The new USP Chapters <232> and <233> will become official on January 1, 2018 and bring significant changes in sample preparation and analysis of pharmaceutical samples both in excipients and APIs. Certain APIs pose challenges to acid digestion because they are very stable compounds and not easily broken down.

	USP methods <232> and <233> call for total digestion of pharmaceutical samples and quantification of individual elements. Many pharmaceutical materials can be easily digested but APIs with multiple aromatic ring structures can be very difficult to completely break down and obtain a clear digestion, as prescribed in the new chapters. Microwave digestion can provide a solution to many of these compounds.

	This application note will focus on the use of the CEM MARS 6 microwave digestion system and new high performance iPrep vessels to completely digest very difficult APIs. The ring structures of the APIs are shown to illustrate the high stability of these compounds. The sample sizes listed represent the maximum allowable in order to achieve a complete digest.

	Three APIs were prepared using a CEM MARS 6 microwave digestion system equipped with iWave technology and iPrep vessels.

	iWave is a novel Light Emitting Technology(tm) that measures the temperature of the actual sample solution inside the vessel and does not require an internal probe.

	iPrep is a simple to use three-piece vessel which uses a simple hand torque device to seal. The patented dual-seal design provides for higher temperatures and fine control of the vent and reseal process necessary for these sample types.

	Each API sample was weighed and added to an iPrep liner with 9 mL HNO

 and 1 mL HCl. The vessels were capped, assembled, and placed in the MARS 6 for digestion. Digestion parameters are recorded in Table I.

	The MARS 6 with iPrep vessels was able to completely digest each of the API materials and provide a clear solution. Of note is that the TrixiPhos digestate is a bright yellow color. This is a result of the Pd catalyst used when synthesizing the compound. Each API sample was run in duplicate in order to confirm the success of the sample preparation. All samples were completely digested as shown in Figure 1.

	The MARS 6 system with iPrep vessels is an ideal option for working with these difficult pharmaceutical materials.


	P.O. Box 200, Matthews, NC 28106
	tel. (704) 821-7015, (800) 726-3331
	Website: 

Articles

Digestion of Difficult APIs in Accordance with USP 

Atomic

It is only in the last two decades that Raman spectroscopy has begun to realize its potential as an almost universally applicable analytical technique from materials and life sciences applications to point of care analysis. This is primarily thanks to the availability of compact laser sources, high sensitivity cameras and high resolution compact spectrometers. In this application note the use of a stable high power NIR 785 nm laser is shown to be critical in achieving high time resolution and high signal to noise ratios while allowing for both fast and slow processes to be captured simultaneously.

	The weakness of detectors in the NIR region (800–1050 nm) with quantum efficiency dropping off over this range from ca. 40% to 0% is a key drawback of 785 nm Raman spectroscopy. Typically long exposure times and spectral averaging are required to achieve a good signal to noise level, however this approach seriously reduces the time resolution achieved (to minutes) and means that weak signals for compounds that are present for only a few 100s of milliseconds cannot be observed. Using more efficient NIR detectors (such as back thinned CCDs) does not usually solve this problem due to etaloning that can be difficult to suppress. This drawback can be circumvented by a combination of efficient collection optical arrangements and above all high laser light fluxes. With a stable high powered laser (500 mW at sample) and a large confocal volume for collection to limit sample damage, time resolutions as low as 100 ms per spectrum can easily be achieved.

	As an example, a oxidation reaction in which H

 is added to a solution containing a precatalyst is chosen. In this reaction only 0.25 mM precatalyst is present (far below that which can be detected by Raman spectroscopy but its absorption at 785 nm means that its spectrum is resonantly enhanced) and H

 is added to give a final concentration of 250 mM which decreases slowly over time. Within 1 s of addition of H

, however, the precatalyst becomes activated with an intermediate that is present only with a maximum concentration of 0.05 mM for 0.5 s and has an absorption band near 785 nm which results in enhancement of its Raman spectrum by ca. 10,000 times through resonance. However, it is observed only because high signal to noise spectra can be recorded at 200 ms intervals.

