Foraging has become an increasingly popular past time, the ability to find food in the wild could assist with survival but also offers an immense sense of achievement and satisfaction. It is important that the practice is carried out with care and attention to ensure that potential food sources are correctly identified. Edible mushrooms are a great source of nutrition, however, there are some that are poisonous that could make you sick or in the worst-case scenario lead to death.
Figure 1 – The field mushroom, Agaricus campestris
There are three main groups of mushrooms that foragers will look for, these are the parasites, which parasitise trees, plants and even insects, the saprobic mushrooms, which rot down old wood or other dead substrate, and the mycorrhizal fungi which attach themselves to the roots of host organisms transferring nutrients, among other things between themselves and the host. All interact heavily with their environments and as is the case with living organisms, the environment in which they develop and grow will have a direct influence on their exposome which by definition encompasses life-course environmental exposures.
For this study, we have focussed on one specific species of saprobic mushroom that commonly grows in grassland. The Field Mushroom, Agaricus campestris is probably the most commonly collected edible mushroom in the UK. It is also one that is commonly found on roadside verges, and is also regularly mentioned as a mushroom that can absorb pollutants from its environment. It’s genus has many edible members that grow in the same environment and it is likely that the results from this experiment would be reflected by those species. The sampling was completed by Marlow Renton from Wild Food UK who is an expert forager and recently published a Foraging pocket guide.
It is proposed that a fruiting body of a mushroom that developed and grew in a polluted environment would absorb some of that pollution and become polluted. Fruiting bodies that develop in a clean pollution free environment should reflect that in their respective exposomes.
When foraging close to urban areas there may well be times that foragable items are spotted close to a busy road. These fruits may appear to be clean or pollution free but it is difficult to make that assessment using just eyes alone. The following case study aims to investigate the influence of pollution on the fruiting bodies of mushrooms as a function of distance picked from a busy road. Roads by their very nature are a serious source of pollution, the vehicles that use them exhaust a mixture of gases including water and carbon dioxide, mixed in with the major combustion products are other more toxic gases and also some metal particulates from the wear and tear of the engine. Modern vehicles are equipped with catalytic converters that serve to chemically convert many toxic emissions into a more inert and less harmful gases. It is hypothesised that concentrations of the catalysts used in the catalytic converters may well be higher in the soil and/or mushrooms found closer to the road.
Inductively Coupled Plasma Mass spectrometry (ICP-MS) is a sensitive analytical technique used to measure ultra-trace levels of metals present within a sample. Samples must be water soluble. Solid samples may be analysed by ICP-MS following on from sample preparation that usually involved acid microwave digestion. Once in solution the samples are sprayed into an argon plasma which could be up to 10,000 ºC, the water will evaporate and the high energy of the plasma will cause any metals present in solution to ionise which are charged versions of the atoms. The mass spectrometer will then separate the different masses of the ions and record their abundance which is proportional to their concentration within the sample.
Aims and Motivation
In order to assess the uptake of various metallic toxins, a simple experiment was conducted to test the hypothesis that mushrooms picked closer to the road contained a higher level of toxicity than those picked further away from the road. The influence of the substrate on the mushrooms would also require careful consideration by analysing soil substrate samples collected from the same sampling locations as the mushrooms.
Mushroom samples were picked along with soil samples from the same spot at various intervals moving away from a busy road in Hereford. The collected samples were stored in sterile containers and transported to the Artemis Analytical laboratory for analysis. Once received the samples were dried in a drying oven for 24 hours at a temperature of 80 ºC. The samples were ground up in a pestle and mortar, ~0.1 g samples from each was weighed accurately, the same sample preparation was used for the mushroom and soil samples. A supermarket-bought field mushroom was also prepared in the same way and analysed as a point of reference.
The powdered samples were each digested with 10 mL of concentrated nitric acid in a microwave digester on full power for 2 hours until the solids had fully dissolved and everything was in solution. The acid digestate was made up to 100 mL, filtered and then analysed using ICP-MS.
The ICP-MS instrument was setup and calibrated using external standards, a broad suite of elements was measured including the precious metals found in catalytic converters.
The concentrations of the metals are expressed in parts-per-million (ppm) for the dry soil samples. The concentration in the mushroom samples is expressed in parts-per-billion (ppb) and has been calculated for hydrated mushrooms assuming that the 99.18% of a mushroom is water.
Limitations 1. It is worth noting that these tests are by no means exhaustive, only the metallic elements were screened and measured. The vitamin content and nutritional value was not assessed in this study. This is possible using a different type of mass spectrometry, it would make an interesting future study.
2. It was impossible to separate the grass roots and other organic matter from the soil samples. Therefore, where we refer to soil it is really the soil and growing substrate.
3. Samples were taken from as regular intervals as possible from the road, though as they were growing naturally precision was impossible.
4. Only 1 species of edible mushroom was used for the test, this can be assumed to be reflected by other members of the genus, but these results do not apply to all fungi, edible or otherwise.
5. Other variable natural factors that may influence these results such as wind direction have to be assumed to be homogeneous in these results.
6. Assumptions have to be made about whether the source of the pollution was the growing substrate or airborne particles as the process itself was not observed, though the data collected informs those assumptions with reasonable veracity.
