Current Trends in Analytical Spectroscopy: Technique and Instrument Modifications for both New and Improved Measurements

Progress continues to develop in analytical spectroscopy as improvements are made to instrument sensitivity, limits of detection, and accessibility. This article discusses some of the advancements in techniques and modifications to technologies that are improving measurements and enabling well-established techniques to break into new and exciting fields of research across five spectroscopic disciplines: X-ray fluorescence spectroscopy, infrared spectroscopy, inductively coupled plasma–optical emission spectroscopy, Raman spectroscopy, and laser-induced breakdown spectroscopy.

Since their respective inceptions, spectroscopic techniques have been modified to adapt to new applications and to improve analytical performance. Recent development lies in instrumental fine tuning and slight alterations to allow new types of measurements. Advancements in the fields of biology and nanotechnology have created demand for new methods of measurement using instruments already accessible, pushing the boundaries of what was previously available. The following sections track some of these trends and more across five major techniques: X-ray fluorescence (XRF) spectroscopy, infrared (IR) spectroscopy, inductively coupled plasma–optical emission spectroscopy (ICP-OES), Raman spectroscopy, and laser-induced breakdown spectroscopy (LIBS).

IR Spectroscopy: Measurements at the Nanoscale

Standard IR spectroscopy shines infrared light on a material and measures the fraction of light that is absorbed at a particular wavelength. Light is absorbed when the vibrational frequency of a bond in a molecular structure is the same as that of the incoming light. IR spectroscopy is a valuable and mature technique, but challenges arise when trying to measure nanomaterials or very small amounts of samples. Typically, IR spectroscopy can measure down to 1 mg of sample; however, to be free from the interference effects of water, the sample must be completely dry-a preparation that can take days to complete (1). A new technique called nanomechanical infrared spectroscopy (NAM-IR) has emerged in recent years to measure engineered nanomaterials and very small amounts of sample. As nanotechnology continues to advance, this technique holds an immense amount of promise to assist in many applications, such as pharmaceutical quality control, precision diagnosis, and even drug delivery. In NAM-IR, nebulized samples are collected on nanomechanical resonators, such as strings or membranes, through diffusion and inertial impaction. The higher the velocity of the incoming particles, the more efficient the collection is. When irradiated with infrared light, the captive particles change the resonance vibrations of the resonator via photothermal heating. This resonance shift is directly proportional to the absorbed energy (1). Previously these NAM-IR measurements had to be performed in a specialized laboratory, but recently a group (2) has designed a NAM-IR system based on magnetic transduction. This instrument uses 10-µm gold electrodes in between two Halback arrays of neodymium magnets to increase the magnetic field over the sensor chip in which the resonator is embedded. In this case, the nanomechanical resonator used is a perforated membrane. An alternating current is applied to two electrodes to drive an initial vibration of known frequency through the resonator. To test their setup, indomethacin, a common oral anti-inflammatory drug, was analyzed. Approximately 100–250 pg of sample was calculated to have deposited onto the membrane, and the resultant measured spectra were in good agreement with those obtained from traditional Fourier transform infrared (FT-IR) spectroscopy.

Another technique that has emerged recently to study objects at the nanoscale is a combination of atomic force microscopy and infrared spectroscopy (AFM-IR). In this approach (1), a pulsed, tunable laser causes a localized thermal expansion of the sample that displaces an AFM tip and cantilever. The resulting oscillation is measured with a second laser and is directly proportional to the amount of IR radiation absorbed by the sample. Because the displacement of the AFM tip is not limited by the diffraction limit of the IR wavelength (about 5 µm), the spatial resolution of this technique is substantially higher than that of standard IR spectroscopy. This method has proven especially valuable in the biological sciences by providing a wealth of information about the nanoscale chemical, structural, and mechanical properties of biological materials. AFM-IR has been used in attempts to understand the pathways of neurodegenerative diseases. These are thought to be caused by the misfolding of proteins in the brain that then trigger the formation of more misfolded proteins. The malformed proteins clump together, forming aggregates of nonfunctional brain tissue. Standard IR techniques are not able to elucidate heterogeneous tissue at a single species level and therefore can't provide a link between the individual component of an aggregate and its toxicity. Ruggeri and colleagues (3) used AFM-IR to determine that the ataxin-3 protein, which is responsible for spinocerebellar ataxia-3, misfolds during protein aggregation and not before as previously thought. Another group, also led by Ruggeri (4), showed that the quality of the intermolecular hydrogen bonds and the structural organization of an amyloid sheet structure are fundamental to determining toxicity in polyQ diseases such as Alzheimer's and Huntington's. Obtaining a better understanding of these pathways presents the possibility of human intervention to disrupt or delay the onset of such diseases.

