PIKE Technologies, Inc.

Fourier-transform infrared–attenuated total reflection (FTIR–ATR) sampling has become one of the most popular techniques for mid-infrared measurements. With its short pathlength, typically there is no sample preparation required. It is often thought of as non-destructive. However, when measuring non-particulate, solid samples by ATR, most often a high-pressure clamp is required to create intimate contact between the sample and ATR crystal surface, which is necessary for the collection of a high-quality spectrum. With applied pressures by the clamp of up to 10,000 psi, the clamp tip can leave a dimple in many softer, pliable samples.

In this application, the feasibility of measuring a polypropylene face mask by diffuse reflection as a non-destructive alternative to ATR was investigated.

Mid-infrared spectra of a 3-ply polypropylene face mask were collected using two sampling techniques, ATR and diffuse reflection. The single-reflection MIRacle ATR was equipped with a germanium crystal plate. The UpIR accessory was used for the diffuse measurement, and a diffuse gold disk served as the background reference. Both accessories are manufactured by PIKE Technologies. Then 30 s of data were co-added for each measurement at a 4 cm

The optical layout of the UpIR is shown in Figure 1. Unlike other diffuse reflection accessories, the UpIR has a large collection mirror that is positioned underneath—rather than above—the sampling platform, thereby allowing the sample to be placed face-down on top of the accessory. The UpIR is 23 cm tall (excluding baseplate), and extends above most FT-IR spectrometers. The optical design and height make the UpIR an ideal tool for measuring larger samples.

The UpIR diffuse reflection accessory and its optical design with simulated beam path.

Diffuse reflectance spectra may appear different from their transmission equivalent by exhibiting stronger-than-expected absorption from weak IR bands. A Kubelka-Munk (K-M) conversion can be applied to a diffuse reflection spectrum to compensate for these differences. This mathematical conversion is available in most FTIR software packages. Figure 2a and 2b show the spectrum before and after the K-M conversion, respectively.

Mid-infrared spectra of a three-ply face mask collected via (a) diffuse reflection, (b) diffuse reflection after the Kubelka-Munk conversion (blue) and ATR (red).

Comparing the face mask spectrum collected by diffuse reflection after the K-M conversion to the spectrum collected by ATR spectrum in Figure 2b, the fingerprint region of both techniques shows the characteristic bands of polypropylene, and are similar.

Mid-infrared diffuse reflection sampling may be a viable, non-destructive option for measuring pliable solids.


6125 Cottonwood Drive, Madison, WI 53719
Tel. (608) 274-2721, Fax (608) 274-0103
Website: www.piketech.com

Articles

For analysis of non-particulate solids, the diffuse reflection sampling technique may offer an easy, non-destructive method for mid-infrared measurements. Spectral results of a polypropylene face mask collected via diffuse reflection and attenuated total reflection (ATR) were compared.


Non-Destructive FT-IR Measurements via Diffuse Reflection Sampling

Mixtures of methanol in ethanol were investigated using an automated ATR accessory. The spectral bands of methanol and ethanol were evaluated showing the expected absorbance changes. 

	Quantitation of contaminants and toxins within human-consumable products is a critically important aspect of ensuring consumer safety. Methanol contamination of consumable alcoholic beverages is particularly concerning due to the acute toxicity of methanol, with 4 mL of ingested methanol leading to irreversible vision loss and 30 mL of methanol potentially resulting in death (1). Recently, headlines discussing deaths and injuries linked to methanol contamination of commercially available liquors at popular resorts have emphasized the importance of methanol screening and quantitation methodologies.

	Here, we use PIKE Technologies' automated ATR accessory, the AutoATR, to observe the spectral changes over various concentrations of methanol in ethanol (Figure 1). The AutoATR uses 24 unique Si ATR elements, which have a thickness of 500 microns. The thin profile of the ATR crystal minimizes absorption of the IR source by the Si phonon bands which, in combination with the accessory's base optics, results in high energy throughput and reproducibility.


	Figure 1: PIKE AutoATR automated ATR accessory.

	Sampling automation is made possible by the AutoPRO7 software package. AutoPRO7 streamlines the collection of all background and sample spectra, reducing the required user involvement to the distribution of samples after the background spectra acquisition has completed.

	A series of five concentrations of methanol in ethanol were investigated, including 0, 10, 20, 30, and 40% 

 methanol in ethanol. Each mixture was created using an Eppendorf micropipette and high-purity methanol and ethanol. After each mixture was created, a clean micropipette tip was used to distribute 120 μL of each solution into the AutoATR well plates after the background spectra acquisition was completed.

	The ATR spectrum of each methanol/ethanol sample is shown in Figure 2. The dominant spectral bands of ethanol correspond to the 1087 and 1046 cm

, while methanol exhibits a characteristic spectral band at 1033 cm

. The complementary change in concentration of the two species is highlighted by the isosbestic point at 1036 cm


	Figure 2: ATR spectra of each % v/v methanol in ethanol, collected with the AutoATR.

	Quantitation of contaminants and toxins such as methanol is critically important to ensuring the well-being of consumers. In order to streamline these measurements, automated technologies such as the AutoATR provide a uniquely accurate and high-throughput approach to ATR-based chemical identification. The AutoATR has proven capable of spectrally distinguishing methanol from ethanol and is well-suited for quantifying individual species in mixed solutions.

Clinical Chemistry, Immunology and Laboratory Quality Control

 (Elsevier, San Diego, California, 2014), pp. 317–335.