Results and Discussion
The results presented below for the soil and mushrooms sampled at 0 metre distance from the road samples were collected from the centre of Hereford right next to an extremely busy A road, these data are marked with the red star on the plots below. The other samples were collected at locations of increasing distance from a road in a more rural locality close to Hereford. The green star data point is from the supermarket-bought mushroom and is included as a point of reference.
For the vast majority of the elements measured in both the mushroom and soil samples it was found that their respective concentrations were higher in the samples collected closer to the road than those collected further away. The palladium and platinum results presented in figures 2 and 3 respectively clearly demonstrate the inverse relationship between collection distance from the road and concentration.
Figure 2 – Palladium measured in mushrooms & soil
Figure 3 – Platinum present in mushrooms & soil
Iron is an ubiquitous element present in high concentrations in many locations on the earth’s surface and is an essential element of life. There was not a clear cut relationship between the concentration of the iron measured in the mushrooms collected closer to the road when compared to mushrooms collected further away. This would suggest that the uptake of iron expressed in the mushrooms was not effected by their proximity to the road. It was noted that the concentration of iron within the soil sampled close to the busy road was significantly higher, this would be expected due to the build up of rust from road vehicles. When comparing the soil and the mushroom results for iron there does not appear to be such a pronounced correlation. The iron results contrast significantly with the platinium and palladium results suggesting that perhaps the uptake of these metals may be less selective with respect to the iron.
Figure 4 – Iron present in mushrooms & soil
It is worth noting that the mushrooms analysed during this study were not washed prior to them being dried and prepared for ICP-MS. This leaves the question unanswered as to if the exact source of the metal contaminants measured, had been adsorbed onto the surface or were present inside the mushroom’s fruiting body. Additional work is required in order to determine which part of the mushroom contained the highest concentration of the metal contaminants, skin, flesh on the top or flesh in the stalk.
Figure 5 – lead present in mushrooms & soil
The use of lead compounds as an additive to petrol fuel has been common place since the 1920’s due to the increase in engine performance. In the UK alone it is estimated that 140,000 tonnes of lead was released into the atmosphere from the 1970’s until its use was finally banned in 1999. The lead pollution still lingers and this was observed with a high concentration in the soil of 50 ppm next to the road. The lead concentration in the soil does decrease as a function of distance from the road with a slight elevation in concentration at the 70 metre distance. That increase in the lead concentration in the soil at 70 metres is also reflected in the mushroom sample collected from the same spot which would suggest there is a significant link between the concentration of the lead present within the mushroom and that of the substrate or soil on which it grew. That trend was not observed with the iron concentrations, the concentration within the soil did not directly correlate with that of the concentration observed within the mushroom, this could be indicative of selective uptake. As the use of lead in fuel is banned the concentration of lead in the air should be relatively low which again is evidence that the lead present in the mushrooms much have been taken up from the soil substrate.
Figure 6 – uranium present in mushrooms & soil
Uranium is a very toxic heavy metal and is found naturally, it has been reported in the past that significant quantities of uranium have been measured in the Hereford area by the Hereford Time, the radioactive decay product is radon gas which can pose health risks. The results we present in this article clearly demonstrate that a higher concentration of uranium was measured in the mushroom samples collected next to the busy road. It is unclear why the levels of uranium are significantly higher closer to the road, this does require further investigation with additional samples taken at different locations.
The mushrooms that were foraged as far as their heavy metal content was concerned did not pose a health risk with excessively high concentrations. It could therefore be concluded that they were safe to eat, however, from the concentration plots for most elements under investigation there is a clear drop off in the concentrations of the heavy metals measured as a function of distance picked from the road.
As with any food that is foraged, it is always advisable to wash it prior to consumption.
There is plenty of scope for additional studies to be carried out as mentioned in the discussion, a repeat of the experiment with additional sampling points would be very useful. It would also be interesting to wash a sample set of mushrooms prior to analysis to determine if the contaminants were only present on the skin, the water used to wash the mushroom samples could also be analysed.
Manchester has, throughout the ages, been at the forefront of research and development for the ever-evolving landscape of science and technology. Through both the agricultural and industrial revolutions the contributions made by Manchester are too many to list, from the evolution of the textile industry right through to that of the mass spectrometry instrument manufacturers of today to name but a couple of examples.
Map of Mass Spectrometry locations in Manchester (Dates for people are for their time spent in Manchester)
John Dalton and Sir Joseph John Thompson
The unified atomic mass unit is named after John Dalton who lived in Manchester during the early 1800s, it was during this time that Dalton conducted several important investigations including his works on atomic theory. The ‘Dalton’ as a unit of atomic mass is often used in mass spectrometry circles, this leads on well to the introduction of Sir Joseph John Thompson who is the Great, Great Grandfather of mass spectrometry and whose name has also been adopted by some as the unit of mass-to-charge ratio (Th). Thompson was a Nobel prize winning physicist born in Manchester in 1856 whose notable achievements included the discovery of the electron and the invention of the mass spectrograph with assistance from Aston in 1912 at the Cavendish laboratory, University of Cambridge. The pioneering mass spectrometry experiments on neon gas led to the discovery of a lower abundance isotope. In the following year the idea of isotopes was simultaneously proposed by Soddy and Fajans through their independent study of radioactive decay chains. Soddy had previously worked with Rutherford, another Nobel laureate, at what is now the University of Manchester back in 1902 on the transmutation of the elements and so the link continues.