XRF: Space Exploration and Cultural Heritage Analysis

XRF is broadly used for elemental analysis. When excited by an external energy source, individual atoms emit X-ray photons of a characteristic energy. This technique is commonly used in astrophysics and planetary science to study the geologic composition of celestial bodies. Standard X-ray spectrometers pose a few problems when sent into space because of their large masses and volumes. Previously launched models intended to investigate objects in space have been based on simple collimators, which is fine if the bodies of interest are large enough to fill the instrument's field of view. However, the sky is full of X-ray sources that are often stronger than the X-ray output of nearby small bodies of interest and can easily interfere with measurements. Problems occur when the space and weight available to the instrument is considered. The primary factor in the design of multi-instrument probes is how much the instrument components weigh because of the enormous cost of launching each additional gram into space. If full advantage is to be taken of these instruments with respect to observing small bodies, a new lightweight design is necessary. A team at Harvard and the Smithsonian Astrophysical Observatory (5) has recently developed prototype XRF instruments based on focusing optics to better study small planetary bodies, such as asteroids. Named MiXO, short for miniature lightweight Wolter-I focusing X-ray optics, this instrument is based on hybrid X-ray mirrors consisting of metal and ceramic. The mirrors are crafted through a combination of electroformed nickel replication and a plasma thermal spray process; a ceramic layer provides stiffness, and a nickel-cobalt layer maintains the smoothness needed for X-ray reflection. In this prototype, much of the nickel has been replaced with alumina to reduce the weight by a factor of 30. A few example configurations are described that illustrate a threefold improvement in light-collection power, and at least 10 times improvement in angular resolution, detection sensitivity, and energy band coverage over the current leading system for observing the X-ray fluorescence of astronomical objects.

Closer to home, XRF spectroscopy continues to expand into the analysis of historical and cultural heritage objects. The big advantages of using XRF in this field are that it is a nondestructive technique and can be used in situ, without having to physically move the object of interest to a laboratory. While the use of XRF continues to expand into this area, specialized modifications to the technique are being made. Much of the recent literature is focused not on analytical developments, but on new ways to gain insight into the origin of the materials, mostly though the use of multiple techniques. For example, to fully understand chemical changes that have occurred over time within a painting, X-ray techniques (such as XRF, X-ray diffraction, and X-ray absorption spectroscopy) are combined with FT-IR or Raman spectrometry (6). When measuring heritage objects, conventional XRF only gives a two-dimensional (2D) pigment distribution because of the depth to which the X-ray beam penetrates. A group in Japan (7) designed an instrument that combines a confocal XRF spectrometer and two polycapillary lenses to image multiple layers and generate elemental three-dimensional (3D) maps of artwork. A half lens sits in front of a silicon drift detector and a full lens is attached to a compact X-ray tube. The excitation and detection focusing spots are adjusted to be the same location, called the confocal point. Using a micro X-ray beam to irradiate the sample, the team successfully located a hidden black cat underneath multiple paint layers in a replica work of Daubigny's Garden originally by Vincent van Gogh. This system had a resolution depth of about 54 µm and can successfully be used to identify pigments in multiple layers.