	By far the most popular wavelength used for Raman spectroscopy is 785 nm, as it offers the best balance between avoiding fluorescence, absorption of the laser light (and Raman scattering) by the sample and therefore heating effects, and the limits to detector sensitivity. The lasers available at 785 nm are diode lasers, however, a narrow line width (<3 cm

 for condensed phases such as solids and solutions and even narrower for gases) is essential for Raman spectroscopy in order to resolve individual Raman bands. The Cobolt 08-NLD 785 nm laser addresses all of the important performance features in a compact footprint and ensures reliability thanks to the proprietary manufacturing technique called HTCure™.


	Vretenvägen 13, 17154 Solna, Sweden
	tel. 46 8 545 912 30
	Website: 

Molecular

Blink and You Miss It—Catching Fleeting Catalytic Intermediates by High-Speed 785 nm NIR Raman Spectroscopy

This application note demonstrates the ability of the NIC MA-3000 direct mercury analyzer to analyze coal samples with accuracy and precision, in compliance with ASTM D-6722-01 (Standard Test Method for Total Mercury in Coal and Coal Combustion Residues by Direct Combustion Analysis).

	Since mercury occurs naturally in coal and other fossil fuels, when these fuels are burned for energy, the mercury becomes volatilized and airborne into the atmosphere. In the United States, power plants that burn coal to create electricity account for about half of all man-made mercury emissions. In nature, elemental mercury can go through a series of chemical transformations that convert it to highly toxic forms that are concentrated in fish and birds.

	The most toxic form of mercury is methylmercury, an organic form created by a complex bacterial conversion of inorganic mercury. Mercury from the food chain is known to bioaccumulate in humans, as bioaccumulation in fish and birds carries over into human populations, where it can result in mercury poisoning. Mercury is dangerous to both natural ecosystems and humans because it is highly toxic, especially because of its ability to damage the central nervous system. Mercury poses a particular threat to human development in utero and in early childhood. Thus, to prevent mercury poisoning, it is necessary to accurately quantify total mercury in coal so that mercury emissions into the atmosphere may be carefully controlled.

	Coal samples are ground with a mortar to avoid segregation effect.

	Calibration is done using certified aqueous ionic-mercury standard solution diluted to the required concentration. The least-squares regression method is used to create and complete the calibration curve.

	Analysis was performed using the NIC MA-3000 dedicated direct mercury analyzer that selectively measures total mercury by thermal decomposition, gold amalgamation, and cold vapor atomic absorption spectroscopy. The MA-3000 analyzer measures mercury in virtually any sample matrix-solid, liquid, and gas-and offers quick results without time-consuming or elaborate sample preparation processes.

	This report demonstrates that NIC MA-3000 is able to reproduce good STD addition recovery of coal. NIC MA-3000 analyzes coal samples with accuracy and precision. It is a perfect solution to today's increasing laboratory demand for easy, fast, and accurate mercury measurements.

http://www.who.int/ipcs/assessment/public_health/mercury/en/

https://www.epa.gov/air-emissions-inventories/2011-national-emissions-inventory-nei-technical-support-document

https://pubs.usgs.gov/fs/fs095-01/fs095-01.html


	14-8, Akaoji-cho, Takatsuki-shi, Osaka. 569-1149
	tel. 72-694-5195, fax 81-72-694-0063
	Website: 

Total Mercury in Coal Using Direct Mercury Analysis

Dealing with a sample matrix high in total dissolved solids (TDS) is a common challenge for ICP spectrometry. Most "real samples" contain considerable concentrations of TDS including soils, sludge, waste water, brines, high acid digests, and fusions. The challenges encountered include interrupted runs, signal drift, clogged nebulizers, shortened torch life, and greater interferences. To overcome these challenges, it is essential you select a proper sample introduction system so that you can achieve the best performance your ICP-OES or ICP-MS can provide. In this article, a complete sample introduction package is presented to improve ICP performance in a high TDS sample matrix. 

	An argon humidifier is essential and one of the most important accessories for ICP-OES and ICP-MS analyses involving samples with high TDS. The added moisture in the nebulizer gas helps to reduce salt deposits in the nebulizer and torch injector, allowing uninterrupted operation. In addition to the argon humidifier, a large bore injector is recommended, typically, greater than 1.5 mm ID for radial view ICP-OES instruments and greater than 2.0 mm ID for axial view ICP-OES and ICP-MS instruments. Due to the rapid devitrification of quartz in the presence of high TDS, a demountable ICP torch is preferred to reduce replacement costs. A nebulizer capable of handling high TDS should be chosen and combined with a baffled cyclonic spray chamber to eliminate large droplets from reaching the torch. Below we will describe in detail the characteristics of each of these essential high TDS components.