Sir Joseph John Thompson
AEI and the Metropolitan Vickers MS1
The initial collaboration between Thompson and Aston lead to many innovations in the field of mass spectrometry as the technology rapidly evolved in many laboratories around the world. The next major milestone in the list of Manchester mass spectrometry achievements occurred in 1946 with the Metropolitan Vickers electrical company developing the world’s first commercial mass spectrometer outside of the USA known as MS1. The Metrovick company had a diverse portfolio of products ranging from vacuum gauges, large electric motors through to the Lancaster bombers used in the second world war. MS1 was sold to James Chadwick, a physicist at the University of Liverpool who received his Nobel prize in 1935. Chadwick had been credited with the discovery of the neutron 3 years prior, he was born very close to Manchester and was also a student of Rutherford. The MS1 was equipped with an electron ionisation ion source and had a practical m/z range up to 300 Th. Metrovick went on to develop the world’s first commercial double-focusing mass spectrometer known as the MS7 in 1955, along with a whole suite of other products including the infamous MS9. Metrovick became AEI which eventually became Kratos Analytical who are still based in Trafford Park today. The AEI MS30 was the first double beam high resolution magnetic sector that enabled an independent mass scale to be used for peak matching through the use of a lock mass. The double beam instrument was developed by Brian Green who later joined VG Micromass, the origins this technique must have inspired what is now known as the lockSpray ion source used on accurate mass Waters electrspray products.
Metropolitan -Vickers MS7 – World’s first commercial double focusing mass spectrometer
1960s – 1970s
Grenville Turner used an AEI magnet on the instrument he developed for the argon-argon dating technique in 1966, this instrument was also named MS1. In 1969 it was used to date the lunar samples returned to earth from the Apollo 11 mission, MS1 was moved to the University of Manchester and is now in the department of earth and environmental sciences.
VG Micromass was formed in 1969 with a staff of 7 including Robert Craig who was a key member of the magnetic sector business at AEI. They developed a variety of small radius magnetic sectors and a TIMS instrument that were used for uranium/plutonium isotope ratio measurements and rubidium-strontium dating of geological samples. By the end of 1972 more than 20 staff had left AEI to join VG Micromass which resulted in the development of a faster scanning magnetic sector that could be used for GC-MS applications. As the demand for high mass resolving power GC-MS instruments that could keep up with quadrupole products increased, fully laminated magnets were developed by a team lead by Brian Green. A decade of mass could be scanned with 10 ppm mass accuracy in 0.1 seconds which resulted in the company receiving the Queens Award for innovation. The new magnet technology was incorporated into the 7070 and ZAB product lines.
It is also important to recognise the influence Manchester has had on the development of countries through the provision of mass spectrometry instrumentation, for example, the very first mass spectrometer purchased by Pakistan in 1974 was a VG Micromass 12 which now has pride of place in the instrumentation museum at the International centre for chemical and biological sciences (ICCBS) at the University of Karachi.
Until the 1980s, the ion sources available for the analysis of large biomolecules were non-existent, electron ionisation is too hard and it is difficult to volatilise a compound with a mass greater than 500 Da for separation by gas chromatography. In 1981 at the University of Manchester Institute of Science and Technology (UMIST) Michael Barber developed a technique with co-workers known as fast atom bombardment (FAB). This must have been influenced in part through his interest in surface analysis techniques and the research into secondary ion mass spectrometry (SIMS) carried out by John Vickerman’s group also based at UMIST. FAB was born by modifying a primary ion gun originally intended for SIMS to neutralise the primary ions prior to impact on the target where the sample was applied with a glycerol matrix. It was possible using a FAB ion source to ionise large biomolecules such as the protein tripsynogen with a mass of 23,978 Da using the ZAB-SE developed by Brian Green with extended geometry for high mass applications. The introduction of FAB as an ion source option revolutionized the field of mass spectrometry and lead to early peptide sequencing experiments.
The MALDI technique was not far behind, this also employed the use of a matrix modifier in a similar vein to that of glycerol used in FAB. The breakthrough for large biomolecule MALDI MS was made by Koichi Tanaka from Shimadzu who managed to analyse the protein carboxypeptidase-A using a sample matrix modifier consisting of 30 nm cobalt nano particles suspended in glycerol.
The electrospray ionisation technique first proposed by John Fenn was the next big innovation for mass spectrometry; the mass range of this technique is unrivalled. One of the main challenges faced by the instrument manufacturers was the development of an atmospheric pressure inlet, the eventual Micromass solution to this problem still in use today involves dual off-axis orthogonal sampling of the ion beam known as Z-Spray.
Meanwhile Kratos continued with the development of their magnetic sector instruments and were the first to use an electrical array detector in a commercial mass spectrometer, this enabled up to 4% of the mass range to be monitored simultaneously on a 1 inch micro-channel plate that incorporated 1024 channels.
In 1989 VG Elemental developed the PlasmaTrace which became the world’s first high resolution ICP-MS instrument that successfully addressed the issue of isobaric interference.
Kratos Analytical are a subsidiary of the Shimadzu Corporation with much of the MALDI instrument development work still being carried out in Trafford Park.
Early 1990s Kratos Kompact MALDI TOF
VG Analytical (aka Micromass) at a similar time also began work on a MALDI product known as the TofSpec led by John Hoyes which incorporated the first time-of-flight mass analyzer developed by the company.