ICP-OES: Sample Introduction and Cost of Operation

ICP-OES ignites argon to create a plasma that ionizes nebulized samples to determine elemental composition. Most of the recent advances in ICP-OES have been improvements to its analytical capabilities, primarily through increasing the efficiency of sample introduction. Most samples that are analyzed by ICP-OES are aqueous solutions, which presents a few drawbacks when it comes to instrument sensitivity: The sample introduction depends on the nebulization efficiency, less energy is available for sample atomization because of the vaporization cost of the solvent, and the introduction of matrix effects (8). Much work has been done in recent years to find different ways of introducing samples into ICP-OES instruments as gaseous species rather than aqueous ones. Electrothermal vaporization (ETV) has been used as a sample introduction method for many years now, but only recently has the number of reports using ETV in conjunction with ICP-OES increased (8). A major advantage of this sample introduction technique is that it can be used to directly analyze solid material, which increases sample throughput and decreases both the time and possibility of contamination during sample preparation. It also significantly improves the transport efficiency versus a traditional pneumatic nebulizer from <5% to at least 60% (8). A significant limitation of this technique, however, has been relatively slow data transfer that results from a slight incompatibility between the signals produced during sample vaporization and the detection system of the ICP-OES. Luckily, researchers are finding ways to overcome this signal lag. Chaves and colleagues (9) have written a MATLAB program to increase data transfer time. This program can generate 3D spectra while simultaneously performing fast background correction and signal integration for as many as eight analytes. All necessary calculations are performed offline, further reducing the time it takes to process the data.

The use of chemical modifier gases during vaporization has been investigated to improve sensitivity. Hassler and colleagues (10) investigated the detection of trace elements in high-purity copper samples using fluorinated gases or halogenation reactions for most elements. They had a high rate of success using this method and were able to elucidate 22 elements present in the samples.

Standard ICP-OES using the current Fassel-type torch design uses around 12–20 L/min of argon, making it expensive to operate (8). In recent years, a couple of new designs have been proposed and tested to reduce the costs of argon consumption. A redesigned plasma torch was introduced (11) in 2005 called the static high-sensitivity ICP torch (SHIP), consisting of a quartz tube with a spherical excitation zone and an external air cooling system. This design significantly reduces argon consumption to around 0.05–1.00 L/min. Though first proposed more than 10 years ago, recent studies utilizing the new torch for comparison with standard ICP-OES are still ongoing. Nowak and colleagues (12) studied the arsenic content in three kinds of fish and had an argon flow rate of 0.66 L/min, compared with the same test using 14 L/min with a conventional ICP torch. Another approach (13) to reducing argon consumption focused instead on the amount of argon used to purge the instrument of molecular oxygen and water vapor before testing. In this sealed optical system, argon is recirculated through a small purifier cartridge. Though this approach saves a much smaller amount of argon compared to that necessary during torch operation, no refill is required-once a system has argon inside for purging, no more is needed. This sealed design was also found to improve analytical performance in the vacuum-ultraviolet (UV) region (less than 190 nm) and potentially allows for fewer matrix effects than the SHIP torch system.

Raman Spectroscopy: Measurements in Biology and at the Nanoscale

Raman spectroscopy is based on the inelastic scattering of monochromatic light to reveal the molecular composition of a sample. This technique has two inherent disadvantages that prevent it from being used unmodified for high-resolution measurements: low sensitivity and diffraction-limited spatial resolution. Surface-enhanced Raman spectroscopy (SERS) is often used to overcome the low sensitivity. Substrates for analysis usually consist of nanostructures or a rough metal surface. When the adsorbed sample particles are irradiated with particular wavelengths of light, the Raman scattering is enhanced by this surface. Though able to overcome low sensitivity problems, SERS alone does not provide spatial resolution on a nanometer scale. Tip-enhanced Raman spectroscopy (TERS), however, does. TERS exploits a resonance between metallic nanostructures at the apex of a probe and an incident light to generate a strong, localized electric field that enhances Raman scattering.

The expansion of TERS into new application areas, specifically the biological sciences, has made it necessary to find a way to prevent sample damage because of the Joule heating that occurs during electric field generation. Using thiophenol molecules with known thermal desorption profiles, Mochizuki and colleagues (14) were able to monitor desorption and accordingly adjust the incident laser intensity during testing to prevent a damaging increase in probe temperature. This group also looked at the dissipation of generated heat into the surrounding medium through probe oscillation during measurement. For temperatures up to the thermal desorption of thiophenols (60–100 °C), the combination of these two methods have proven to effectively prevent damage to heat-sensitive samples such as organics and biomolecules.