	The Elegra™ argon humidifier, released in February 2016 (1), utilizes highly efficient membrane technology to add moisture to the argon as it flows through the inert metal-free construction. The device runs at atmospheric pressure and does not require heating or electric power, as required by other manufacturers. The Elegra is also configured with a bypass switch so that the operator can turn humidification on and off without connecting or disconnecting any tubing. The elegant design results in a very compact accessory that is simple to install.

	A nebulizer stress test was completed aspirating a 25% NaCl solution while monitoring the nebulizer gas flow with and without the Elegra (Figure 1). A decrease in gas flow is a good indicator that a nebulizer is clogging. Note a nebulizer with a TDS tolerance rating of only 5% was used to illustrate the advantage of the Elegra. As a worst-case test, the salt solution was aspirated continuously, with no rinsing. As expected, without the Elegra, the nebulizer was completely clogged after only 5 min. In contrast, the same nebulizer with the Elegra, held a relatively constant gas flow throughout the entire test (over 30 min). A similar result was also observed with the injector (1).

	The combination of high temperature and salt deposits causes a quartz torch outer tube to devitrify, resulting in frequent replacement of the entire torch. The D-Torch™ offers an affordable, robust demountable ICP torch design which greatly reduces consumable costs. With the D-Torch the outer tube can be replaced when worn and the optimum injector can be selected for each application. The D-Torch also incorporates a ceramic intermediate tube for greater robustness.

	Unique to the D-Torch is an optional ceramic outer tube, which does not suffer from devitrification. In Figure 2, a quartz outer tube is compared to a ceramic outer tube, both of which have been exposed to 6 h of running 10% NaCl. You can clearly see that there is no change to the integrity of the ceramic outer tube, whereas the quartz is severely devitrified.

	A detailed performance evaluation of the fully ceramic D-Torch was presented in a 2010 application note (2). In this report, the D-Torch exhibited exceptional stability in the presence of 3% NaCl and provided equivalent analytical performance to a standard quartz torch.

	Glass Expansion spray chambers are specifically designed to provide high performance and fast washout (3). The Twister™ cyclonic spray chamber is the ideal choice for high TDS applications due to its central transfer tube (baffle). The baffle acts as a secondary droplet filter to reduce droplet size and plasma loading without compromising detection limits yet improving precision (%RSD). The reduced sample load helps to increase torch life, slow salt build-up at the injector tip, and decrease the frequency of cleaning ICP-MS interface cones.

	The SeaSpray™ nebulizer comprises a unique self-washing tip designed to prevent crystal growth, providing a tolerance of up to 20% TDS. The SeaSpray's popularity is due to its ability to offer outstanding nebulization efficiency for trace level analyses combined with excellent precision and a high tolerance to TDS.

	To evaluate the performance of the SeaSpray DC nebulizer a 1 ppm multielement standard was prepared in a 3.5% NaCl matrix and aspirated at 1 mL/min for 9 h with no rinsing. The combination of the SeaSpray DC nebulizer, Twister spray chamber, ceramic D-Torch, and Elegra argon humidifier provided exceptional stability (Figure 3). A measurement was taken approximately every 3 min over a period of 9 h, while a precision of less than 1% was maintained throughout the experiment.

	An easy way to eliminate drift, interrupted runs, and frequent maintenance to your ICP sample introduction system is to add the Elegra argon humidifier. The D-Torch offers an affordable, robust ICP torch design while greatly reducing torch consumable costs. The optional ceramic outer tube for the D-Torch provides a significant improvement in torch life and stability in the presence of high TDS. Combining the SeaSpray nebulizer with the Twister spray chamber will provide optimum sensitivity and exceptional long-term precision even in the presence of high TDS.

	(1) Glass Expansion February 2016 Newsletter, "The Elegra Argon Humidifier: Uninterrupted and Maintenance-Free ICP Operation."

