Choosing the aqueous phase for chromatography experiments: deionised water or ultrapure water?

**Author: Original from Internet**

Chromatography experiments, particularly precision analyses such as high-performance liquid chromatography (HPLC) and ion chromatography (IC), impose almost exacting requirements on the purity of the water used. The use of unsuitable water can lead to a range of issues, including baseline drift, ghost peaks, wear on the piston rod, column clogging and reduced sensitivity, which directly affect the accuracy and reliability of the data.

In accordance with the national standard GB/T 6682(China Standard), laboratory-grade water is classified into three grades: Grade 1 water / Grade 2 water / Grade 3 water. Grade 1 water is the ‘entry point’ for chromatography

The core process is distillation. Water is heated to vaporise, then condensed and collected; in theory, this removes most non-volatile salts, particles and microorganisms. According to Watsons’ documentation, the process involves distillation at a high temperature of 105°C followed by multi-stage filtration.

① Effective at removing inorganic salts and hardness ions.

② Suitable for general laboratory applications with low requirements.

③ Readily available on the market and low-cost.

① Volatile organic compounds, ammonia, CO₂, and certain low-boiling-point impurities may be carried in with the vapour.

② CO₂, plasticisers, microorganisms, etc., may be introduced during packaging, storage, and after opening.

③ Lacks laboratory-grade CoA; TOC, endotoxins, nucleases, and metal background levels are not guaranteed.

The core process typically involves multi-stage filtration + RO reverse osmosis + disinfection. Data indicates that it employs multi-layer deep filtration, activated carbon adsorption, RO reverse osmosis deep purification, secondary reverse osmosis and ozone sterilization, amongst other methods.

① Low electrical conductivity; some batches can approach the range of laboratory Grade 3 or even Grade 2 water.

② Low cost and readily available.

③ In many laboratories across the country, there is indeed a tradition of using it as a ‘temporary substitute water’.

① It meets food and drinking water standards, not laboratory reagent standards; ② Batch, origin, packaging and storage duration can affect water quality; ③ Low conductivity does not guarantee compliance with TOC, microbial, endotoxin, metal ion or silica limits.

The core process involves ion exchange. Cation and anion exchange resins are used to remove ions such as Na⁺, Ca²⁺, Mg²⁺, Cl⁻ and SO₄²⁻ from the water.

EDI (electrodeionisation) or mixed-bed systems may also be employed to further increase the resistivity. Deionised water effectively reduces conductivity, but cannot completely remove non-ionic organic compounds, microorganisms, particles or endotoxins.

The core lies in a combination of multiple technologies. A typical process involves: pre-treatment → RO → EDI/mixed-bed → UV oxidation → ultrafiltration/final 0.22 μm filtration → online resistivity/TOC monitoring. The quality specifications for ultrapure water include parameters such as 18.2 MΩ·cm, TOC <5 ppb, bacteria <10 CFU/mL, and undetectable or extremely low endotoxin levels.

1. Initial or intermediate rinsing of glassware;

2. Topping up water baths, ultrasonic cleaners and certain cooling circulation systems;

3. General teaching experiments and qualitative experiments;

4. Preparation of standard reagents that are not sensitive to ionic background;

5. Temporary emergency substitute for Grade 3 water.

1. HPLC / LC-MS;

2. ICP-MS / ICP-OES / AAS trace metal analysis;

3. TOC, ion chromatography, ultra-trace cation and anion analysis;

4. PCR, qPCR, RNA experiments, cell culture;

5. Semiconductors, photolithography, precision cleaning;

6.Preparation of standard solutions, reference reagents and calibration solutions.

Resistivity reflects only ionic impurities; it does not indicate compliance with standards for organic matter, microorganisms, endotoxins, silica, particulates or metal background levels.

Even if ultrapure water achieves a resistivity of 18.2 MΩ·cm, it will rapidly absorb CO₂ upon exposure to air, causing the resistivity to drop; therefore, it is best to prepare and use it immediately.