Answering Life's Questions with Spectroscopy

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  • Published: Jun 28, 2017
  • Author: Jon Evans
  • Channels: HPLC / Gas Chromatography / X-ray Spectrometry / Infrared Spectroscopy / Atomic / Base Peak
thumbnail image: Answering Life's Questions with Spectroscopy

Spectroscopy has been a common analytical technique in laboratories for many years. Recently, however, it has begun to emerge from the laboratory, both figuratively and literally, as more and more researchers discover that it is ideally placed to answer the questions they want to ask.

The term spectroscopy actually encompasses a wide range of different techniques, including Raman spectroscopy, infrared spectroscopy and nuclear magnetic resonance spectroscopy. It even encompasses techniques that aren’t really spectroscopy at all, because they don’t involve interactions between matter and electromagnetic radiation, with mass spectrometry being the prime example.

Nevertheless, all these techniques generate spectra and thus can be encompassed within spectroscopy, and these spectra can be used to identify specific compounds in a sample. Because each of these spectroscopy techniques works in a different way, they possess a wide range of different analytical abilities and properties, and can reveal a host of interesting information about those samples and compounds. This means that there is almost always a spectroscopy technique available for answering whatever question is being posed.

The trick then is for researchers is to find the spectroscopy technique that is best able to answer the questions they want to ask. Usually this depends on the information the technique can reveal about a compound, the amount of the compound required for the technique to reveal that information and what other compounds are present as well. When the questions involve determining whether an athlete has taken a performance-enhancing drug, the technique needs to be able to detect very small concentrations with great accuracy within a complex sample such as urine. This generally calls for mass spectrometry.

Mario Thevis

“The fact that mass spectrometers can provide considerable information from minute amounts of a substance with enormous specificity have always been among the most important aspects,” says Mario Thevis, professor in the Center for Preventive Doping Research at the German Sport University Cologne. Thevis doesn’t use mass spectrometry on its own, though, but in conjunction with gas or liquid chromatography. This allows him to detect performance-enhancing drugs or their metabolites in complex biological samples such as urine, by separating the drug or metabolites from the many other compounds that are present.

By using tandem mass spectrometry, which involves conducting multiple rounds of mass spectrometry on compounds as they are broken down into fragments, Thevis can also probe the structure of the drug molecules. “The generation of structural information on drugs and corresponding metabolites is supported by mass spectrometric data, and those are further utilized to develop and/or expand doping control analytical assays,” he says.

The sheer number of different performance-enhancing drugs and metabolites that need to be screened for presents another major challenge, especially as this number is growing all the time. In the past, this has required employing several different analytical techniques, but the latest mass spectrometers are able to take on more and more of the responsibility.

"Our work would be significantly more complex without sensitive mass spectrometers"

Mario Thevis

In 2016, Thevis and his colleagues at the Center for Preventive Doping Research showed that a combination of liquid chromatography and a state-of-the-art hybrid quadrupole orbitrap mass spectrometer could detect over 200 performance-enhancing drugs and metabolites in urine at nanogram to microgram concentrations.1 This encompassed a wide range of different drug classes, including diuretics, stimulants, β2-agonists, narcotics and anabolic androgenic steroids, as well as newer compounds like hypoxia-inducible factor stabilizers, selective androgen receptor modulators and selective estrogen receptor modulators. It was even able to detect growth hormone-releasing peptides, which usually need to be detected with laborious and time-consuming stand-alone assays.

According to Thevis, this could only have been achieved with mass spectrometry. “Our work would be significantly more complex and several projects could not be conducted without having sensitive mass spectrometers available,” he says.

The ability to detect and identify tiny concentrations of compounds is what also attracted Matthew Loxham, a fellow in respiratory biology and air pollution toxicology at the University of Southampton in the UK, to mass spectrometry. In this case, however, Loxham was interested in using it to determine the composition of the tiny particles released into the air by modern forms of transport. These airborne particles are known to be toxic if inhaled and have been linked with the development of a wide range of diseases, including respiratory diseases, lung cancer and cardiovascular disease.

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Matthew Loxham

Matthew Loxham

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The toxicity of these particles does vary quite a bit, however, depending on their size and elemental composition. Smaller particles are more toxic because they can travel further into the body, while particles made up of transition metals such as iron, chromium and zinc, which are produced by vehicles braking, are more toxic than the carbon particles produced by combustion. This is because transition metals can generate damaging oxygen species inside the human body.

