Water Used for HPLC

Tips for Daily Analysis

In HPLC analysis, water is essential for use in preparing mobile phases and samples. If impurities are present in a mobile phase, however, they can increase background signals that can increase baseline noise, drift, or cause other phenomena, such as negative peaks or unknown peaks during gradient elution.
Similarly, if impurities are present in sample solutions, they can cause unknown peaks. If impurities include a target component being analyzed, they can even affect quantitation values. To prevent struggling with such problems and to increase data reliability, this article discusses some basics about water.

What Is the Purity of Water?

What purity level is necessary for HPLC analysis? The standard JIS K 0124: 2013 "General rules for high performance liquid chromatography" specifies the following (excerpt).


Water used for this standard shall be purified by a combination of reverse osmosis, distillation, ion-exchange, ultraviolet irradiation, filtering, or other methods, and shall have a water quality level that does not interfere with analysis. Water quality shall be evaluated based on index values such as specific resistance, total organic matter (TOC), or absorbance values.


The following discusses water purity in more specific terms.

What Water Should Be Used in Reversed Phase Chromatography?

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. Note that JIS K 0211: 2013 "Technical terms for analytical chemistry (general part)" defines ultra-pure water as follows (excerpt).

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).

What Water Should Be Used for Ion Chromatography?

Given that ion chromatography is basically an analytical technique used for measuring ionic substances, the most important point is to use water with ions sufficiently removed. It is best to use water from an ultra-pure water production system with specific resistance of 18 MΩ·cm or greater.
The quality of commercial HPLC grade distilled water is guaranteed for use in analysis using UV absorption detectors, as mentioned above, but it is not necessarily guaranteed for use in analyzing inorganic ions. Therefore, you should confirm that the water does not cause any analysis problems before using it. In general, commercial water marked as ion-exchanged water or purified water is not recommended, because the quality can vary depending on the manufacturer. Based on the above points, it can be said that for ion chromatography, using freshly purified water from an ultra-pure water production system will provide more trouble-free and worry-free results.

What Is the Proper Way to Handle Water?

The higher the purity of water, the greater the care required to handle it. Water is able to dissolve a wide variety of substances, so contamination starts the instant it contacts the air. If commercial water is used, it should be consumed as soon as possible. It is important to keep ultra-pure water production systems regularly maintained. It is also important to minimize contact with air by not generating bubbles when acquiring either commercial or purified water from an ultra-pure water production system. Also try to add water directly into the preparation container and minimize the use of wash bottles. Water in wash bottles can become contaminated easily and may contain plasticizers leached from the plastic bottle inner walls. (Replace water in wash bottles often.) Needless to say, always pre-wash equipment with fresh water before use.

For Research Use Only. Not for use in diagnostic procedures.

This page may contain references to products that are not available in your country. Please contact us to check the availability of these products in your country.

Top of This Page