Bob Bateman through his time working at AEI and then VG Analytical was also key to many of the worlds firsts and innovate developments in mass spectrometry instrumentation including the reverse geometry ZAB with a collision cell for ion activation. Bob Bateman also improved the mass range of a 70 70 magnetic sector instrument by changing the ion beams angle of entry into the magnet; if the angle is halved, the radius doubles and the mass rage is extended by a factor of 4. The AutoSpec mass spectrometer with its unique EBE geometry set the gold standard for dioxin analysis was also one of his projects. Bob and Brian Green were both awarded the British Mass Spectrometry Society’s Aston Medal in 2008 and 1996 respectively for outstanding contributions to the field of mass spectrometry.
The requirement for high sensitivity tandem mass spectrometry products resulted in some interesting hybrid instruments developed by VG Analytical and Kratos including the coupling of a quadrupole or even a TOF mass analyzer to the end of an AutoSpec magnetic sector instrument post collision cell. The challenges associate with this development was the necessity to float the final mass analyzer with its associated electronics used for product ion scanning at up to 8 kV to match the AutoSpec’s beam energy.
The first tandem quadrupole instruments including the Quattro 1 and BioQ used a dual detector system that enabled data dependent acquisitions (DDA) to be run. The first quardupole would scan for an ion above a threshold intensity by diverting the ion beam to the first detector before the collision cell. Once triggered the ion optic known as the Altrincham lens would switch the ion beam back on-axis through the collision cell so that a product ion scan could be run on the high intensity pre-cursor ion.
In 1995, the development of the world’s first commercial hybrid quadrupole time-of-flight (QTOF) mass spectrometer was led by John Hoyes and Bob Bateman by coupling a single quad Platform LC mass spectrometer to a TofSpec through a collision cell, this proved to be a game changer with most of the major vendors following suit with their respective offerings. The Micromass QTOF was released at ASMS in Portland, Oregon in 1996 and went on to receive a Queen’s Award for technological achievement in 2000.
Micromass Q-TOF2 LC-MS/MS, 1998
Meanwhile the research team at Kratos in Trafford Park started work on what would become the world’s first hybrid ion trap time-of-flight (IT-TOF) instrument that was later developed in Japan.
Alexander Makarov joined HD Technologies based in Manchester in the mid-1990s and went on to develop a completely new mass analyzer that later became known as the Orbitrap. The development of the Orbitrap technology using FT-ICR technology with a mass resolving power of more than 150,000 at m/z 195 with no cryogens was revolutionary. It was not long before Thermo acquired HD Technologies but it is interesting to note their location on the Atlas Business park was practically next door to one of the Micromass sites.
Inorganic mass spectrometry also received its fare share of Manchester innovations with the introduction of the VG Analytical Platform ICP elemental mass spectrometer developed by a team led by Jim Speakman. This was the first ICP-MS instrument to use a hexapole collision cell before the quadrupole mass analyzer for the removal of polyatomic interference ions.
Micromass Platform ICP-MS 1996
The world of mass spectrometry is a friendly one, however is can be highly competitive when it comes to the speed, sensitivity, selectivity and specificity top trumps game. Competition from overseas has been fraught especially between the Manchester firms and the other big players such as Agilent, Thermo, Bruker and ABI Sciex. As with everything in life there are highs and lows; in 2003 Micromass Uk Ltd. was found guilty in a US court of law of patent infringement due to the differential pumping system employed on the high-end tandem quadrupole instruments (Quattro Ultima) being very similar to that originally developed by ABI Sciex. This resulted in a damage award of $52.6 million in favor of the plaintiff.
The last of those Quattro Ultima instruments including the platinum variant from the early noughties still hold their own in terms of sensitivity with some instruments available today, they are also rock solid with robust electronics and no surface mounted components.
Another innovation available as an option on the Quattro Ultima series of instruments implemented by Brian Green was a 128-bit DAC used to digitise the detector output, with careful calibration and if there was no isobaric interference it was possible to use this quadrupole-based instrument for accurate mass measurements to within <5 ppm.
The next tandem quadrupole instrument to be released by Waters after the Ultima series was the Quattro Premier, it was not possible to use the dual stage differential pumping on the front end but instead the ion transfer optics and collision cell were improved to include a travelling wave ion guide or TWIG. A TWIG consists of a stack of ring electrodes that is used to confine the ion beam with RF, superimposed onto the RF is a DC pulse that propagates through the elements of the optic in the direction of travel on which the ions can surf. The original T-Wave concept used in the Quattro Premier was developed at Waters by Kevin Giles as a fast, high efficiency collision cell with 5 ms dwell times. The Quattro Premier was also the fastest tandem quadrupole instrument at the time capable of switching from positive to negative ion mode in 20 ms which made it possible to acquire both ion modes in a quasi simultaneous manner when analysing a complex multi-residue sample.
T-Wave ion optics later evolved into TriWave technology used for ion mobility spectrometry (IMS), the Waters SYNAPT mass spectrometer was released in 2006 and employed a Q-q-IMS-q-TOF geometry, the addition of the Drift scope software made IMS easily accessible to the masses. The innovative ScanWave collision cell was another world first, used on the Xevo TQ tandem quadrupole instruments it improved the sensitivity of a product ion scan by linking the ejection of the product ions from the ScanWave device with the scanning of the second quadrupole mass analyzer.