In order to determine the elemental composition of airborne particles, Loxham turned to a specialized form of mass spectrometry known as inductively-coupled plasma mass spectrometry (ICP-MS). In conventional mass spectrometry, compounds are ionized and then identified based on their passage through electric and magnetic fields, which depends on their charge and mass. Various ionization methods are used with mass spectrometry, which vary in the degree to which they fragment the compounds into smaller molecules.

Determining elemental composition requires an incredibly harsh ionization method that can blast the particles into their component elements. This is what ICP-MS does, by ionizing the particles with a plasma of charged argon ions and electrons at a temperature of around 10,000K. The plasma vaporizes the particles into their component atoms, which are also ionized, allowing them to be identified by mass spectrometry. No other technique does this as effectively.

“For me personally, because of my background as a toxicologist and cell biologist, my main interest is in characterizing the composition of these particles and so really ICP-MS is the main technique I use,’ says Loxham.

The sensitivity and versatility of ICP-MS is what makes it particularly appropriate. “Its ability to detect not just the common elements like iron, magnesium, barium, tin and zinc, but also to detect low levels of more unusual elements adds more information so that we can better understand where things have come from,” Loxham explains. “This extra information on some of the elements that you wouldn’t detect with less sensitive techniques just adds a bit more confidence to our findings.”

In 2012, Loxham and colleagues used ICP-MS to study particles collected at a railway station beneath Amsterdam airport and compare them with particles collected from other sources, including a wood stove and a road tunnel.2 They found that particles from the railway station were richer in metals, especially iron, copper, chromium, manganese and zinc, than those collected from the other sources. This is probably because the railway particles are likely generated by braking, as well as arcing between highly-charged electrical conductors, rather than combustion.

More recently, Loxham has received funding from the UK Biotechnology and Biological Sciences Research Council to study the particles emitted at Southampton docks, for which he will again be using ICP-MS. “There’s a big question mark over docks and ships and what they emit and how their emissions impact upon local health and air quality,” he says. This study will involve collecting particles from various sources around the docks, like ships, trains and metal scrap piles, and then using ICP-MS to see how the particles emitted by these sources differ in their composition.

By their very nature, airborne particles are obviously small and present at comparatively low concentrations, but sometimes samples are very small out of choice or necessity, as when analyzing valuable historical artifacts. Because scientists don’t want to damage these artifacts, they can only take tiny samples for analysis. Ideally, they would use a non-destructive technique that doesn’t require taking any samples at all. Spectroscopy provides options for both approaches.

Barbara Berrie

Barbara Berrie

This is why Barbara Berrie, head of the Scientific Research Department at the US National Gallery of Art in Washington, DC, utilizes a whole range of spectroscopy techniques to study the gallery’s collection. These include Raman spectroscopy, Fourier transform infrared spectroscopy and x-ray fluorescence spectroscopy.

“We use methods that help us answer the questions conservators and art historians ask: ‘What is this blue? From the trace elements can you tell me which trade routes the colorant may have come along? Was this colorant a new invention when the artist used it? Is it possible this color has faded or changed since the artist laid it on hundreds of years ago? What is the paint binder? Is the texture I’m seeing here related to alteration of the paint or is it original?’” says Berrie. “The techniques are applied to minute samples from works of art, or adapted to use without sampling.”

For example, in 2016 Berrie studied a tiny sample taken from a painting entitled Madonna and Child by the 13th century Florentine artist Giotto.3 The sample was taken from the bottom of the painting, near to existing damage, in a section depicting the Madonna’s robe, which contained blue, yellow and green pigments.

Together with colleagues, Berrie used various different analytical techniques to investigate the sources and nature of these pigments. Scanning electron microscopy (SEM) and energy-dispersive x-ray analysis (EDX) revealed that the yellow pigment was a compound containing lead and tin commonly used at the time, but that the blue pigment was a less commonly-used copper-based mineral called azurite. They also detected separate green-blue particles in the azurite pigment, which analysis with Raman spectroscopy revealed was a rare copper bismuth arsenate mineral called mixite. Raman spectroscopy identifies molecules based on the characteristic way their molecular vibrations scatter incoming laser light.

"We could not answer the questions we are asked without modern spectroscopic methods. The samples we look at are extremely small and very complex chemically."

Barbara Berrie

This mixite was probably naturally present in the azurite paint, rather than added deliberately, as it is a secondary mineral that is commonly found in conjunction with azurite, especially in certain geographic regions. This suggests that the mixite could help to reveal where the azurite used to produce the paint came from originally.

