Susan Berets and Jeffrey Christenson

	Heating foods and beverages can lead to a variety of chemical changes. These changes can effect storage lifetime and nutritional value. Hence there is a continuing need for highly sensitive methods to detect these changes.

	This application note explores the potential of using a high-throughput single-reflection diamond ATR to detect structural changes in egg yolk upon heating.

	Infrared spectra were collected on an FT-IR spectrometer equipped with the Harrick DiaMaxATR™ single-reflection diamond ATR accessory with its Temperature Controlled Liquid Cell (Figure 1). The system was purged to remove water vapor and CO

 resolution and signal averaged over 32 scans. A background was collected from the clean ATR crystal at ambient temperature.


	Figure 1: The DiaMaxATR™ with Temperature Controlled Liquid Cell.

	The egg yolk was separated from the egg white, and ~0.3 mL of the yolk was injected into the heatable cell. A sample spectrum was collected at ambient temperature. Then the cell was heated to 100 °C using the compatible Temperature Controller. Another spectrum was collected when the cell temperature stabilized and at 2 min intervals thereafter. Data collection was controlled by Harrick ATC-Link™ software.

	The resulting spectra are shown in Figure 2. With increasing temperature, the OH stretching band around 3500 cm

 decreases in intensity as the water evaporates from the yolk and begins to appear as two underlying broad bands, centered around 3390 cm

. The former indicates the presence of amines and the latter carboxylic acids, both of which become more concentrated in the yolk as the water is driven off. In addition, changes are evident in the 1500 to 1700 cm

 region, as expected from changes in the proteins induced by heating. The amide I bands at 1650 cm

 are likely from the α and β helixes, respectively. The broad underlying band in that region may indicate unordered amides. The peaks at 1560 cm

	Single reflection ATR spectroscopy is valuable for examining thermally induced changes in materials. Changes in the water concentration and amide bands of egg yolk were observed using the Harrick DiaMaxATR accessory. Further analysis is possible to obtain more rigorous identification of the heat-induced structural changes.


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Articles

Single reflection ATR spectroscopy is valuable for examining thermally induced changes in materials. Changes in the water concentration and amide bands of egg yolk were observed using the Harrick DiaMaxATR accessory. Further analysis is possible to obtain more rigorous identification of the heat-induced structural changes.

An FT-IR Spectroscopic Study of Cooking an Egg Yolk on a Single-Reflection Diamond ATR

	Many polymers and thin films on semiconductors are designed with specific molecular orientation to take advantage of mechanical, chemical, or physical properties. Some plastics, for example, exhibit different mechanical strength depending on the orientation. Molecular orientation can be extracted from polarized ATR measurements.

	This application note demonstrates determining the correct orientation of the polarizer for these measurements and illustrates the importance of polarized measurements in specific examples.

	For all measurements, a Harrick Wire Grid Polarizer (KRS-5 substrate) was used. The polarizer was oriented in the same position for both background and sample measurements. All spectra were collected using an FT-IR spectrometer at an 8 cm

	Harrick's Horizon, Seagull, and VariGATR accessories were used here. Such accessories redirect the infrared to and from the sample via mirrors, changing the direction of the electric field which makes it tricky to ascertain the polarizer position for s- or p-polarization. For ATR, the polarizer orientation is straightforward to determine. The effective thickness for p-polarization is nearly twice that for s-polarization at a 45° incident angle (1), which is directly reflected in band intensity.

	Three samples were investigated. An unoriented liquid, water, was used to demonstrate the method used to determine polarizer orientation with the Horizontal multiple reflection ATR. Then two oriented samples were examined: Tunicin was measured with the Seagull at a 45° incident angle and an oriented thin film on silicon was examined with the VariGATR at a 65° grazing incidence.

	Figure 1 shows the ATR spectra of water, measured with the two perpendicular orientations of the polarizer. Clearly, the 0 setting gives bands that are roughly twice as strong as those for the 90 setting, indicating that the former gives p-polarized radiation.


	Figure 1: ATR spectra of water recorded with the polarizer set to 90 (red) and 0 (blue).

	Figures 2 and 3 show spectroscopic differences resulting from polarized measurements. In Figure 2, the O-H and C-H stretching regions change with polarization, suggesting different orientations of functional groups on the surface. Figure 3 shows differences at 1500 cm

. These bands are not visible with s-polarization but are with p-polarization, which probes bonds perpendicular to the interface. This shows the preferential orientation of functional groups on the surface.


	Figure 2: ATR spectrum of Tunicin measured with s-polarization (red) and p-polarization (blue).

	Combining a polarizer with ATR measurements provides valuable information about the sample, and determining the polarization delivered is straightforward. Polarization can enhance band intensities and provide information on oriented functional groups.


	Figure 3: ATR spectrum of a 16 Å thick photoresist ultra-thin film on a silicon wafer, measured with unpolarized (black), s-polarized (red), and p-polarized (blue) incident radiation.


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For all measurements, a Harrick Wire Grid Polarizer (KRS-5 substrate) was used. The polarizer was oriented in the same position for both background and sample measurements. All spectra were collected using an FT-IR spectrometer at an 8 cm-1 resolution averaged over 32 scans.

Polarization in FTIR ATR

Grazing angle Ge ATR spectroscopy is extremely sensitive to monolayers and thin films on high refractive index substrates such as Si. As with any ATR method, the sensitivity increases as the critical angle is approached, as demonstrated herein.

