Solutions for Mass Spectrometry: Tips and Tricks to Optimize Quality Spectra
By Dr. Egidijus Machtejevas, Product Management Analytical Chromatography, Merck Millipore, Darmstadt, Germany
Mass spectrometry (MS) is a common technique used to identify and quantify molecules in complex mixtures because of its high sensitivity and extreme versatility. It is used across diverse research areas, ranging from systems biology and proteomics to environmental analysis. Electrospray ionization (ESI) is by far the most popular ionization technique used for mass spectrometry.
There are many factors that can influence the quality of spectra obtained with mass spectrometry. This article will present tips and tricks for successful mass spectrometry, including choosing the right solvents and buffers, solvent storage, buffer preparation, maintaining the recommended water content in eluent, mobile phase quality and contamination, equipment cleaning and column washing.
Tips & Tricks
Electrospray ionization has two ion modes: positive and negative. In positive ion mode, the analyte becomes protonated using volatile acids; in negative ion mode the analyte becomes deprotonated using ammonium formate or acetate, ammonia or volatile bases. Solvents that can be applied to ESI include acetonitrile with water, isopropanol, methanol and n-propanol. Strong acids such as hydrochloric acid, sulfuric acid or nitric acid should be avoided because they tend to form ion pairs with analytes, making the analyte unsuitable for any type of ionization. Additionally, these strong acids, at least in part, have unfavorable oxidizing properties. Many laboratories tend to use trifluoroacetic acid (TFA) in order to form ion pairs with peptides and proteins; however, TFA can cause strong ion suppression of the analyte, and may contaminate the mass spectrometer. If use of TFA is necessary, then a weak acid or isopropanol should be added to help decrease the signal suppression effect.
The viscosity for methanol or isopropanol is quite high compared to acetonitrile. If acetonitrile is replaced by either of these two alcohols, the system and the columns must be suitable for the replacement, as the backpressure will increase. Chromolith® HR media (EMD Millipore, Billerica, MA) produces similar chromatographic results as Core-Shell particle packed columns, but at much lower backpressures (40 bars versus 100 bars, respectively). This pressure difference provides an opportunity to add viscous eluent and to increase spray stability with the monolithic column.
All solvents, both water and organic, should be stored in surface treated amber glass or borosilicate glass (if solvents are to be decanted). Standard clear glass bottles are not recommended because dissolution of silica and alkali from the glass is possible, resulting in ion suppression or adduct formation. For bottle caps and adapters, professional equipment directly mounted to the original brown glass bottle is highly recommended. Decanting should also be avoided, if possible, because it may result in solvent contamination.
The use of any type of plastic device for storage or handling of sample, including bottles, funnels or beakers can result in ghost peaks and increased background noise (Figure 1). This contamination is the result of plasticizers, antistatic agents or stabilizers that can be leached into the solvent.
Figure 1: Spectrum of ultrapure water stored in clean brown glass bottle and a polypropylene bottle.
Figure 2: Buffer purity. a) Comparison of ammonium bicarbonate and ammonium acetate buffers.
Figure 2. Buffer purity. b) Polyethylene oxide spectrum (PEO) when sodium and potassium are present in buffer.
Figure 3: MS grade solvents provide highest quality. a) Combined total ion current (TIC) of the analysis of three different acetonitrile qualities using flow injection analysis.
Figure 3: MS grade solvents provide highest quality. b) Analysis of acetonitrile qualities using reserpine testing.
Figure 3: MS grade solvents provide highest quality. c) Analysis of methanol qualities using LC. Four different methanol samples were compared using a gradient run, starting with 2% organic: 2% methanol going up to 100% methanol within 30 minutes.
With respect to buffers, it is important to avoid working with ammonium bicarbonate because this salt is typically highly contaminated, which can result in strong background noise and ghost peaks on the spectrum as compared to ammonium acetate (Figure 2). In general, the preparation of buffers should not include dissolving the salt in water, but rather by titrating the respective acid and base. If the use of salts is necessary, then an MS analysis should be performed prior to use to determine if and what type of contaminant will be present in the liquid chromatography-mass spectrometry (LC-MS) run.
Buffers and Eluent pH
To prevent contamination of the mass spectrometer, only volatile buffers and additives should be used to adjust eluent pH. The use of nonvolatile buffers or solvents may cause precipitation and ultimately tedious cleaning procedures. The choice of buffer for adjusting eluent pH strongly depends on the application. Formic acid works in both positive and negative electrospray mode, and the intensity and sensitivity of the signal is high. For a higher pH, trimethylamine and hydrochloric acid are commonly used. Additionally, the pH of the eluent must match stationary phase characteristics. For silica-based stationary phase, the pH of the eluent should be in the range of 2 to 7.5. If the pH is below 2, the bonded modification from the silica skeleton or the silica particles will detach; if the pH is above 7.5 the silica backbone will dissolve.
