The Ocean Optics Curie Spectrofluorometer is designed to measure fluorophores with excitation and emission properties across a wide 200-850 nm UV-Vis wavelength range. Our objective is to demonstrate the capabilities and sensitivity of the system in measuring DNA and protein concentration in solution using three Molecular Probes Quant-iT Assay Kits. Detection limits are reported for each kit.

The Ocean Optics Curie is a high-sensitivity cuvette emission spectrofluorometer that can detect picomolar-range concentration of fluorophores in solution from 200–850 nm. 

The Curie features a CCD-array detector and is distinguished by internal linear variable filtering technology. The patented filtering helps to discriminate between powerful pulsed xenon wavelengths from the onboard excitation source and the weak spectral emissions from samples. The Curie is the only spectrofluorometer with linear variable filtering technology that can be used to spectrally shape excitation energy and eliminate the need for scanning monochromators. Each filter's transmission or blocking band can be moved throughout the 230–500 nm or 300–750 nm wavelength range.  

Three Molecular Probes Quant-iT Assay Kits were used to review the Curie's performance. The Quant-iT PicoGreen dsDNA Assay Kit (P7589) and the DNA Broad Range DNA Assay Kit (Q33130) were used to measure double-stranded DNA (dsDNA) in solution. The Quant-iT Protein Assay Kit (Q33210) was used to measure protein in solution. All working solutions and standards were prepared per the instructions provided with each kit. Sufficient volumes were prepared to allow for measurements in a standard 1-cm quartz fluorescence cuvette. 

Figure 1 shows the fluorescence spectrum acquired for the Pico-Green Assay Kit. Using this ultra-sensitive kit, the Curie was able to detect dsDNA to 10 ng/mL concentration at a five-second integration time. Even lower detection limits are possible with longer integration periods. 

 Figure 1. Fluorescence spectra acquired for the PicoGreen Assay Kit (Molecular Probes Quant-iT PicoGreen dsDNA Kit [P7589]).			

Figure 2 shows the fluorescence spectra acquired for the Broad Range DNA Assay Kit. Using this kit, the Curie was able to detect dsDNA to 5 μg/mL concentration at a 1-s integration time. 

Figure 2. Fluorescence spectra acquired for the Broad Range DNA Assay Kit (Molecular Probes Quant-iT Broad Range dsDNA Kit [Q33130]).			

Figure 3 shows the fluorescence spectra acquired for the Protein Assay Kit. Using this kit, the Curie was able to detect protein concentration to 25 μg/mL at a 0.5-s integration time. 

Figure 3. Fluorescence spectra acquired for the Protein Assay Kit (Molecular Probes Quant-iT Protein Assay Kit [Q33210]).			

The Curie Spectrofluorometer is sensitive enough to detect the full range of fluorescence standards included in the Quant-iT DNA and Protein Assay Kits. Using the ultra-sensitive PicoGreen Assay Kit, detection limits to 10 ng/mL were achieved. This data illustrates that the Curie is capable of excitation and emission detection throughout the UV-Vis region. The Curie is a cost-effective CCD-array platform that provides a less complex alternative to more sensitive PMT-based technologies for dsDNA and protein analysis.  

830 Douglas Avenue, Dunedin, FL 34698 USA

Articles

DNA and Protein Concentration Measurements Using Fluorescence Analysis

The combination of an FTIR spectrometer with an optical microscope offers a valuable resource to investigate a variety of complex samples. There are a wide range of applications that have taken advantage of the attractive qualities of FTIR imaging systems including product development, biomedical research, forensics, polymers, and proteins. 

Numerous methods for collecting the imaging data exist, many of them requiring expensive step-scan instruments in combination with a focal plane array (FPA) detector that involves sophisticated electronics for reading the multiple detector elements. An FTIR imaging system that is coupled with a linear array detector does not require a step-scan instrument but provides a fast and efficient analytical method to study various systems while collecting complete infrared spectra. FTIR imaging is used to describe the collection of a chemical profile of the sample area by expressing the intensity of a single absorption peak, or ratio of two absorption peaks, using an image "map". 

 Figure 1.  Hair sample image with FTIR microscope.			

