Liquid chromatography mass spectrometry (LC-MS) is a powerful analytical technique which combines the separation capabilities of either high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography (UHPLC) with mass analysis by mass spectrometry. The need to ensure that standards, organic solvents and HPLC mobile phases are of the highest purity is widely recognized, yet water quality, which may have an impact on chromatographic performance and the quality of mass spectral data, is frequently taken for granted.
LC-MS is an extremely versatile, highly sensitive and selective technique, which is commonly used in pharmaceutical, bioanalytical, food and beverage, environmental, clinical, forensic, proteomic, metabolomic and drug development laboratories.
Good chromatographic performance and the acquisition of high quality mass spectral data depend on the purity of the water1. Water may contain a variety of impurities, which can adversely affect chromatographic performance by impacting on resolution, integration and baselines, introducing ghost peaks and altering the selectivity of the stationary phase. Particulates found in impure water may cause damage to the LC system and create column blockages resulting in increased downtime, while ionic contaminants can make interpretation of mass spectra more complicated by interfering with spectral identification and quantification of low-level analytes.
Organic contaminants present in water can cause a number of chromatographic problems. When present in the mobile phase, organic compounds may compete with the analyte to bind to the active sites of the stationary phase. This reduces the amount of analyte retained on the column and subsequently eluted, with a corresponding reduction in method sensitivity. If organic compounds accumulate on the column surface they can restrict analyte and solvent access to active sites, resulting in mass transfer issues and loss of resolution, while any accumulation at the head of the column can cause ghost peaks. Additionally, the accumulation of organic contaminants on the column may result in increased back pressure and, ultimately, a shorter column lifetime. In situations where organic contamination levels are very high, it is possible for the organic compounds to accumulate with time and act as a new stationary phase, causing peak tailing and retention time shifts.
Bacteria produce organic by-products such as pyrogens, nucleases or alkaline phosphatase, which may result in column and frit blockages and chromatographic issues, such as those described above.
Ionic contaminants may modify the ionic strength of a solution, which can affect some chromatographic separations. Significantly, analytes may form adducts with metal ion contaminants such as Na+ and K+, which can complicate mass spectral interpretation.
Particulates and colloids
Particulates may cause damage to the HPLC pump and can also cause columns and frits to block. This effect is even more significant for UHPLC users, as the very small particle sizes and decreased diameters of these columns make them more susceptible to premature blocking than their HPLC counterparts. Colloids can be irreversibly adsorbed onto the stationery phase, resulting in a change to the separation efficiency of the column.
Ultraviolet (UV) radiation
Passing water through a beam of ultraviolet light breaks down organic compounds. A wavelength of 185 nm effectively breaks down and oxidizes carbon-containing molecules, yielding ionized fragments for subsequent removal by ion exchange, whereas longer wavelength UV radiation (254 nm) disrupts the activity of bacterial enzymes, preventing replication. To maximize breakdown of organic molecules, both the PURELAB Ultra Analytic and PURELAB flex use a full spectrum UV lamp.
The media cartridges in both the PURELAB Ultra Analytic and PURELAB flex contain synthetic, activated carbon beads, which adsorb a wide variety of organic compounds, and high purity ion exchange resins to minimize the release of impurities.
Purifying wat er for LC-MS
The high sensitivity of LC-MS necessitates the use of ultrapure water for the preparation of all reagents, buffers, mobile phases and any sample pre-treatments2. For ultra-sensitive LC-MS applications requiring very low levels of total organic carbon (TOC), ELGA’s PURELAB Ultra Analytic (Type 1+) water – with, typically, a resistivity of 18.2 MΩ .cm, a very low TOC value of less than 2 ppb (figure 1) and bacteria levels below 0.1 CFU/ml – is highly recommended. ELGA’s award winning PURELAB flex, with a TOC of less than 5 ppb (figure 1), is suitable for all other LC-MS applications.
Ultrapure water with a high resistivity (>18.2 MΩ .cm), free from particulates, organic and ionic compounds, should be used for all LC-MS applications to ensure good chromatographic performance and high quality mass spectral data. To find out more about ELGA LabWater’s water treatment technologies and solutions for life science applications, visit www.elgalabwater.com
For more information please visit www.elgalabwater.com