Water content in eluent
The recommendation for water content in eluent lies between 5-80%. If the water content is below 5%, then the buffer is at risk of precipitating in the system. In this case, a countermeasure is to utilize a suitable organic solvent to dissolve the buffer. If the water content is above 80%, then there is a risk that the MS detection spray will breakdown. There are several countermeasure options to avoid such a breakdown, including decreasing the surface tension of the eluent by adding a volatile organic solvent, decreasing the flow rate from the LC to the MS, increasing the dry gas temperature or the flow of the dry gas, or decreasing the LC flow rate.
Microbial contamination in the system is a significant issue if the water content is above 95%. In this case, the recommendation is to regularly flush the LC system with an organic solvent—preferably isopropanol, not acetonitrile—because acetonitrile can polymerize and block the valve.
Figure 4. Regular flushing of the water system device can increase water quality. a) Spectrum of ultrapure water tapped on Monday after a standby of the system over the weekend, and after discard of 4 litres.
Figure 4. Regular flushing of the water system device can increase water quality. b) Total ion current (TIC) of ultrapure water tapped on Monday after a standby of the system over the weekend, and after discard of 4 litres
Figure 5. Total ion current (TIC) of water stored in a clean borosilicate glass bottle and water stored in bottles that were cleaned via dishwasher.
Mobile phase quality
The quality of the mobile phase (organic eluents or water) strongly influences the sensitivity of MS detection. Organic solvents for high performance liquid chromatography such as acetonitrile and LiChrosolv® methanol (EMD Millipore) are available in three qualities: isocratic grade for liquid chromatography; gradient grade for liquid chromatography, and; hypergrade for LC-MS gradient runs). Hypergrade quality material should be used for MS analysis because it has the highest purity and lowest signal suppression (Figure 3).
There are several options for using water as an eluent, including demineralized tap water, bottled water, and ultrapure water. Demineralized tap water is acceptable for LC-UV, but is not recommended for MS because of potential contamination. Bottled water is recommended for low water consumption and ultrapure water from a purification system is recommended for higher consumption. Ultrapure water systems, such as Milli-Q® (EMD Millipore), deliver type I water and are meant to be used for LC-MS; however, regular flushing of the device—and regular use—can further improve water quality (Figure 4).
Mobile phase contamination
Contaminants in solvents and additives can accumulate on the stationary phase and elute as distinct ghost peaks on the chromatogram. This scenario may occur when the column is equilibrated under highly aqueous conditions or during a gradient run under aqueous conditions. To avoid ghost peaks, column equilibration should be kept as short as possible and flushed with approximately 10 column volumes in isocratic mode. Under gradient conditions, equilibration is not necessary if the first one or two runs are discarded.
Figure 6. Column washing for removal of unbound organic entities. Twenty gradient runs were performed going from 5% to 95% acetonitrile in 3 minutes, and keeping the 95% acetonitrile for 2 minutes at a flow of 0.4 millilitres per minute.
The simplest way to clean bottles is by evaporation in a fume hood. Contamination can occur if glass vessels are cleaned using a dishwasher. The conditions of dishwashing are harsh and the solutions used are strongly basic, which may result in the dissolution of silica and alkali from the glassware. Additionally, detergents or plasticizers can deposit on the glass surface of the vessel, which will then be visible in the solvent (Figure 5). If a dishwasher is used to clean glass vessels, it is critical that the vessels are flushed afterwards with MS grade solvent.
Column bleeding and washing
Another source of contamination is the column itself. New columns should be washed to remove unbound organic entities, also known as column bleeding. A new column directly out of the box should undergo between 7 and 8 gradient runs before it is ready to be used in MS (Figure 6). Another option is to wash the column with 0.1% formic acid in isopropanol at half optimum flow (0.2 mL/min) for about 30 minutes (Figure 6). This washing process eliminates contamination and prepares the column for use.
Mass spectrometry is a powerful technique for identifying and quantifying molecules within complex mixtures. The success of mass spectrometry relies in large part on reducing the likelihood of contamination, which can occur anywhere throughout the process, from sample preparation to equipment cleaning. An important first step in this process is to only use the highest quality materials for MS, including buffers, reagents, water and columns.
Egidijus Machtejevas was born in Lithuania where he studied chemistry and biotechnology at Kaunas University of Technology. After gaining his PhD in analytical chemistry in 2001 he worked as a post-doc with Professor Unger at Mainz University. In 2008 he joined Merck as a researcher in a group of Chromatography & Bioseparation/Bio-Analysis R&D. After 1 year he changed to PM as Marketing Manager of chromatography products in North America. Currently he is a global product manager of chromatography focusing on monolithic silica columns. He has twenty scientific papers and nine book chapters to his name, and his major research areas include multidimensional liquid chromatography, proteomics and the development of monolithic stationary phases for chromatography.
Stephan Altmaier received his PhD in inorganic chemistry from Hannover University (Hannover, Germany) in 2003. In 2006, he joined Merck as a researcher in a chromatography and stationary phase development lab. Today, he is head of the application lab in the instrumental analytics section of Merck Millipore. His focus is on the development of applications on monolithic and particle-packed silica HPLC columns of various dimensions and selectivities using MS detection.