyme active sites, while they also produce various compounds such as nucleases and endotoxins that reduce the effectiveness of lab assays and reactions. Dissolved compounds account for the majority of water impurities. Dissolved inorganic compounds are the most abundant and typically exist as ions, which can affect both protein solubility and the successful formation of protein-protein and protein-lipid interactions. Organic compounds dissolved in the water are principally biological in origin and can greatly enhance microbial growth, contributing to many of the problems discussed above. They can even help contribute to the degradation of other experimental targets, such as proteins and nucleic acids, reducing the reliability of your data. Finally, dissolved gases mostly cause problems creating ionic instabilities – carbon dioxide, for example, will dissociate in water to form carbonic acid, which can alter pH by up to 1.1 pH units. This is important, as changes in pH can disrupt a broad range of reactions. As we have seen, there is a lot to consider when choosing the right water supply and purity for your experiments: nucleases, dissolved gases, colloidal matter, organic content and more. Fortunately, a set of discrete categories have been created in order to facilitate simple management of your lab water’s purity. Referred to as Types I, II and III, these classes of water have been subjected to rigorous purification processes to achieve reproducible standards. For advice on which type to use for your specific lab processes and applications, please see our new infographic. These grades of water have been created using a series of purification steps such as filtration, reverse osmosis, UV-light treatment or electrodeionisation. This means that each type of water has a set of established physical and chemical parameters than you can use to match with the requirements of your experiments. Hopefully, it’s now evident that water purity should form an integral part of your experimental design. With instrumentation becoming increasingly more sensitive and many labs frequently working with infinitesimal concentrations, the purity of lab water is now more important than ever. Don’t let poor water quality get in the way of your work – make sure you select the right water type for your application.

References

1. European Commision (DG ENV0). June 2009. Final Report, Study on water performance of buildings. Available at: https://bit.ly/1iY3e0S
2. Good Campus Guide, S-Lab Briefing 5: Reducing Water Consumption in Laboratories. Available at: https://bit.ly/1oZaHOZ
3. University of Oxford and AECOM Ltd., University of Oxford Water Management Strategy Report. January 2011. Available at: https://bit.ly/1m3JEjD
4. Whitehead, P. 1998. Ultra-pure water for HPLC. Why is it needed and how is it produced? Laboratory Solutions, December Issue.
5. Based on decades of experience obtained by the water purification experts at ELGA LabWater.
6. Lane A.N., Arumugam S. 2005. Improving NMR sensitivity in room temperature and cooled probes with dipolar ions. Journal of Magnetic Resonance, 173(2), pp.339-33.
7. Rezaei, K., Jenab, E., Temelli, F. 2007. Effects of Water on Enzyme Performance with an Emphasis on the Reactions in Supercritical Fluids. Critical Reviews in Biotechnology, 27(4), pp.83-95.

Author

Jim Keary is Laboratory and Process Manager for ELGA Labwater. Jim is a chemist, who along with his team, conducts research and development into new products and processes.