Effect of water quality on HPLC

High Performance Liquid Chromatography ( HPLC ) is an effective and reliable technology that has become an important tool for many laboratory studies. One of the most common problems with HPLC is the effect of contaminants in the solvent on the analytical results.


Particles can damage the pump and the syringe. Particles can also block the column and melt it, which can result in increased back pressure. The particles will also appear as another solid phase, possibly changing the composition of the sample.

Organic matter

Organic matter in ultrapure water may affect the resolution and integration of chromatographic peaks, may lead to ghost peaks, may also alter the selectivity of the stationary phase, and affect the chromatographic baseline.


Changes in ion concentration can affect the separation results, and some of the ions that absorb UV will have an effect on the peak. Certain corrosive ions also reduce the life of accessories such as high pressure infusion pumps.


The colloid will be irreversibly adsorbed on the stationary phase, affecting the separation of the column.


Microorganisms can clog the column, and its metabolites increase pollutants such as organic matter and particles.

Bottled purified water and distilled water are the pure water used in some HPLC analysis. Since the bottled purified water and distilled water are only ordinary pure water instead of ultrapure water, the water still contains a small amount of impurities such as ions and organic substances to prepare liquid phase and wash. Deliquoring not only can foul expensive columns, but also affects the HPLC to set a flat baseline.

The purification process of bottled purified water usually includes adsorption, filtration, reverse osmosis, etc., and the removal of particulate organic matter is better, but the removal of trace ions and organic matter cannot meet the requirements of high sensitivity HPLC experiments, and because of its The packaging is usually mostly PVC bottles, and the oxygen permeability is high. At the same time, there is a certain amount of chemical dissolution, which pollutes the pure water in the bottle. Especially after storage for a period of time, the water quality drops more, so it is expressed on the HPLC chromatogram. Specific absorption peaks, but there are more severe unstable disturbances at the baseline.

Distilled water refers to pure water prepared by distillation. The water can be distilled once and several times. After the water is distilled once, the non-volatile components remain in the vessel and the volatile components enter the initial fraction of the distilled water. Usually only the middle part of the fraction is collected, accounting for about 60 %. To obtain purer water, An alkaline potassium permanganate solution is added to the distilled water to remove organic matter and carbon dioxide; and a non-volatile acid (sulfuric acid or phosphoric acid) is added to make the ammonia a non-volatile ammonium salt. By HPLC detection of double distilled water, it was found that strong absorption peaks appeared at 254 nm and 214 nm at 22-27 minutes, which indicated that there was a strong hydrophobic organic matter, which was caused by the azeotropic phenomenon of the distillation process. Some volatile organic compounds are not completely removed.

The ultrapure water system combines various purification processes such as reverse osmosis, ion exchange, activated carbon adsorption, membrane filtration, ultrafiltration and ultraviolet oxidation, and the water conductivity reaches 18.2 M Ω• cm, the water quality of the produced water exceeds the national standard water standard and is stable and measurable. The ultra-pure water is ready to use, no pollution is introduced due to storage, and the water quality is guaranteed. It is more suitable for users than using bottled purified water or distilled water. The need for high precision instrumental analysis.

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