For the first time, small molecules inside a cell have been mapped using TERS. Previously, the best-known technique to map small biomolecules nondestructively was super-resolution fluorescence microscopy. However, this relies on the attachment of fluorophores to molecules, which can have an impact on their behavior and function. Additionally, only those molecules of interest that have been tagged can be seen with this technique-all others are invisible. Using excised cultured mouse pre-adipocyte cells, Kumar and colleagues (15) mapped the growth of newly synthesized phospholipid accumulation. Deuterated stearate ions were metabolized by the cells to provide a fatty membrane tail that could be imaged because of the carbon–deuterium bonds visible in the Raman spectra. The resolution obtained was less than 20 nm, effectively demonstrating another analytical tool for nano-imaging that the authors hope will help pave the way for better design of therapeutic drugs that need to target specific cell areas.

Side-illumination TERS is a variant in which the incident laser light comes in at an angle from the side, providing very efficient near-field enhancement when paired with a metal substrate. However, this method presents two challenges. First, the focal spot projected on the sample tends to be quite large, leading to a high level of background noise. Second, the use of a low numerical aperture objective lens limits the collection efficiency. To overcome these obstacles, Shen and colleagues (16) proposed a new probe design to increase the signal-to-noise ratio and collection efficiency. The latter criteria is essential for a fast acquisition time to reduce potential image distortion. The proposed design is theoretical and was mathematically demonstrated to increase the localized electric field by 25 times when compared to a regular tip. The presence of concentric grooves on the theoretical tip surface are shown to strongly enhance the field intensity without affecting its spatial resolution. Two asymmetrical nanorods at the tip assist in unidirectional emissions. The researchers note that the nanofabrication of the two nanorods would be very difficult, but can be done. Overall, these two modifications allow more light to be directed toward and collected by the collection objective. This design has the potential to be very useful for single molecule measurements in a variety of sample types.

LIBS: 3D Mapping, Molecular Signatures, and Instrument Sensitivity

LIBS measures atomic and ionic compositions by using short pulses of laser light to create a dense, high-temperature plasma. Discrete spectral peaks are emitted when excited atoms fall back down into their ground states. This is a technique for measuring the atomic and ionic signatures of a material in the UV to near infrared (NIR) spectral range. Recently, it has been demonstrated that it is possible to also obtain intact molecular information by analyzing the spectra in the mid- and long-IR wavelength range (LWIR-LIBS) (17). Current detection systems are comprised of mercury-cadmium-telluride (MCT) arrays that can acquire a full LWIR spectrum (5.6–10 µm) in under 5 s (18). One group (17) that has done extensive work with this new technique tested a setup to acquire both LWIR and UV–NIR LIBS spectra simultaneously by simply connecting the two different detection systems into a linear array. The test was successful, and the known ingredients of two major over the counter medications were easily identified.

As previously noted in this article, nanoparticles have received a lot of attention in the medical field lately as a vector for drug delivery and immunotherapy. Gimenez and colleagues (19) recently developed a technique to image manufactured nanoparticles consisting of gadolinium, calcium, or gold using LIBS in biological tissues. Using these nanoparticles, developed specifically for image-guided radiotherapy and which are smaller than 5 nm in diameter, they successfully created a 3D image of excised rodent renal tissue. Traditional methods of tissue imaging rely on the use of labels to mark the introduced nanoparticles so they can be distinguished, but this approach can have a drastic influence on the shape, size, or charge of the particle and affect its distribution, or even detach from the nanoparticle entirely. LIBS circumvents this challenge because it relies solely on the excitation emissions of the nanoparticles themselves. LIBS may not be as sensitive or as high-resolution as other spectroscopic techniques (though it is still excellent), but it has the advantage of an acquisition rate up to 100 times higher, making the creation of 3D maps of large samples (that is, entire organs) a relatively quick process. This study used nanosecond infrared laser pulses of 500 µJ focused through a 15x microscope objective at epoxy-encased tissue to obtain a 2D map. Two approaches to creating a 3D map were studied and found to be complementary. The first was to slice the sample into many 200-µm-thick layers, image them all independently, and then construct a 3D map from the individual results. The second was to repeatedly scan the same ablation area, adjusting the focus for the new depth between each scan. The latter approach obtained a spatial resolution of about 10 µm in all directions. Because of the (relatively) destructive nature of the laser-ablation process, its use is limited to excised tissue and could only be used for imaging removed samples, such as during biopsies.

