What Water Should Be Used in Reversed Phase Chromatography?

a) Purified water with an extremely low TOC value and resistance of 18 MΩ·cm or greater (electrical conductivity of 0.056 μS/cm or less).

b) Water purified using a combination of methods, such as reverse osmosis membranes, ion-exchange polymers (including continuous ion-exchange substances), activated carbon, ultraviolet rays, and ultrafiltration, so that resistance is 18 MΩ·cm or greater (electrical conductivity of 0.056 μS/cm or less).

Since reversed phase chromatography is typically used as a technique for analyzing or separating organic substances, the water used to prepare mobile phases or samples should be sufficiently free from any organic matter, but the purity level required depends on the specific detector used.

If using an ultraviolet absorption detector, it is normally recommended to use commercial HPLC grade distilled water. Impurities with absorbance in the UV region are removed from commercial HPLC grade distilled water and absorbance levels in the shorter wavelength regions are guaranteed. Therefore, HPLC grade water generally can be used without worry. If you purify water yourself, however, distillation and ion-exchange filtration alone are normally insufficient. Instead, an ultra-pure water production system is strongly recommended. In recent years, the trend has been to use ultra-pure water production systems that can reduce the total organic carbon content (total organic carbon or TOC) by using ultraviolet irradiation to decompose organic matter. In particular, in the case of gradient elution, the difference in TOC levels can significantly affect the appearance of ghost peaks. On the other hand, if a fluorescence detector and mass spectrometer are used, commercial HPLC grade distilled water may be inadequate. In such cases, either use fluorescence analysis grade or LCMS grade water, or use an ultra-pure water production system.