HPLC ASTM Type I Water

Abstract

High performance liquid chromatography (HPLC) is a powerful analytical technique used to separate, identify and quantify compounds with a wide range of differing properties. The need to ensure that standards, organic solvents and HPLC mobile phases are of the highest purity is widely recognised, yet water quality, which may have a major impact on chromatographic performance, is frequently taken for granted. For ultra-high performance liquid chromatography (UHPLC), a technique using sub-2 µm particles and high pressures to enhance analytical speed and sensitivity, water quality is very crucial.

HPLC Water Introduction

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.

WHY is HPLC Water a Crucial requirement?

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 slowdown of mobile phase.

Reduction of column life-time

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

Organics

Reduction in access of sample and solvent 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.

Leachables

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.

Gases

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.