What is the right quality of water for HPLC analysis?

Quality Water for HPLC Analysis

Quality Water for HPLC Analysis

Water for HPLC is used in most mobile phases and also in sample preparation. Water has been rightly termed as a ‘Universal Solvent’ because it dissolves most substances, is easily available and has non-toxic nature but at the same time ordinary tap water is not used for laboratory applications because of its potential for interference due to presence of contamination.

Water contaminants include dissolved atmospheric gases, natural minerals and organic substances, dissolved solids and suspensions and bacteria or microorganisms if the necessary nutrients and environmental conditions are present to support them.

Water for laboratory use requires different degrees of treatment for required applications. Reagent grade water is defined as water suitable for use in a specified procedure such that it does not interfere with the specificity, accuracy and the precision of the procedure. General laboratory applications include glassware washing and rinsing, reagent and buffer solution preparation, making blanks and standard solutions for calibration purposes, etc.

Applications like water for HPLC and trace metal analysis require water conforming to highest purity specified under Type 1 by ASTM. Such water requires mixed bed deionization and filtration with 0.2μ membrane filters. The typical specifications for Type 1 water are outlined here.

Resistivity >18 megohms(mΩ)at 250C
TOC < 5ppb
Silica <3ppb
Sodium <1ppb
Chloride <1ppb

Water for HPLC is need at time of preparation of aqueous mobile phases, buffers and solutions of samples and standards. HPLC grade water can be sourced in bottles from solvent suppliers or a water purification system can be installed in the laboratory.

Influence of Water contaminants on HPLC operation

Let us briefly examine in what ways contaminants in water for HPLC can affect your runs :

Solid Suspensions

Solids of size above permitted limits can adversely damage pumps and affect column life by increased wear and tear of seals,valves and pump piston.

Blockage of column frits leading to increased column back pressure and either complete stoppage or slow down of mobile phase.

Reduction of column life-time.

Colloids in suspensions if any absorb on stationary phase and reduce efficiency of column separation.


Reduction in access of sample and solvent to molecules to binding sites on stationary phase resulting in loss of resolution.

Contribution to extraneous ghost peaks.

Ionic species

Modifications resulting in ionic strengths due to presence of ionic species can affect sequence of separation of polar molecules.

Certain UV absorbing ions such as nitrates and nitrites can contribute to ghost peaks.


Storage of high quality of water often introduces leached compounds from containers. Plastic containers can leach out organics whereas glass containers can introduce ions on long term storage of HPLC mobile phases or buffers.


Dissolved air in mobile phase results in formation of minute bubbles under high pressures encountered in the HPLC system. Such bubbles contribute to detector noise and voids in the column.

It is therefore absolutely necessary to filter the mobile phase and buffers at time of preparation and before pumping using on line filters. Degassing using on-line degassing or external techniques is equally important.

Commercial Sources of water for HPLC

Reputed suppliers of chemicals and reagents provide water for HPLC in bottles but in the long run it is advisable and economically viable to install commercially available dispensing units which will cater to your water requirements for other sophisticated analytical techniques besides HPLC. Such systems are designed for large volume outputs and provide other useful features such as hand held controlled dispensing, sensors to indicate levels inside internal storage tank and prevent external spillages.

System components normally include pre- and post filtration capsules for removal of dissolved solids above 0.2 μ size. Activated carbon filters provide freedom from organics and dissolved chlorine. Mixed –bed deionizers control conductivity and UV irradiation source removes organic traces. Reverse osmosis system and a water storage tank complete such systems.

What source of water for HPLC do you use in your lab? Have you faced any challenges with water quality? Share with us in the comments below.

About Dr. Deepak Bhanot

Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise beginning from sales and product support of analytical instruments. After completing his graduation and post graduation from Delhi University and IIT Delhi he went on to Loughborough University of Technology, UK for doctorate research in analytical chemistry. His mission is to develop training programs on analytical techniques and share his experiences with broad spectrum of users ranging from professionals engaged in analytical development and research as well as young enthusiasts fresh from academics who wish to embark upon a career in analytical industry.


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  2. Hi! I was wondering if you have any references for the 16-18 megaohms resistivity range? Thx!

  3. So distilled and/or de-ionized water are not qualify to substitute HPLC grade water even for use in a tC18 cartridge before the HPLC analysis?

  4. Dear Sir,

    I am interested in setting up an HPLC water plant

    I have reference of Evoqua water system

    Can you help me for this plan?

  5. Catherine Clark says:

    What would be the effects of tap water on the flow cell?

    • You should not even think of using tap water. The impurities present will result in peaks which would be difficult to explain. Secondly the dissolved solids in tap water will over a time period clog the fine passage of the flow cell

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