	(2) Thermo Fisher Scientific Technical Note #43053, "Radial Demountable Ceramic Torch for the Thermo Scientific iCAP 6000 Series ICP Spectrometer" (2010).

	(3) Glass Expansion October 2014 Newsletter, "ICP Spray Chamber Update."


	4 Barlows Landing Rd, Unit 2A, Pocasset, MA 02559
	tel. (508) 563-1800, fax (508) 563-1802
	Website: 

Considerations When Analyzing Real-World Samples by ICP Spectrometry

	Surface enhanced Raman spectroscopy (SERS) is an extension of Raman spectroscopy in which gold or silver nanoparticles amplify the Raman signals. The technique works via an electromagnetic effect where molecules come into proximity with gold or silver particles. When incident laser light strikes the nanoparticulate surface, localized surface plasmons can be excited, greatly enhancing Raman signals. The enhancement can be significant, making SERS well-suited to trace level detection of illicit drugs such as cocaine, heroin, methamphetamine, and tetrahydrocannabinol (THC).

	To test how well Ocean Optics SERS substrates can detect trace drugs, measurements were performed using gold and silver nanoparticles with an Ocean Optics modular Raman setup. As we discovered, detection of several illicit drugs using gold nanoparticles is a rapid, reliable technique that requires only a few milliwatts of laser power.

	Heroin hydrochloride, cocaine hydrochloride, Δ

-tetrahydro-cannabinol, and (±)-methamphetamine were prepared in 100 ppm solutions in methanol.

	For the Ocean Optics SERS substrates, which comprise analyte-sensitive nanoparticle chemistries applied to a treated glass surface, we used 15 µL of 100 ppm solutions for the gold and silver nanoparticle substrates. We tested at 100 ppm to determine feasibility, although results suggest lower concentrations are possible.

	For measurements with the gold and silver SERS substrates, we used a modular Raman system comprising the QE Pro high-sensitivity spectrometer, a 785 nm laser for Raman excitation, and sampling optics. The 785 nm excitation produces excellent Raman spectra for most chemicals, with limited interference from fluorescence. These systems also offer very good spectral resolution, making them a preferred wavelength choice for Raman spectroscopy of chemicals and organic materials. Three measurements for each sample were taken from multiple spots on the same substrate, which then were averaged.

	As demonstrated in the spectra, SERS methods utilizing Ocean Optics substrates can detect ppm levels of illicit drugs, which Raman techniques alone would not be able to accomplish.

	Also, the gold nanoparticles appear to work best for detecting these drugs. Certain peaks have higher Raman cross-sections, and each peak is enhanced by the gold nanoparticles differently depending on how the molecule is oriented with respect to the gold surface.


	8060 Bryan Dairy Rd., Largo, FL 33777
	tel. (727) 733-2447
	Website: 

SERS Methods Detect Trace Levels of Cocaine, Heroin, Methamphetamine, and THC

	Diamond is an important material choice for sampling optical elements in Fourier transform-infrared (FT-IR) spectroscopy. The multi-phonon absorption in the mid-infrared, although temperature dependent, is weak for thin optical elements and can be referenced to an empty cell background. However, the cost of diamond optics requires the sampling area to be very small. As a result, the signal-to-noise ratio (SNR) advantages of the FT-IR systems are frequently stretched to the limit and more energy throughput is usually quite helpful.

	Frequently, the sampling technique of choice is attenuated total reflection (ATR) for its benefits of ease of analysis. ATR elements can be manufactured from most optical materials of relatively high refractive index. Ge, Si, ZnSe, ZnS, KRS-5, and diamond are often used for this application. For most of these materials, a broad band anti-reflection (BBAR) coating can be applied to both surfaces of a window or the entrance and exit facets of an ATR element and can significantly increase the energy throughput of the sampling accessory or system.

	Since diamond and ZnSe have similar refractive indexes a similar BBAR can be used on diamond once the binding layers of the coating are appropriately modified.

	Spectral Systems has developed its Diamond XP-BBAR(tm) coating with spectral performance as shown in Figure 1.

	It is seen in Figure 1 that the energy and thus the SNR is about 30% better through most of the analysis region of a typical FT-IR instrument.

	Since there are no suitable coating materials for the very long wavelength portion of the spectrum the coated element is not as good, but still usable, below 600 cm

. If a liquid nitrogen cooled MCT detector is used, this portion of the spectrum is usually not recorded by the spectrometer.