Hair analysis can provide vital information concerning important chemicals that may be in the body. The presence of various toxins and nutrient deficiencies or abundances can be determined using this process. Infrared microscopy is one tool that can be used to examine the interior of the hair and differentiate passive contamination from drugs absorbed into the hair from ingestion. By slicing the hair specimen as either a cross section or laterally, infrared spectra can be obtained of the cortex and medulla of a single hair with a spatial resolution as high as 6.25 μm. 

Figure 2.  Infrared spectra of the medulla (green) and cortex (red).			

A piece of hair was sliced using the HW-1 Multi-Angle SliceMaster and the sample was embedded into a KBr crystal for measurement. A 100 × 100 μm area was imaged with a JASCO FT/IR-6100 integrated with the IMV-4000 imaging microscope. An instrument resolution of 16 cm

  was used to collect 32 scans for the single-beam background and sample spectra. Complete infrared spectra with a spectral range from 4000–750 cm

Figure 3.  Infrared image of percent alpha helix (left) and beta sheet (right) distribution.			

Initial investigation of the hair sample found that there is a greater amount of lipid content in the medulla while primarily protein exists in the cortex of the hair sample. When the spectral data is input into the Secondary Structure Estimation software, it is found that a higher amount of alpha helix is present in the cortex while there is more beta sheet found in the medulla portion. 

Infrared imaging was able to identify the different components of a hair specimen and also provide information concerning distribution of the secondary structures of the proteins present in the hair sample. 

Hair Analysis Using an FTIR Imaging System

ASD's NIR technology can be used to qualitatively distinguish between various thermoplastic resins.

With plastics pervading virtually every aspect of manufacturing (from appliances to apparel), the need to refine products and processes becomes increasingly significant. To those ends, this study purposes to use NIR technology to qualitatively pinpoint the differences between thermoplastic resins 

Spectra were obtained using ASD's LabSpec®Pro and High Intensity Source Probe (Muglight). The baseline (or reference standard) was derived using Spectralon — a commercially available polytetrafluoroethylene with a nearly 100% reflective surface across the entire NIR region. The resins were then placed in the sample puck on the sapphire window of the Muglight and measurements recorded. 

 Figure 1. Spectrometric difference between six different resins.			

The sample spectra were collected using LOG 1/Reflectance — a math pretreatment commonly used for linearizing reflectance data. Six different thermoplastic resins (described below) were acquired. Applying the full-range wavelength (350 nm to 2500 nm) with ten spectra averaging, were gathered for each of the following samples: 			

Figure 2. Difference occurring between 1700 nm and 2300 nm.			

The chemometric software Grams 32/Al™ version was employed to compile all spectra and correlating data points (the data was calculated via Grams discriminate mode). Using cross-validation and self-prediction methodologies, an effective calibration model was achieved. Analysis of the data shows that an accurate model was successfully generated for predicting quantification of thermoplastic resins. As confirmed by these findings, ASD's NIR technology can be readily adapted to qualitatively distinguish between various thermoplastic resins. 

5335 Sterling Drive, Suite A, Boulder, Colorado 80301

NIR Discrimination of Thermoplastic Resins

Fluorescence polarization methods have been used in biochemical applications since the 1970's (1). Dedicated instruments that provide simple, speedy, and highly sensitive results have been used in applications ranging from the quantitative analysis of drugs in blood to PCR products (2). Here, enzyme activity of trypsin is measured using the Hitachi F-4500 fluorescence spectrophotometer equipped with the automated polarization accessory.

In the fluorescence polarization measurement system, the excitation beam is polarized and projected to the sample, and only molecules in the same orientation as the polarizer will fluoresce. The emitted fluorescence is measured in its parallel and perpendicular components separately relative to the incident polarization angle. The fluorescence polarization degree, P, is calculated by the formula in Figure 1. A smaller molecule is easier to rotate with a lower polarization degree, and a larger molecule takes a higher polarization degree. 

This experiment utilized the EnzCheck™ Polarization Assay Kit for Proteases by Molecular Probes containing a fuorescence-labeled casein (BODIPY FL-casein) and a dilution buffer (3). Pancreatic trypsin from cattle supplied by Wako Pure Chemical Industries was diluted with PBS (phosphate-buffered saline) (4). Using the conditions listed in Table I, the P value 20 min after start was measured consecutively five times with polarization degree measurements at intervals of 10–30 s. Trypsin concentrations ranged from 10–100 ng. 