	6125 Cottonwood Drive, Madison, WI 53719
	tel. (608) 274-2721, fax (608) 274-0103
	Website: 

An Observation of Methanol in Ethanol Using Automated ATR Spectroscopy

Protein secondary structure during thermal unfolding and aggregation is readily acquired using IR spectroscopy and a temperature-controlled mid-IR transmission accessory. Myoglobin was used as a model system to illustrate the method.

	FT-IR spectroscopy is a powerful tool for measuring protein secondary structure. Using a temperature-controlled transmission accessory, the evolution of protein secondary structure through thermal transitions is readily accessible. In fact, the experiment and analysis are relatively quick; therefore, a set of proteins can be rapidly screened for stability and temperature-dependent conformational changes. This information is particularly important when evaluating multiple protein mutants.

	To demonstrate the simplicity of secondary structure evaluation using FT-IR spectroscopy, the well-characterized protein, myoglobin (1), was unfolded in D

O (3.5 µM, 50 µm pathlength) from 25 °C to 90 °C in 5 °C increments using the Falcon Mid-IR Transmission Accessory from PIKE Technologies, Inc. The accessory’s Peltier elements allow for quick ramp rates and temperatures ranging from 5 °C to 130 °C. Temperature profile and spectral data collection was executed by PIKE TempPRO

 software. The amide I band was deconvolved by Fourier deconvolution (bandwidth 50 cm

, enhancement 2) and frequencies were obtained from second derivative spectra.

	Four peaks dominate the 25 °C spectrum. The largest peak at 1649 cm

 is assigned to α-helix and random coil structure. From the structure obtained by X-ray diffraction (Figure 1 inset), myoglobin is comprised of several α-helix and random coil regions (2). Two other peaks are observed at 1630 and 1679 cm

 and correspond to extended chain and turn regions, respectively (1). The last peak at 1612 cm

 is not amide I vibration but instead tyrosine side chain absorption (3). Thus, from quick spectral manipulation and inspection, the secondary structure at 25 °C is readily determined.

					Figure 1: FT-IR spectra of myoglobin between 25 °C and 90 °C. Myoglobin is rendered with UCSF Chimera (4).

	As the temperature increases, significant changes in the myoglobin secondary structure are observed. Between 25 °C and 70 °C, the 1649 cm

 peak height decreases and linewidth increases. These changes in the amide I band reflect increased protein structural disorder. A temperature increase from 70 °C to 75 °C causes a drop in the 1649 cm

 band intensity. Simultaneously, two peaks grow in at 1683 and 1637 cm

. These changes indicate conversion of random coil/α-helix to intermolecular β-sheet. Further conversion to β-sheet is observed as the temperature rises to 90 °C. The final β-sheet structure is an insoluble aggregate. This aggregation process is not reversed when the temperature is driven back to 25 °C.

	Using the PIKE Falcon accessory makes monitoring the thermal conversion of myoglobin from α-helix to random coil to β-sheet simple.

		F. Meersman, L. Smeller, and K. Heremans, 

	6125 Cottonwood Drive, Madison, WI 53719

Simple Method for Monitoring Protein Secondary Structure During Thermal Unfolding and Aggregation

Yeast suspensions were dried and analyzed via FT-IR using an automated transmission sampling accessory. Up to six-fold sampling capacity increase may be realized through automation. 

FT-IR spectroscopy has been shown to be an effective tool to classify bacteria, yeast, and fungi at the strain, species, and genus level. Compared to traditional microbiological techniques, IR sampling is less time-consuming. Microbiological applications for FT-IR analysis include a rapid method of identification and screening, quality control for food and pharmaceutical fields, and assessment of growth environment on cell and cell byproduct physiology.  

An IR plate-reader for biological samples is desirable for laboratories confronted with high-throughput demands, and the need to save resources through automation. This application note illustrates the use of an in-compartment XY plate reader to analyze yeast and discusses the resource savings involved through accessory automation.  

Yeast suspensions (30 µL) were pipetted onto a sample well of a 96-well reusable Si plate, and dried at 45 °C for 1 h for subsequent infrared transmission measurements. The 96-well Si plate was loaded into the PIKE Technologies XY Autosampler Accessory, an FT-IR sample compartment automated transmission/diffuse reflection accessory. The spectral range spanned from 4000 to 600 cm

 . Data collection time was 1 min. For transmission measurements the accessory's integrated DTGS detector is used to maximize the signal-to-noise ratio. Automated plate movement to each sample position is controlled by PIKE AutoPRO software, which also triggers data collection via the FT-IR spectrometer. 

Figure 1 shows a typical transmission spectrum of the dried yeast suspension. Some main spectral features include the C-H stretching vibrations from fatty acids between 3100–2800 cm

 , the amide I and amide II bands between 1800–1500 cm

 , and C-O-C and C-O-P stretching vibrations of polysaccharides between 1200–900 cm

 . A comprehensive list of absorption band assignments may be found in Naumann (1). Application of multivariate analysis allows differentiation between unique biological materials (2,3). 

Figure 1: Infrared transmission spectrum of dried yeast suspension.			

Through automation the number of IR samples analyzed per day may be greatly enhanced and potential sample and data entry errors decreased. Due to variability in biological materials, replicate sample measurements are often required. If four replicate measurements are made for one sample, 84 samples may be analyzed in a typical eight-hour work day. This equates to over 330 individual spectra collected without end user input, except to load the 96-well plates once the process is started by the AutoPRO software. 

In addition to biological applications, an automated XY plate reader may be used for polymer solutions and solvent residuals analysis. 

Infrared transmission spectra of microbiological suspensions may be collected by drying the suspension on an IR transparent plate. The use of an in-compartment XY plate reader accessory provides means for automated measurements via transmission or reflectance to increase laboratory capacity. 