	35 Corporate Park Drive, Hopewell Junction, NY 12533
	tel. (845) 896-2200
	Website: 

Diamond Anti-Reflection Coating

	Confocal Raman imaging is an ideal method for studying 2D materials because it reveals their molecular characteristics through a nondestructive and fast procedure. It is used to describe the orientation of layers and to investigate defects, strain, and functionalization of these materials as their Raman properties are determined by molecular bonds, relative orientation, and number of layers. Their morphological details can be visualized with scanning electron microscopy (SEM). By correlating the information of both approaches with RISE™ (Raman Imaging and Scanning Electron) microscopy, the sample can be more thoroughly analyzed at high resolution.

	Here the stacking, folding, and twisting of bilayer graphene was investigated. These factors strongly influence its phononic and electronic properties.

	Correlative Raman-SEM (RISE™) imaging was performed with a combined WITec/ZEISS microscope for the analysis of bilayer graphene that was obtained by chemical vapor deposition and placed on a Si/SiO

	The SEM image shows that the sample consists of monolayer and multi-layered graphene areas (Figure 1). Whether and how the multi-layers are twisted was established by analyzing the position of the G and 2D peaks of graphene, the peaks' intensities, and their full widths at half maximum (FWHM) (Figure 2).

	Additional 2D' and R bands allowed for the determination of the twist angles. Figure 3 shows the color-coded RISE® image derived from the spectra.

Lise-Meitner-Stra e 6, 89081 Ulm, Germany
	

tel. +49 (0) 731 149 700, fax +49 (0) 731 149 70 200
	

RISE™ Raman Imaging and Scanning Electron Microscopy Investigation of Twisted Bilayer Graphene

Recent advances in single-bounce ATR accessories provide an easy way to acquire a high-quality infrared spectrum from very small amounts of sample in less than 1 min. This has resulted in increased use of this technique in the pharmaceutical industry, making accessory qualification a concern. We suggest an adaptation of the Pharmeuropa guidelines as an excellent starting point.

	FT-IR spectrometry is a key technique used throughout the pharmaceutical industry and most of the pharmacopoeias in the world contain a section on how to qualify an infrared spectrometer for use. However, there is no clear guidance from regulatory agencies on how to qualify an FT-IR spectrometer system with an ATR accessory. In fact, there is some question whether this falls under “Analytical Instrument Qualification” or “System Suitability.” While the US Pharmacopeia defines appropriate measurements for wavelength accuracy for transmission spectroscopy, it clearly states that ATR spectra are different. The European Pharmacopoeia recommends the same polystyrene peak positions but adds a resolution test. A recent Pharmeuropa report (29.3) describes some proposed changes to the European Pharmacopoeia and actually proposes ATR peak positions for polystyrene as well as pointing out that a different resolution test is also necessary. One challenge with ATR spectra is the relative differences in peak intensity. While some of the peaks in a transmittance spectrum of 35 µm thick polystyrene film are highly absorbing and not appropriate for verifying wavelength accuracy, they are much weaker in an equivalent ATR spectrum, particularly in the C-H stretching region. In this application note, we will describe an evaluation method based on the Pharmeuropa proposal that we believe will verify the performance on an ATR accessory in FT-IR instruments.

	Spectra were acquired on various FT-IR spectrometers with different single-bounce ATR accessories at 2 cm

 instrument resolution using a NIST traceable polystyrene standard that had been qualified in a calibration laboratory. A workflow was created using the four peak locations proposed for ATR qualification. We chose to use the full width at half maximum on polystyrene peaks at 1154 cm

 to determine spectral resolution. The instrument resolution was confirmed using water vapor peaks with the accessory mounted in the system. Figure 1 shows the spectra from a polystyrene film in transmission (blue) and the ATR spectrum (red) acquired with the diamond iD5 Accessory on a Thermo Scientific™ Nicolet™ iS™ 5 FT-IR spectrometer.

	The results (targets and actual) of the proposed pharmacopoeia measurements are tabulated (inset) for the diamond ATR spectrum. While we obtained good results using the peaks described in the Pharmeuropa proposal, the peak near 3060 cm

 is quite weak, a situation which would be even worse for Ge-ATR (shallower depth of penetration).