Innovations in mass spectrometry also include applications of the technique to answer real world questions. Mike Morris who was supervised for his Ph.D by Michael Barber led the clinical operations team at Waters which developed neonatal screening, thereputic drug monitoring and clinical toxicology applications using mass spectrometry. These techniques are now used routinely around the world and have increased the speed and accuracy of the results when compared to the methods they replaced.
Once the ABI Sciex patent expired the possibility to use dual stage differential pumping became possible to the other mass spectrometry vendors; this resulted in the release of the Waters Xevo TQ-S that incorporated a StepWave ion guide on the front end to improve sensitivity. The StepWave off-axis ion optic device was developed by David Gordon with Kevin Giles and others and is now used on all the Waters high sensitivity LC-MS/MS product lines.
The contributions made to the field of surface analysis using SIMS at the University of Manchester by John Vickerman’s group which later became Nick Lockyer’s group have also been world leading. These include the first use of gold clusters and C60 as a primary ion beam which have both contributed to improving the sensitivity of SIMS to the analysis of higher molecular weight analytes with less fragmentation. A new SIMS instrument design was conceived by Vickerman known as the J105 3D Chemical Imager this was developed in Manchester in collaboration with Ionoptika and SAI based in Old Trafford who assisted with the design of a novel axial TOF cooler buncher. The latest development to emerge from this group include the invention of a water cluster primary ion gun that again improves sensitivity of the technique to higher mass biomolecules.
Ionoptika J105 3D Chemical Imager at the University of Manchester
The Michael Barber Centre for Collaborative Mass Spectrometry (MBCCMS) now headed by Perdita Baran develops native mass spectrometry techniques and instrumentation used to investigate conformation, conformational change and aggregation of large biomolecules. Jakub Ujma developed a novel hybrid ion mobility instrument based on a second generation Micromass QTOF which incorporated a high-resolution temperature programmable IMS drift tube with a range from 150-520 K. Bruno Bellina modified a Waters SYNAPT G2-Si to couple laser spectroscopy to ion mobility which was another unique instrument and experimental technique completed in Manchester at the Photon Science Institute.
The Waters Corporation have donated a number of mass spectrometry systems to the Recycling Organisation for Research Opportunities (RORO) charity which has reconditioned them and then installed them at universities in developing countries. This initiative has put the first LC-MS/MS systems (Waters Acquity TQDs) into Ghana and Nepal and other Manchester mass spectrometry products into Pakistan, Ethiopia, Kenya, Oman and Mongolia. The Quattro Ultima systems installed at JKUAT in Kenya along with an LCT donated by the University of Manchester have been used in collaboration with the Royal Society of Chemistry and GSK to train more than 100 scientists from across Africa in practical LC-MS/MS techniques.
Waters later moved their mass spec headquarters to Wilmslow in 2014 which still counts as Manchester; one of the latest evolutionary steps of the TriWave technology is found in the Waters SELECT SERIES cyclic IMS mass spectrometer. The cyclic ion mobility TriWave device took many years to perfect with many scientists working on the project including Kevin Giles and Jakub Ujma who had moved to Waters after completing his PhD. The experimental capabilities possible with cyclic IMS are described in detail elsewhere, they are a world first and recognised as a significant evolutionary step in the field of ion mobility spectrometry.
The nuclear physics group in the department of physics at the University of Manchester, led by Kieran Flanagan, are now looking to develop bench top instrumentation that will take mass spectrometry to the next level of specificity through its hyphenation to collinear resonance ionisation spectroscopy (CRIS). The CRIS technique has been developed over the years at ISOLDE, CERN for the fast characterisation of exotic radionuclides with low production rates and short half-lives, where every second counts due to the high demand of beam time. Instrumentation currently under development include high abundance sensitivity instrumentation for carbon dating using the CRIS technique. An ICP-MS-CRIS instrument is also being developed for the detection of radionuclides such as strontium-90 which has proved difficult to quantify at the levels required using even the latest ICP-MS/MS techniques.
Manchester is the natural home to many 3rd party mass spectrometry service providers such as Providion and MS Tech Solutions who keep the legacy products up and running.
Carbon Dating Instrumentation under development at Artemis Analytical Ltd
The information presented in this blog article has been inspired by the Author’s personal experience with Manchester mass spectrometry products, starting with the completion of a master’s project analysing carotenoids using a brand new Micromass LCT (oaTOF LC/MS) that was installed by Almas Khan almost 20 years ago. Following on from this the same LCT was used almost daily with a Kratos MS25 RFA magnetic sector GC-MS for the next three years when running the mass spectrometry service at Liverpool John Moores University. Five years working for Micromass UK Ltd. (Waters Corporation) ingrained the Manchester mass spectrometry legacy . The latest Manchester mass spectrometry perceptions have originated from experiences gained working and studying at the University of Manchester and more recently with Artemis Analytical Ltd.
Elemental analysis by definition covers the whole periodic table and includes both qualitative and quantitative analysis of samples covering a wide range of concentrations. Organic elemental analysis may also be determined for a range of small or large molecule applications. It is possible to analyse a multi-component mixture of compounds using chromatographic techniques. If the mass spectrum of the separated components is determined accurately with high mass resolving power then each compound’s molecular formula may be deduced by software. Inductively coupled plasma mass spectrometry (ICP-MS) or x-ray fluorescence (XRF) instrumentation for the analysis of heavy metals are now ubiquitous in the modern analytical laboratory but what methods were available to chemists several hundred years ago for the determination of elemental composition let alone isotope analysis without a mass spectrometer, was it even possible?
