High Purity Water for HPLC and LC-MS Applications
by Cecilia Regnault and Stéphane Mabic, Millipore Corp.
Figure 1. Overlay of plots extracted at 210 nm from PDA data and total ionic plot (ESI + mode) from m/z 200 to 1000 extracted from MS data of SDI, double distilled and Mill-Q Gradient water.
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Water is the most widely utilized analytical laboratory reagent and not always well characterized. Due to the high volumes of water used in sample preparation and liquid chromatography, extreme care must be taken with the water quality. Organic contaminants in the water may affect resolution and integration, introduce ghost peaks, alter stationary phase selectivity and impact baselines.
Various types of high purity water can be produced via water treatment technologies such as double distillation water, service deionization (SDI) water, or a purification system adapted to HPLC and LC-MS analyses. Although these are considered high purity water, when concentrating them and comparing them using PDA (2996, Waters) and ESI-Single Quadrupole (ZQ2000™ detector, Waters) detection (Figure 1), the freshly produced water shows the lowest baseline and no organic contaminant peak. In this study, water was pre-concentrated on a pre-column (Atlantis® dC18 3 μm × 2.1 mm × 150 mm column, Waters) for 1 hr at 1 mL/min. The run was achieved on a C18 column (X-terra® C18 3.5-μm × 4.6-mm × 30-mm column, Waters). The gradient went from Water/ACN 100/0 to ACN/Water 100/0 in 30 min, with a flow rate of 0.25 mL/min.
Distillation is a well known process during which water contained in a boiler is heated up. Water vapor is then condensed and the purified water is collected in a receiving flask. During this process it is expected that contaminants initially present (ions, organics, particles and bacteria) will not distill but remain in the boiler. However, a number of organic molecules may distill more easily than water or co-distill with water.
Based on ion exchange resins, service deionization provides very good water quality in terms of ionic purity (18.2 MΩ·cm). A large part of charged organic molecules are retained as well by the ion exchange resins. However, neutral organics (small and large molecules) and particulates have no affinity for the ion exchange sites and flow directly through the SDI bottle. Important variations of organic levels are expected, due to the changes in the tap water composition.
A water purification chain designed to produce high-purity water for HPLC and LC-MS applications combines several technologies. The initial purification system is a pretreatment unit. Water is processed through a semi-permeable reverse osmosis (RO) membrane to remove the bulk of contaminants, including organics, present in the tap feed water. Osmosed water is then directed to an electrodeionization (EDI) module. Purified water has low ionic (>10 Ω·cm) and low total organic or oxidizable carbon (TOC <30 ppb, measured in-line). It feeds a polyethylene reservoir chosen for its low release of organic extractables. The final polishing unit removes traces of residual organics and ions. Reservoir water feeds an ion-exchange resin (Jetpore) and activated carbon before undergoing the UV photo-oxidation. This process employs a dual-wavelength (185 nm and 254 nm) low-pressure mercury UV lamp that causes organic molecules to break up. The final polishing step removes any trace ions and organic fragments thanks to a mixture of ion exchange resins and activated carbons.
Conclusion
The presence of organics greatly impacts UV and MS detection. It is important to use high purity water with a very low organic contaminant level to prepare samples and run the LC. This is achieved when using freshly produced water with an adapted water purification system. Therefore, utilization of an ultrapure water system with on-line TOC analysis is most efficient for monitoring these organic contaminants at the point of use. Additionally, good maintenance and monitoring ensure a more reliable HPLC or LC-MS analysis for the chromatographer.
Cecilia Regnault is LC-MS support scientist at Bioscience Division, Millipore Corp. Stéphane Mabic is worldwide applications support manager. Cecilia may be contacted at cecilia_regnault@millipore.com or by phone at +33 1-30-12-70-34.