	We conclude that the guidelines recommended by Pharmeuropa provide an excellent starting point for this long overdue development. However, we strongly recommend replacement of the 3059.7 cm

 peak with either of the stronger signals near 3025 cm

, which will provide a more robust measurement, regardless of the ATR crystal type.

	Questions about this application note may be directed to: 


	5225 Verona Rd., Madison, WI 53711
	tel. (800) 532-4752
	Website: 

Qualifying ATR Accessories for Pharmaceutical Applications

We monitor potential-induced changes at a Au film electrode using the ATR-SEIRAS sampling technique. Benefits are discussed.

	Attenuated total reflectance surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS) provides in situ surface-sensitive IR spectra of molecules at the electrode-solution interface. A Au thin film electrode is deposited on the reflecting plane of the ATR optic. Excitation of surface plasmon modes in the film provides up to 10-fold electric field enhancement at the interface, enhancing IR cross-sections of adsorbed species up to 100-fold. Controlling the applied potential on the film allows vibrational characterization of potential-induced changes. Such studies reveal fundamental details of electrochemical processes and inform the development of next generation energy and sensing solutions.

	The VeeMAX III with ATR and electrochemical cell option (PIKE Technologies, Madison WI) was used as the FT-IR sampling accessory. The electrochemical cell is designed to be easily modified to meet varied requirements in the field. We modified the cap of the VeeMAX III electrochemical cell to accept a Au coil counter electrode, a glass bubbler for Ar purging, an exhaust bubbler, and a Au bead electrode. The Au bead was pressed against the Au film to ensure potential control. In the cell body, a hole was bored to fit a reference electrode arm.

	We sputtered a ca. 30 nm Au film electrode on a 60° angled Si ATR crystal. We then cycled the potential of the film in 50 mM KClO

 supporting electrolyte between -100 and +1100 mV versus Ag/AgCl. This cleans the film and textures the surface to improve SEIRAS intensity. The VeeMAX III was set to 60° to match the crystal face angle. FT-IR spectra were collected at 4 cm

 resolution, 128 scans, and 40 kHz scanning velocity using an MCT detector.

	We used the potential-dependent adsorption of 4-methoxypyridine (MOP), a nanoparticle shape-directing ligand, as a test system. We collected a reference spectrum of supporting electrolyte, then a sample spectrum with 0.1 mM MOP, both at the open circuit potential. The result is shown in Figure 2a. We observe two strong ring vibrations (1) and the methoxy C-O-C asymmetric stretch (2). A third ring vibration (3) has its transition dipole moment (TDM) mostly parallel to the electrode-solution interface when adsorbed, so (3) is very weak. Only vibrations with their TDM normal to the interface are IR active. This confirms the surface sensitivity of the measurement.

	Figure 2b reveals changes in interfacial composition upon potential changes. The negative absorbance band (4) represents loss of interfacial water. This band is absent in (a) because, at open circuit conditions, water adsorbs in a flat state on Au, rendering the H

O bending mode inactive in SEIRAS. We see also the characteristic MOP bands, which shows that we have potential control over the adsorption of MOP.

	With PIKE's variable angle VeeMAX III ATR accessory and electrochemical cell, we have demonstrated a surface-sensitive technique which can resolve minute changes in interfacial composition.


	6125 Cottonwood Drive, Madison, WI 53719
	tel. (608) 274-2721
	Website: 

Application Notebook-09-01-2017

Digestion of Difficult APIs in Accordance with USP

Digestion of Difficult APIs in Accordance with USP

Blink and You Miss It—Catching Fleeting Catalytic Intermediates by High-Speed 785 nm NIR Raman Spectroscopy

Total Mercury in Coal Using Direct Mercury Analysis

Considerations When Analyzing Real-World Samples by ICP Spectrometry

SERS Methods Detect Trace Levels of Cocaine, Heroin, Methamphetamine, and THC

Diamond Anti-Reflection Coating

RISE™ Raman Imaging and Scanning Electron Microscopy Investigation of Twisted Bilayer Graphene

Qualifying ATR Accessories for Pharmaceutical Applications

Surface-Sensitive Spectroelectrochemistry with ATR-SEIRAS