The Early Years
The combustion of a sample to change the colour of a flame must have unwittingly been the oldest method employed for elemental analysis, very little in the way of sample preparation is required. Although not officially recorded Early man must have made these observations since dawn of time when having a fire and inadvertently burning various rocks containing interesting ores. Greek philosophers back in the 3rd century BC proposed that all mater consisted of four elements in nature – earth, water, air and fire. The occupation of Egypt by the Arabs in the 7th century resulted in the first use of the word alchemy in Eurpoe through the combination of ‘al’ from Arabic with ‘Khemia’ the Greek word for Egypt. It was proposed by the Arabian alchemists in the 8th century that all metals were made up of various proportions of mercury and sulfur with gold being the perfect metal. The transmutation of base metals into gold was apparently facilitated by the use of substance known as the pholosphor’s stone. It was not until 1901 Rutherford and Soddy discovered that radioactive thorium was transmutating into radium however Rutherford was apparently not so keen on using the same language as the alchemists.
In 1556 a German metallurgist named Georgius Agricola published the first paper and noted that different ores when placed into a flame changed “the colour of fumes” this information was used for qualitative analysis. In the early 1700s Newton investigated light and developed the prism as a means of separating light into its constituent colour or wavelengths. Another century passed before the prism was used to observe and characterise the spectral lines emitted from the elements present in the sun, Foucault noticed that the sodium lines were present in both the sun and a flame. The collaboration between Bunsen and Kirchhoff who are credited with founding the field of analytical chemistry accelerated the development of a flame excitation instrument by combining the hot Bunsen burner flame with Kirchhoff’s spectroscope. Sample introduction was simple and involved placing small quantities of a solid sample directly into the flame usually on a wire loop. Excited atoms or ions generated in a hot flame emit characteristic photons or coloured light during the radiative de-excitation process. The spectroscope was used to record the wavelengths of the emission lines that were characteristic of the element under investigation, and so optical emission spectroscopy was born. For quantitation purposes it is necessary to measure and record the intensity of the emission for each calibration standard prior to running the unknown sample, this however proved difficult with the technology available at that time. The fundamental research was critical for qualitative analysis but it was a number of years before these prototype instruments matured into a product available on the elemental analysis market for quantitation.
The Flame Photometer
The first invention of an instrument resembling a flame photometer was developed by Champion, Pellet and Grenier in 1873. The instrument consisted of two flames one for the analysis of the unknown sample the other for a calibration standard of known concentration, the operator would view displaced emission spectra from each flame through a spectroscope for a single element sodium. Visual photometry was used to compare the intensity of the emission from the unknown sample with that of the emission from the calibration standard, the concentration of sodium was determined using this method to within 5 %. Their instrument design was later improved by Gouy in 1877, who demonstrated that the intensity of the emission was proportional to the size or temperature of the flame and the amount of sample present. In order to account for this Gouy developed the first pneumatically assisted atomizer for liquid sample introduction, an early ancestor of the modern nebuliser and spray chamber sample introduction techniques, this ensured that the amount of sample entering the flame was controlled therefore improving the accuracy of the analysis. Lundegardh during the 1920s, made further improvements to the sample introduction atomiser for flame photometry instrumentation and also incorporated a photographic plate for recording the results.
Gustav Kirchhoff (left) and Robert Bunsen (right)
Atomic Absorption Spectroscopy
Atomic absorption spectroscopy (AAS) was also developed by Kirchhoff using his spectroscope, he observed that when a bright light was shone through a flame that contained excited atoms of an element, characteristic wavelengths associated with electronic transitions of the element were absorbed leaving dark bands confirming its presence. Kirchoff published “the relation between the powers of emission and the powers of absorption for rays of the same wavelength is constant for all bodies at the same temperature.”
Limitations of the instrumentation required to accurately monitor and record the absorption or emission of light hindered the development of the above spectroscopic techniques for quantitative analysis. The integration of photographic plates by Lundegardh did improve the accuracy and precision but calibration was difficult for absolute quantitation. Spectral interference and a lack of sensitivity of these techniques for certain analytes made them impractical for certain trace analysis applications. The main techniques employed in practice up until the early 20th century and indeed today in some cases for elemental analysis use either gravimetric or titrimetric methods of analysis. What was really needed was the development of a reliable electronic detector for recording the instrument response, once this was achieved a calibration curve could be plotted using external standards.
Following on from Gouy’s initial observations related to the size of the flame and intensity of emission it was later concluded that this relationship was due to an increase in the population of the excited state of the analyte in the flame as a function of temperature. Over the next decade various flames were used including hydrogen in air, acetylene in air and acetylene in nitrous oxide, each of these mixtures produced a hotter flame with an associated increase in detection limit. Flame atomic absorption spectroscopy (FAAS) instruments were developed during the 1950s, for the analysis of aqueous samples, the addition of nitrous oxide acetylene flames improved the sensitivity for certain analytes in challenging environmental samples. The most sensitive AAS instruments available on the market today employ graphite furnaces that can detect an analyte concentration with a detection limit of down to ppb for some assays. Sample preparation often involves microwave digestion of a solid sample in nitric acid, biological samples may also be prepared using a similar method prior to sample introduction at a suitable concentration.