Improving the sensitivity of LIBS continues to be a popular topic of investigation. Valjanen and colleagues (20) have shown that by coupling a 2.45-GHz microwave with the generated plasma, the signal lifetime is greatly increased from just a few microseconds to hundreds of microseconds. This allows for a longer signal integration time and a significant enhancement of the signal. In particular, the copper content of a copper–alumina solid was analyzed and the limit of detection found to be 8.1 ppm-an improvement of 93 times over conventional, single-pulse LIBS. The effects of self-absorption were also mitigated, which could be promising for measuring high concentration elements. Wang and colleagues (21) investigated sustaining the LIBS-generated plasma in a different way. They have shown that increasing the diameter of the ablation crater increases the persistence of the Cu(I) line, possibly because of shock waves from the ablation itself compressing the plasma and increasing its temperature enough to cause further atomic excitation. They note, however, that the depth does not seem to have the same effect on the Cu(I) line.

Conclusion

The full potential of analytical spectroscopy has yet to be realized. As technology advances and its utility continues to grow, old laboratory staples are being improved upon, refined, and used in new ways to helps us understand the world around us.

References

(1) M. Unger and C. Marcott, in Encyclopedia of Analytical Chemistry (Wiley Online Library, 2017), pp. 1–26.

(2) M. Kurek et al., Angew. Chem. Int. Ed. 56, 3901–3905 (2017).

(3) F.S. Ruggeri, Nat. Comm.6, 7831 (2015).

(4) F.S. Ruggeri et al., Curr Pharm Des. 22, 3950–3970 (2016).

(5) J. Hong et al., Earth Planet Sp. 68, 35 (2016).

(6) S. Carter et al., J. Anal. At. Spectrom. 32, 2068–2117 (2017).

(7) K. Nakano et al, Microchem. J. 126, 496-500 (2016).

(8) G.L. Donati et al., J. Anal. At. Spectrom. 32, 1283–1296 (2017).

(9) E.S. Chaves et al., J. Anal. At. Spectrom. 26, 1833–1840 (2011).

(10) J. Hassler et al., J. Anal. At. Spectrom. 31, 642–657 (2016).

(11) W. Buscher et al., J. Anal. At. Spectrom. 20, 308–314 (2005).

(12) S. Nowak et al., Talanta129, 575–578 (2014).

(13) M.S. Wheal et al., J. Anal. At. Spectrom. 25, 1946–1952 (2010).

(14) M. Mochizuki et al., Nanoscale 9, 10715–10720 (2017).

(15) N. Kumar et al., Chem. Commun. 53, 2451–2454 (2017).

(16) H. Shen et al. J. Raman Spectrosc. 47, 1194–1199 (2016).

(17) C. Chaffee, "LIBS Continues to Evolve," Optics & Photonics News, 2017, https://www.osa-opn.org/home/articles/volume_28/may_2017/features/libs_continues_to_evolve/.

(18) C.S.C. Yang et al., Appl. Opt.54(33), 9695–9702 (2015).

(19) Y. Gimenez et al., Sci. Rep. 6, 29936 (2016).

(20) J. Viljanen et al., Spectrochim. Acta, Part B 118, 29–36 (2016).

(21) Y. Wang et al., Phys. Plasmas 23(11), 113105 (2016).

Nicole Olson is a freelance science writer. She works in research and development in St. Paul, Minnesota. Direct correspondence to nicolerose5482@gmail.com