Liquid chromatography-mass spectrometry (LC/MS) is a common analytical technique that couples high-resolution chromatographic separation with mass spectrometry for identification and quantification. Water quality is a crucial and often underrecognized concern for LC/MS.1
Type I water is recommended for various analytical techniques, including high-performance liquid chromatography, gas chromatography, and mass spectrometry.2 In LC/MS, ultrapure water is necessary, not only for the mobile phase but also for sample dilution, standards, blanks, sample preparation, rinsing, and instrument cleaning.
The most widely accepted specifications for Type I water come from ASTM International (formerly the American Society for Testing and Materials). In August of 2017,3 this organization, which develops consensus standards for a wide range of materials, provided specifications for four classes of water―Types I, II, III, and IV―which vary based on the allowable level of impurities. Type I, often referred to as "ultrapure water"―is the purest.
According to the 2017 ASTM standards, Type I water must have an electrical resistivity of at least 18 MΩ·cm at 25°C, indicating minimal dissolved ions. Type I water should also have a total organic carbon (TOC) concentration below 50 µl/L. The table below shows additional specifications.
0.056
18
50
1
Common impurities affecting LC/MS include organic compounds, bacteria, particulate material, dissolved atmospheric gases, and inorganic ions. These impurities can lead to various problems, including overlapping peaks, baseline instability, high background noise, ghost peaks, potential column damage, and a lack of reproducible results.
Perhaps the greatest concern with ultrapure water for LC/MS is the presence of organic compounds, including humic-based materials, biopolymers, and trace amounts of organic chemicals.4 When present in the mobile phase, organic compounds may compete with the analyte or bind to the active sites of the stationary phase. This competition reduces the amount of the analyte retained on the column, leading to a loss of resolution and sensitivity. With mobile-phase gradients, these organic materials form bands that tend to be eluted later.5
In addition to organic matter, ionic contaminants in the form of alkali ions may also skew results and introduce noise in analyses. Finally, bacteria and biofilm residue in water can create blockages in columns, leading to flow issues and degraded chromatographic performance.
Arium® systems are a common in-house option to provide labs with ultrapure water. Such systems tend to make use of some combination of activated carbon, filtration, electrodeionization, ion exchange, reverse osmosis, and ultraviolet photooxidation.5
Reverse osmosis can efficiently remove over 99% of organic material, resulting in TOC concentrations in the parts-per-billion (ppb) range.4 However, reverse osmosis units often fail to remove specific low-molecular weight organic compounds, such as isopropyl alcohol and methanol.4
The UV photooxidation of organic compounds can reduce TOC levels to below 5 ppb.2 At a wavelength of 185 nm, UV radiation breaks down and oxidizes carbon-containing molecules, producing ionized fragments that can subsequently be removed by ion exchange.4 Conversely, longer-wavelength UV radiation (254 nm) leads to bacterial inactivation.6 The combination of 185- and 254-nm wavelengths induces the generation of hydroxyl radicals from dissolved oxygen and water.1
It is advised that liquid chromatography eluents should never be kept in plastic containers that contain materials (like polyethylene and polypropylene) that may leach out and cause ghost peaks.5 Lab technicians should also be mindful of plastic containers like measuring cylinders, pipettes, washing bottles, and even pH probes. Furthermore, phthalates are a ubiquitous source of contamination that can be detected in trace levels on almost all surfaces.5 Although glass is generally better than plastic for LC/MS applications, it often has active surfaces. Furthermore, it can also be difficult to remove adsorbed contaminants, such as detergent residues, from glass.5
Blanks provide a regular check on an LC/MS system's cleanliness and performance and can help identify the source of troublesome ghost peaks.5 Although mobile-phase ghost peaks can be hard to recognize, they often appear broader and possess atypical shapes.5 Finally, if you are concerned about the quality of your in-house water system, commercially available LC/MS grade water may help you assess contamination issues. A comparison of expenses between freshly in-house produced Arium® Type 1 water and bottled water has shown that it is beneficial financially to purchase an Arium® system (see Figure 1).
Arium® Ultrapure Water Systems from Sartorius offer an extensive range of modular-designed systems for producing Type I ultrapure water for chromatography, mass spectrometry, and many other applications. Click here to learn more.
Figure 1. Comparison of expenses between Arium® Type 1 water (ultrapure water) produced in-house and bottled water (LC/MS grade). Calculations are based on the following assumptions: two liters of water consumption per working day, 20 working days per month, produced by an Arium® Mini Essential UV System incl. annual consumables cost, compared to purchased LC/MS-grade bottled water. This illustrative cost analysis spanning one year demonstrates the benefits of adopting in-house treated water. The acquisition of the device becomes financially advantageous after approximately three months, especially when there is a daily demand of two liters, resulting in significant cost savings within a year. For more detailed information, please contact Sartorius.
1. Regnault C, Kano I, Darbouret D, Mabic S. Ultrapure water for liquid chromatography-mass spectrometry studies. J Chromatogr A. 2004;1030(1-2):289-295.
2. Nabulsi R, Al-Abbadi MA. Review of the impact of water quality on reliable laboratory testing and correlation with purification techniques. Lab Med. 2014;45(4):e159-165.
3. Standard specification for reagent water. ASTM D1193-99e1. In: ASTM International; 2017.
4. Choi J, Chung J. Effect of dissolved oxygen on efficiency of TOC reduction by UV at 185 nm in an ultrapure water production system. Water Res. 2019;154:21-27.
5. Williams S. Ghost peaks in reversed-phase gradient HPLC: a review and update. Chromatogr A. 2004;1052(1-2):1-11.
6. Rose LJ, O'Connell H. UV light inactivation of bacterial biothreat agents. Appl Environ Microbiol. 2009;75(9):2987-2990.