Atomic Emission Spectroscopy
The development of atomic emission spectroscopy did not receive much attention for a number of years due to a lower detection limit being achieved using atomic absorption techniques. A stable plasma source running at more than twice the temperature of the hottest flame was required to realise the full potential of atomic emission spectroscopy. In 1974, the first commercial inductively coupled plasma optical emission spectroscopy (ICP-OES) instruments were developed using argon gas by KONTRON in Germany. The use of inductively coupled argon plasma instrumentation for emission spectroscopy is very popular today and offers a lower detection limit than atomic absorption for elemental analysis especially for the analysis of toxic metals.
Discovery of the Neutron
The only caveat with both of the above aforementioned spectroscopic analysis techniques is their inherent lack of isotopic selectivity. Rutherford and Soddy first proposed the idea of transmutation of the elements back in 1902, that lead to a significant modification to Dalton’s atomic theory however the term or idea of an isotopes was not considered. Some years later Soddy and Fajans simultaneously proposed the existence of isotopes in 1913, through the study of radioactive decay chains and the discovery of three different stable isotopes of lead.
Mass Spectrometry and Isotope Ratio Analysis
Mass spectrometry changed everything for isotope analysis. Thompson and Aston analysed a neon gas sample using a parabola mass spectrograph developed in 1912 at the Cavendish laboratory in the University of Cambridge. Two parabolas or traces were observed when they had both expected only one for the pure sample of neon.
In the bottom right corner of J. J. Thomson’s photographic plate are the separate impact parabola marks for the two isotopes of neon: neon-20 and neon-22.
Thompson had initially thought they had discovered a new element but Aston was not convinced. After learning about Soddy’s discovery and proposed existence of isotopes Aston spent a fellowship distilling liquid air and performing what are now known as isotope enrichment experiments on neon. Neon samples were diffused through porous clay, some enrichment was achieved and measured using a quartz microbalance. Once further developments and improvements by Aston of the mass spectrometry instrumentation came to fruition other isotopes were discovered and the field of isotope ratio mass spectrometry (IRMS) was established. Previous assays using gravimetric analysis for the determination of the (average) atomic mass of the elements on the periodic table were cross validated with results acquired using isotope ratio mass spectrometry with remarkable accuracy. The consequence of the early work carried out by Thompson and Aston was the realisation that the chemical properties of an element are determined by its atomic number rather than its atomic weight. The existence of isotopes was also published independently by Mosley in 1913, who demonstrated the relationship between an element (atomic number) and the wavelength of the corresponding x-ray spectral lines.
Other Methods for Separation of Isotopes
Isotope ratio analysis has really only become a mainstream analytical technique for a variety of sample type due to the use of electromagnetic separation employed by mass spectrometry. The chemical separation of isotopes is difficult because their chemistry is almost identical. Lighter isotopes do tend to react or evaporate more quickly allowing them to be separated however the efficiency is low. Diffusion rates of different isotopes of a gas or liquid across a membrane will be different due to the lighter isotopes traveling more quickly, this method was used by Soddy for his enrichment experiments however the timeframe is such that it is impractical for use as a quick analytical method. Centrifugal separation of isotopes is common for the enrichment of the heavier isotopes containing uranium, the technique was first proposed by Aston and Lindemann back in 1919, again this technique is more suited for large scale preparative applications rather than analysis. Lasers may be used for the analytical separation and detection of a single isotope from a mixture when finely tuned to resonantly excite a hyperfine interaction between an electron and the nucleus, each isotope will have different hyperfine spectra due to the difference in atomic weight of their respective nuclei. Colinear resonance laser spectroscopy (CRIS) when combined with mass spectrometry offers the highest level of sensitivity and selectivity for sub ppq level elemental analysis.
Development of ICP-MS Instrumentation
The realisation that mass spectrometry could be used to determine the mass-to-charge ratio of ions that are generated from an inductively coupled plasma was a huge step change in the development of ultra-trace elemental analysis instrumentation. When compared with ICP-OES analysis the sampling of the ion beam rather than the emitted light offers many advantages, the lack of spectral interference is critical for the analysis of elemental impurities at very low detection limit. The main technological breakthrough came with the development of an atmospheric pressure inlet for a mass spectrometer that could sample the ion beam generated by an argon plasma ion source for subsequent mass analysis.
Sample Preparation and Analysis by ICP-MS
Sample preparation for ICP-MS is the same as the other spectroscopic techniques, aqueous samples are usually prepared in dilute nitric acid. Solid samples may require dissolution in stronger acids such as aqua regia or even hydrofluoric, microwave digestion is also used to speed up the process. Sample introduction involves the nebulisation of the liquid sample into the spray chamber with argon gas. The analysis of a solid sample is also possible with minimal sample preparation using laser ablation, the ablation plume is swept into the argon plasma using a stream of argon gas. Once inside the ICP torch ionization of the sample occurs in the argon plasma to generate the ion beam. The analyte ion will then be selected by the mass analyzer for transmission through to the detector. The most common mass analyzer used in ICP-MS is the quadrupole for both ultra-trace and trace element analysis covering an elemental concentration range of up to 10-orders of magnitude. ICP-MS instrumentation using a single quadrupole mass spectrometer does however suffer from isobaric interference which can mask the analyte ion as a matrix effect. The argon plasma will generate a series of polyatomic interference ions; for example the argon in the plasma will react with oxygen to give argon oxide at m/z 56 which represents a major isobaric interference with the most abundant isotope of iron at the same mass. Chlorine present in a liquid sample can also form an argon chloride polyatomic ion for each of the major chlorine isotopes that constitute a major isobaric interference for the analysis of arsenic. Various methods that have been employed to reduce polyatomic interference from the ion beam the most successful being the use of a collision cell before the quadrupole mass analyzer. The collision cell will fragment the polyatomic interference effectively changing its mass and therefore only let the analyte ion pass through the mass analyzer for detection. Unfortunately, isobaric interference may also present from an atomic ion generated from an isotope of a different element. The only way to remove these interferences is to use a different type of mass spectrometer that incorporates either a high mass resolving power mass analyzer or a tandem quadrupole mass spectrometer with an additional collision cell in between the two quadrupoles. The collision or reaction cell employed for ICP-MS-MS experiments increase the selectivity of the analysis; this is achieved by using a reaction gas that will selectively react with either the analyte or interference ion to increase its mass for subsequent separation and independent detection.
