Pure and simple

Water is the most widely utilised analytical laboratory reagent, and is used daily to make buffers, lab reagents, calibration standards, instrument blanks, as well as culture media and sample dilutions. Here, Julie Akana asks – is your lab water pure enough?

HIGH PURITY water is a vital resource for every scientific application, including those in academia, and the pharmaceutical, cosmetic and food industries.

Ultrapure reagent grade water can be defined as water that is void of material that can potentially interfere with laboratory methods, reagents or standards. Due to the high volumes of water used across numerous experimental methods, including those of sample preparation, liquid and gas chromatography, and mass spectrometry, extreme care must be taken to ensure an ultrapure water supply. This article reviews the significance of water purity in high performance liquid Chromatography (HPLC), outlines the origins of organic impurities in water, the technologies used to remove them and illustrates their effectiveness. System designs and configurations that eliminate interference are also discussed.

Detection limits of advanced laboratory instruments such as inductively coupled plasma/mass spectrometry (ICP/MS), gas chromatography/mass spectrometry (GC/MS), HPLC and fluorescence can be in the part per trillion (ppt) range or lower, and at this level the potential of interference is high. The presence of trace organic compounds in purified water is of increasing concern in many laboratories and can affect resolution and integration, introduce ghost peaks, alter stationary phase selectivity and impact baselines. Reverse phase HPLC is utilised in analytical laboratories for the identification and quantification of organic compounds, so the presence of organic contaminants is especially worrying. These contaminants must be minimised or eliminated to allow researchers to consistently achieve accurate experimental results.

There are also non-organic contaminants that can interfere with experiments indirectly, but are equally as serious; for example, water impurities such as inorganic ions, particulates, or dissolved gasses can react with reagents or analytes during sample pre-treatment, by contamination of columns or detectors, or by effects on the lifetime of standards. The general objective is, therefore, not just to reduce the concentrations of specific impurities, but to provide pure lab water, an essential component to the production of consistent and accurate data.

There is a direct correlation between the purity of the lab water used to

Figure 1: Chromatographic comparison of the Barnstead Nanopure UV reverse phase gradients with bottled HPLC and conventional DI water

conduct an analytical test, such as HPLC, and the accuracy of the test result. For this reason, ultrapure reagent grade water is essential in analytical data  collection. This article explores this relationship using one of the most challenging and demanding lab applications of ultrapure water, an ultra-trace HPLC analysis.

Elimination of organic contaminates begins with the choice of lab water system. An ultrapure water system, also known as a Type 1 system, should be constructed of materials that resist leaching organic impurities into the water. The housing would ideally be made of a natural homopolymer (such as polypropylene) with no fillers, plasticisers or mould release agents. Purification media and resins should be semiconductor grade quality. Properly designed adsorption and ultraviolet (UV) oxidation techniques should be incorporated into the design of the unit to produce the organic-free water.

Leading manufacturers of these ultrapure reagent grade water systems conduct extensive testing to ensure each design and component configuration will work synergistically to produce high purity water, which can be used with confidence when performing reverse phase HPLC applications. By paying close attention to mobile phase interferences and the elimination of ghost peaks that can plague chromatography procedures, a variety of water sources can be compared to detect interfering material content. Developing an awareness of the sources and types of materials that cause interference can provide a reference point to eliminate the potential for contamination.

Here, a variety of water sources were compared and interference material identified using HPLC analysis. The HPLC test results are reported and an outline of the techniques
utilised to eliminate the potential background interference are identified. In addition, mobile phase interferences and the elimination of ghost peaks that can distort chromatography results are addressed in a comparison of HPLC grade bottled water and the water produced by the Thermo Scientific Barnstead Nanopure UV and Easypure ultrapure reagent grade water systems.

Figure 2: Comparison of the first samples drawn without any appreciable rinse up

Mobile phase baseline interferences have generally not been a problem at the higher wavelengths, such as 254nm or above. Most of the interfering organic species found in purified water are invisible to the detector at these wavelengths. However, interferences or ghost peaks can present problems at the lower wavelengths such as 210nm. Multiple water systems have been evaluated and direct HPLC comparison utilising different sources of water, including bottled (HPLC) water has been performed. Figure 1 presents a chromatographic comparison of the Barnstead Nanopure UV reverse phase gradients with bottled HPLC and conventional DI water. Both the Barnstead Nanopure UV and Easypure UV water systems produce baselines that are free of ghost peaks that could potentially interfere with chromatograms.

Plastic filters can potentially be a source of organic contaminants, so the 0.2 micron absolute filter was studied. The challenge is to provide absolute filtration without introducing contaminants from filtration particles, wetting agents, and bubble point chemicals. The amount of water required to rinse a filter, both new and before each use, must be evaluated and compared to the other filter material available. Figure 2 and figure 3 show the baselines from a Barnstead Nanopure UV reagent grade water system utilising both the Thermo Scientific hollow fibre filter and a "comparative" filter used on a different reagent grade water system. Figure 2 shows a comparison of the first samples drawn without any appreciable rinse up. Notice the presence of interfering peaks on both filters. Figure 3 shows the baselines after an approximately 8L initial rinse. The hollow fibre filter nicely rinsed to a flat baseline, while the other filter still showed substantial peaks. It was repeated after a 16L rinse on the other filter material; the peaks were still present.

Figure 3: Baselines after an approximately 8L initial rinse

Peaks in a chromatogram may not just be due to contamination in the water system or other mobile phase solvents: organic solvents present in the laboratory environment can cause airborne contamination. If organic solvents are handled in the laboratory, the results can be affected. Figure 4 also shows the effect of a water sample exposed to Toluene for less than one minute next to the chromatograph. The potential for environmental contamination can be significantly reduced when using a point-of-use water purification system, because the water is used as produced and not stored (such as HPLC grade bottled water).

It is necessary to look at other factors when conducting HPLC analysis. Those factors include the baking of glassware to remove any organic contaminants that may have adhered to the glass after washing. Also, the proper purging of a system; at a minimum, the first chromatogram of the day should be thrown away. The quality of mobile phase solvents is extremely important because if the solvent is not pure enough, interferences will exist as a result of using this solvent. In addition, prompt testing of the samples is necessary. This is required because water of this quality tends to pick up contaminants in a relatively short amount of time.

Figure 4: Effect of a water sample exposed to Toluene for less than one minute next to the chromatograph

The presence of organics greatly impacts UV and MS detection and it is important to use high purity water with a very low organic contaminant level to prepare samples and run HPLC applications. The conclusion of these assays demonstrated that the hollow fibre filtration material utilised on Barnstead Nanopure UV and Easypure UV water purification systems was superior in its ability to rinse out any interfering substances very quickly. This can be attributed to the naturally hydrophilic nature of the filtration material, as well as the material utilised for the filter housing. It is also evident that the filter material from a different manufacturer required extensive rinse-up to produce what could be interpreted as a flat baseline. Requiring such large quantities of water to rinse the 0.2 micron filter (to drain) wastes the capacity of the ion exchange cartridges unnecessarily. It may also take several runs or a column cleaning to remove filter contaminants. This can waste time and solvent.