	Monolayers and other thin coatings on Si and other high refractive index substrates are of key importance in materials science, bioscience, synthetic organic, and semiconductor research. These films are often close to the FTIR detection limit so methods that improve sensitivity are vital. Traditionally, these samples were examined by transmission or grazing angle specular reflectance spectroscopy. Grazing angle Ge-ATR has recently gained wider use because of its higher sensitivity. Grazing angle Ge-ATR measurements have typically been recorded at 65°, an angle sufficiently above the critical angle for the Si-Ge interface, 58.7°.

	This note explores the increased sensitivity gained by optimizing the incident angle and also discusses the potential spectral anomalies if the nominal critical angle of a given sample is exceeded.

	The grazing angle Ge-ATR measurements were carried out in a commercial FTIR spectrometer, using Harrick's VariGATR(tm) accessory at selected incident angles from 60° to 65° (see Figure 1). Spectra were collected at an 8 cm

 resolution and signal averaged over 32 scans. The samples investigated were thin films on Si substrates. Each sample was pressed against the ATR crystal using the same force using the VariGATR's built-in pressure applicator and force sensor.


	Figure 1: The VariGATR™ shown with its Force Sensor.

	Figures 2a and 2b show spectra recorded from two different samples at several incident angles. Both spectra have higher band intensities at the lowest angle measured. However, the spectrum of sample B shows significant dispersion effects at 60° which may complicate interpretation. These include a sloping baseline and a visible step between the high and low wavenumber edges of the bands. These effects are not apparent in the spectrum of sample A.


	Figure 2: (a): ATR spectrum of sample A recorded at an incident angle of 65° (red), 63° (blue), and 60° (black) with an applied force of 722 N. (b): ATR spectrum of sample B recorded at an incident angle of 65° (blue) and 60° (black) with an applied force of 700 N.

	In conclusion, it is clear that selecting the incident angle to approach the critical angle does increase sensitivity. However, for ease of interpretation, the incident angle should be optimized for greatest sensitivity without significant dispersion effects.


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Monolayers and other thin coatings on Si and other high refractive index substrates are of key importance in materials science, bioscience, synthetic organic, and semiconductor research.

Optimized Grazing Angle Ge-ATR of Monolayers and Thin Films on Si

	Fermentation processes have been used since the Neolithic era and are widely used today in diverse fields such as foods, beverages, and pharmaceuticals. Hence, there is a continuing need for more effective and faster methods of analyzing the chemistry behind the fermentation process.

	This applications note explores the potential of using multiple-reflection diamond ATR to examine the yeast-mediated conversion of sucrose into ethanol. Both sucrose and ethanol exhibit strong absorption bands in the mid-infrared spectral region, and thus ATR can be used to track the progress of the fermentation process.

	Two types of Lalvin yeast were examined: EC1118 Dry Wine Champagne Yeast and ICV-D47 Yeast. For each, 1.5 g of sugar was dissolved in 50 mL tap water and then 1.5 g of yeast was added. The mixtures were stirred every 15 min and were covered in the interim to minimize evaporation. All samples were kept at ambient temperature.

	Infrared spectra were collected on an FT-IR spectrometer equipped with the Harrick ConcentratIR2™ multiple-reflection diamond ATR accessory (Figure 1). The ConcentratIR2 is able to measure analytes having very low concentration in aqueous or other liquid solutions, and requires only a very small sample volume of ~10 μL. The system was purged to remove water vapor and CO

 resolution and signal averaged over 128 scans. A background was collected with the accessory in the sample compartment and no sample on the crystal. Then a drop of sample was placed on the ATR crystal and the spectrum was collected. Spectra were collected over the first 3 h with an additional spectrum recorded ~24 h after fermentation began. The spectrum of water, collected under the same conditions, was subtracted from each spectrum.


	Figure 1: ConcentratIR2™ Multiple Reflection ATR.

	The resulting spectra are shown in Figure 2. The set of overlapping bands from approximately 1200–950 cm

 correspond to sucrose. Ethanol has two bands in the same range, which are initially obscured by the sucrose bands but emerge with decreasing sugar concentration. Looking at the changes over time, it is clear that the EC1118 yeast ferments more quickly than the ICV-D47, as expected from its rating (1). This demonstrates that it is possible to observe the fermentation process with multiple reflection diamond ATR. A more detailed analysis could be performed using partial-least squares (PLS) multivariate regression method to predict the concentrations of sucrose and ethanol. Flow-through and heated cells are available with the ConcentratIR2 to facilitate kinetics studies.


	Figure 2: Infrared spectra of ICV-D47 (top) and EC1118 (bottom) yeast fermentation. Black: 1 h, green: 2 h, red: 3 h, blue: 24 h.

	Multiple reflection diamond ATR spectroscopy is a valuable method for analyzing fermentation and other aqueous reactions. Yeast fermentation in an aqueous sucrose solution was observed using the Harrick ConcentratIR2™ ATR accessory. It was immediately possible to see relative rates between the different yeasts. Further analysis is possible to obtain more rigorous quantitative data or kinetics data to derive reaction rates.

	(1) Scott's Laboratories 2013 Fermentation Handbook, 8-11.

Fermentation processes have been used since the Neolithic era and are widely used today in diverse fields such as foods, beverages, and pharmaceuticals.

Susan Berets and Jeffrey Christenson

Articles

An FT-IR Spectroscopic Study of Cooking an Egg Yolk on a Single-Reflection Diamond ATR

Polarization in FTIR ATR

Optimized Grazing Angle Ge-ATR of Monolayers and Thin Films on Si

Analysis of Yeast Fermentation Using Multiple Reflection Diamond FT–IR ATR