Experimental - Spectroscopy

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34 <strong>Spectroscopy</strong> 26(6) June 2011 www.spectroscopyonline.com with an automated well-plate reader was used with the OMNIC Array Automation software to analyze the samples deposited on the aluminum foil surface. The infrared microscope transmission data were obtained with a Nicolet 6700 FT-IR system and a Continuum infrared microscope configured with an automated X,Y stage. Figure 5: Diagram of the reflectance optics module used in the array autosampler. Figure 6: Screen showing the Array Automation results using the amount of lipid as the metric to color the wells. were deposited from aqueous solution onto barium fluoride or silicon windows, a polyethylene microporous membrane, or polytetrafluoroethylene (PTFE) membrane. For the reflectance study, the samples were deposited onto an aluminum foil surface marked with 96 positions consistent with a standard well-plate format. A Nicolet 6700 spectrometer (Thermo Fisher Scientific) Results and Discussion ATR spectroscopy is an excellent technique to rapidly characterize discrete samples. The dried material is pressed against the 2-mm diameter ATR crystal and a spectrum is acquired. The depth of penetration into the sample in the ATR experiment is wavelength dependent and results in relatively lower intensity of the peaks in the higher wavenumber region. The peak intensity is also related to the total amount of sample in contact with the crystal. Thus, sample hardness and particle size can change the intensities of the peaks slightly, and spectra should be corrected before calculating the amount of each component. Although the samples were prepared for automated reflectance analysis, ATR spectra were also acquired from the algae deposited on the aluminum foil surface. These spectra were similar to the ones obtained by placing the dried algae directly on the ATR crystal. Figure 2 shows the ATR spectra from different algae samples that were acquired. One of the objectives of this research was to develop a better understanding of ways that the automated sampling techniques created for pharmaceutical high-throughput screening systems might be applied to the analysis of algae. In particular, can the automated weighing, dilution, and deposition systems designed around the 96-well SBS format improve productivity in FT-IR analysis of biological materials? A number of years ago, 3M (St. Paul, Minnesota) introduced a disposable IR sampling card based on a polymer microporous membrane. A publication by Mosoba and colleagues (8) reports using a polyethylene microporous film with this sampling concept
www.spectroscopyonline.com June 2011 <strong>Spectroscopy</strong> 26(6) 35 to obtain FT-IR spectra from bacteria samples. They created a multiple-sampling method by stretching the film across the holes in an autosampler wheel. A plate with 96 holes in a well-plate format or a microscope slide with 16 holes also could be developed for automated sampling. A major point of these analyses is to measure the amount of lipid in the algae samples using infrared spectroscopy. The analytical method described in the Pistorius publication uses the spectral features in the C-H stretching region to determine the lipid content. However, the spectral features from the polyethylene film are very similar to those from lipids, creating a large cross interference problem. The PTFE polymer shows no interference in the C-H stretch region and has no features in the amide or carbonyl regions, although the strong infrared peak for PTFE near 1200 cm -1 results in some spectral distortion near the region employed for the carbohydrate analysis. Figure 3 shows spectra from the two polymer films and from algae samples after subtraction of the spectra from the reference films. Although some spectral artifacts are observed from the polymer, the use of microporous polymer film as a sample substrate would result in a very low cost disposable sampling system. In a previous project, biological samples were deposited from a saline solution onto small 5-mm squares of barium fluoride (BaF 2 ) and dried before the acquisition of spectra in transmittance (2). Barium fluoride is an excellent IR window and is not soluble in water. However, it is quite expensive and probably would not be considered a disposable sample holder by most laboratories. An alternative approach that looks very promising is to deposit the samples on small squares of polished silicon that have been cut from a semiconductor wafer. A number of surplus double side polished 8-in. silicon wafers were purchased and were diced into 0.25-in. squares. Figure 4 shows the spectrum obtained by depositing an aqueous suspension onto a barium fluoride window and the spectrum from a bacteria sample deposited onto a silicon square after subtraction of a silicon reference spectrum. This figure shows the similarities and differences between the algae and bacteria samples. A plastic holder was designed to hold the silicon and BaF 2 squares. For compatibility with microscope slide handling stages, a 1 × 3 in. silicon sample holder was also designed, allowing use in a high throughput sampling system that matches two columns in the standard 96-well format. Another alternative sampling technique is to use a single piece of silicon with 96 shallow wells created by a gasket. The silicon piece is mounted in a frame consistent with the SBS 96-well form factor. In a related study, samples of bacteria were deposited and dried on a silicon 96-spot plate (Pike Technologies, Madison, Wisconsin). Spectra were acquired from the samples deposited on the plate using a Nicolet 6700 FT-IR spectrometer and a Continuum infrared microscope configured with a high-precision X,Y stage and a modified version of the OMNIC Array Automation software. Infrared microscopy is very valuable for obtaining high quality PIKE PIKE FlexIR GladiATR The GladiATR advances FTIR-ATR technology to the next level. Its monolithic diamond sampling interface and precision optics offer the most energy efficient design, 4000 – 50 cm -1 working spectral range, and the highest quality data. Optional temperature control enables studies of materials at temperatures up to 300°C. The built-in camera allows for simultaneous viewing and data collection assuring that what you see is what you sample. Try this most versatile accessory and simplify your analyses without compromising your results. FTIR sampling made easier PIKE Technologies www.piketech.com tel: 608-274-2721
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