The ability to probe the pigments and paints used in historical artworks without causing too much damage is a fairly recent development, and is heavily reliant on state-of-the-art spectroscopy techniques. “We could not answer the questions we are asked without modern spectroscopic methods,” admits Berrie. “The samples we look at are extremely small and very complex chemically.”

The answers being obtained with these spectroscopic methods are helping to transform researchers’ understanding of how these historical artworks were produced and how the painting process changed over time. “Ultimately the merging of imaging with spectroscopic analysis allows the curator to better understand a painter’s process and see how they applied their paints to a canvas, what changes they made, and which colors they used,” says Berrie. “Scientific art lovers can admire the images produced by spectroscopic analysis and enjoy that their speciality can be used to understand the role of materials in the creative process of art making.”

As well as being minimally destructive, the other great advantage of spectroscopy is that it can often analyze samples in their natural state, without requiring any special preparation steps. This saves time and complexity, and allows spectroscopy-based analysis to be conducted out in the field, rather than requiring samples to be transferred back to the laboratory. It has also led to the development of portable spectroscopy instruments, such as handheld Raman spectrometers, which have proved of great use for both forensics and quality control.

Jacqueline Stair, senior lecturer in analytical chemistry in the Department of Pharmacy, Pharmacology and Postgraduate Medicine at the University of Hertfordshire in the UK uses spectroscopy for both these applications. “We use a range of spectroscopic techniques but in particular we focus on Raman spectroscopy and inductively coupled plasma – optical emission spectroscopy (ICP-OES),” explains Stair. “We use these techniques to characterize novel psychoactive substances/designer drugs and herbal materials.

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Jacqueline Stair

Jacqueline Stair

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“By using these two techniques we can obtain both molecular and elemental information. Raman spectroscopy is a great non-destructive vibrational technique with handheld versions available that you can use outside of the laboratory. In the case of ICP-OES, you can obtain a comprehensive elemental profile or ‘fingerprint’ of substances with concentrations ranging over many orders of magnitude.”

"Raman spectroscopy has many advantages for the in-field detection of designer drugs; it allows measurements through packaging with minimal interference from water"

Jacqueline Stair

In a recent study, Stair and her team used ICP-OES to analyze 54 samples of St John’s Wort, a medicinal herb commonly used for treating mild depression.4 Like ICP-MS, ICP-OES can determine the elemental composition of samples, but does so by monitoring the characteristic wavelengths of light emitted by the elements when exposed to the plasma. Using it, Stair and her team were able to determine the concentrations of elements such as calcium, magnesium and zinc, and use them as a fingerprint to distinguish between three different formulations of St John’s Wort – dry herbs, tablets and capsules.

In another recent study, she used handheld Raman spectroscopy to identify 29 different designer drugs, based on illuminating the drugs with a single wavelength of light.5 “In the case of designer drug detection, there are hundreds of these drugs which can often be very similar in chemical structure and functionality, thus the use of a discriminating spectroscopic technique is key,” says Stair. “Raman spectroscopy, in particular, has many advantages for the in-field detection of designer drugs.”

One of these advantages is that Raman spectroscopy can identify designer drugs while still in their packets, making it even easier to analyze them out in the field. “Raman spectroscopy allows measurements through packaging (i.e. non-destructive) with minimal interference from water,” confirms Stair.

With all these advantages, it should come as no surprise that spectroscopy is beginning to find its way into museums, sports stadiums, dockyards and even dodgy drug emporiums.

References

  1. Journal of Pharmaceutical and Biomedical Analysis, 2016, 131, 482–496. Simplifying and expanding analytical capabilities for various classes of doping agents by means of direct urine injection high performance liquid chromatography high resolution/high accuracy mass spectrometry.
  2. Environmental Science & Technology, 2013, 47, 3614–3622. Physicochemical Characterization of Airborne Particulate Matter at a Mainline Underground Railway Station.
  3. Heritage Science, 2016, 4, 1. Unusual pigments found in a painting by Giotto (c. 1266-1337) reveal diversity of materials used by medieval artists.
  4. Journal of Pharmaceutical and Biomedical Analysis, 2016, 125, 15–21. Elemental fingerprinting of Hypericum perforatum (St John’s Wort) herb and preparations using ICP-OES and chemometrics.
  5. Forensic Science International, 2017, 273, 113–123. Identification of new psychoactive substances (NPS) using handheld Raman spectroscopy employing both 785 and 1064 nm laser sources.

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