Analysis of Difficult Samples
The most sensitive routine mass spectrometry instrument for ultra trace element analysis in use today that minimise isobaric interference for challenging environmental samples uses a tandem quadrupole mass analyzer ICP-MS/MS. These instruments have a variety of sample introduction inlets available for the analysis of a liquid sample, particles or even nanoparticles in suspension is possible. The addition of an external laser sampling system for mass spectrometry imaging applications of a particular element in a solid sample using laser ablation is also becoming popular. Software now available enables isotope dilution methods to be run with ease and the use of an internal standard is now routine to improve the precision and accuracy of the results. ICP-MS is a highly versatile elemental analysis technique however it is not possible to use it for the analysis of the lighter elements such as carbon. The instrument of choice for isotope ratio analysis would be a mass spectrometer using a magnetic sector with multiple detectors one for each isotope, an inductively coupled plasma ion source or ideally a thermal ionisation mass spectrometer (TIMS) will give the most precise isotope ratio measurements.
Organic Elemental Analysis, the CHNS Elemental Analyzer
Organic compounds are amenable to ICP-MS however they would have to be digested usually by microwave in nitric acid prior to analysis. It is not possible to use an ICP-MS to get any molecular formula information or quantify the ratio of carbon present. If molecular weight information is required one needs to employ a different ionization source for mass spectrometry such as electrospray for LCMS. A simple combustion technique such as CHNS analysis is ideal to determine the ratio of carbon, hydrogen, nitrogen and sulfur in solid or liquid samples. The development of the CHNS elemental analyzer as a commercial instrument for combustion analysis started with Carlo Erba in 1968. Typically, milligram quantities of the sample are introduced into a combustion tube in excess oxygen, flash photolysis ensues followed by gas chromatography to separate and quantify the evolved combustion gas products such as carbon dioxide. CHNS analysis may be used to test product quality, food or soil analysis.
Accurate Mass LC-MS for Elemental Analysis
There are many types of mass spectrometer available today for the analysis of solids, liquids or gases. Liquid chromatography mass spectrometry (LC-MS) involves the hyphenation of a liquid chromatograph with a mass spectrometer. The MS system will usually incorporate an electrospray ionization or atmospheric pressure chemical ionization source. The sample must be soluble in the mobile phase that flows through the liquid chromatograph which is usually a polar solvent. Separation of the analyte from the sample matrix occurs in a chromatographic column due to the analyte’s polarity; the analyte will partition between the polar mobile and immobilized non-polar stationary phase (like attracts like). The stronger the affinity of the analyte to the stationary phase the longer its retention time within the column. Each component or compound separated by the liquid chromatograph will have a unique retention time for a given analytical method run by the liquid chromatograph (mobile phase solvents, flow rate and type of column). Once separated each analyte will flow into the mass spectrometer where ionization will occur, ions generated from the ion source are then separated by the mass analyzer followed by detection to generate a mass spectrum. Electrospray ionization is a soft ionization technique and tends to only yield a protonated molecule in positive ion mode or a deprotonated molecule in negative ion mode. It is possible using tandem mass spectrometry for example to fragment the precursor ion in the collision cell to yield a number of characteristic product ion fragments. The mass spectrometry method development process involves tuning the various ion optics and collision energy to generate a characteristic fragment ion that can be used for quantitation or conformation that an analyte is present in the sample. High sensitivity is one of the best features of MS detection, it is also quick and can be one of the most specific (using accurate mass) and selective analytical techniques available today. Applications include metabolite profiling for metabolomics, proteomics, environmental monitoring and drug development. Once the mass spectrum has been acquired with an accurate mass additional data processing may be used to generate the chemical formula, this in turn will give the elemental composition.
This brief review is by no means exhaustive, there are many other techniques that have played their part in the development of analytical chemistry for elemental analysis. In answer to the original question posed regarding the feasibility of elemental analysis several hundred years ago the answer is yes for certain elements using flame excitation methods, however quantitation was not possible really until the late 1870s. In order to determine the isotope ratio of an element this has only been made possible with the advent of mass spectrometery within the last 100 years. The necessity to push detection limits for the analysis of complex samples has never been in such demand as it is today especially for the detection of radionuclides in difficult environmental samples. The new analytical techniques under development by Artemis Analytical Ltd combine the very best that laser spectroscopy and mass spectrometry have to offer meeting the challenges of tomorrow head on regardless of the complexity of the sample type.
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