Figure 1.  Formula for P value calculation, and a graphical representation of its relationship to enzymatic activity. 			

The mean P value and standard deviation were plotted for trypsin concentrations, and a linear relationship was established as shown in Figure 2. A reduction in molecular weight with the incremental addition of trypsin was determined by measuring the decrease in P value; thus, the decrease in P value is directly proportional to the enzymatic activity of trypsin.  

The Hitachi F-4500 fluorescence spectrophotometer equipped with the automated polarization accessory accurately quantifies enzymatic activity using the fluorescence polarization method. 

Figure 2.  Plot of mean P value versus trypsin concentration.			

(3) "EnzCheck™ Polarization Assay Kit for Proteases (E-6658)," Molecular Probes (Carlsbad, California, 1999). 

3100 North First Street, San Jose, CA 95134

Automated Fluorescence Polarization Method for Enzyme Activity Measurement

There are several methods for improving spatial resolution in infrared hyperspectral imaging. These methods and their associated benefits and disadvantages are described in this note.

The early 21st century is the age of digital imaging. From multimegapixel digital cameras to high-definition television monitors, the race is on to provide ever higher resolution imaging devices. In these products, the resolution is defined by the number of pixels, or individual picture elements, in a given image. For example, a computer display of 1280 × 1024 pixels is considered high resolution when compared to one containing a mere 800 x 600.  

In infrared hyperspectral imaging, the same basic rules apply. The more pixels that an image of a given sample area contains, the greater the image fidelity. However, the resolution of an image is ultimately limited by the imaging optics and the wavelengths of light that comprise the image. As infrared light has much longer wavelengths than visible, an infrared image can never have the resolution of a good video micrograph. Then again, a video micrograph can never have the resolution of an electron micrograph obtained with far shorter wavelengths still. To a first approximation, the resolution of an image can continue to be improved until the pixel size approaches the shortest wavelength of the light that generated that image, provided that suitable optics are employed. In the case of infrared imaging, this equates to a few microns. However, high resolution images come at a price. For example, as the number of pixels in an image increases, so does the file size associated with it. Anyone who has received a high resolution picture from a five megapixel digital camera as an e-mail attachment over a 56k modem will be familiar with this. In infrared hyperspectral imaging, the problems associated with high-resolution imaging are somewhat more serious and include, but are not limited to, large file sizes. 

In order to understand the issues surrounding high-definition infrared imaging, consider the following example. A low-resolution image of a sample area of 40 × 40 microns is described by four pixels of 20 microns a side, as shown in Figure 1a. 

Figure 1.  40 Ã 40 sample area represented by (a) four 20 x 20 pixels and (b) sixteen 10 Ã 10 pixels.			

A high-resolution image of the same area is described by 16 pixels of 10 microns square, as shown in Figure 1b. There are three possible hardware solutions to get from the low- to the high-resolution image.  

1. A matched objective and condenser set is used that has twice the magnification of the low-resolution set. The detector views a smaller area of the sample and so, to obtain an image of the 40 × 40 micron sample area, four frames are acquired and stitched together. The advantage of this approach is that the condenser, as its name implies, condenses the light from the source onto the sample. As such, the higher magnification condenser focuses the light onto a smaller sample area. Therefore, each detector element 'sees' about the same amount of light in both cases. 

2. Only one condenser/objective set is used but a 2× magnifying optic is placed between the objective and the detector. As in Figure 1, four frames are acquired to represent the entire sample area. The advantage of this technique is that it is relatively inexpensive and simple to operate. However, the disadvantage is that the magnification happens after the condenser. Therefore the light intensity striking each detector pixel is much lower in the high-resolution case, reducing signal-to-noise and thereby increasing collection times beyond those suggested by simple geometry.  

3. Use one set of optics, but use a camera with as many 4× the number of pixels per unit area. This has the advantage that only one frame is required to image the sample area. However, as in Figure 2, the light striking each pixel is much reduced compared to the low-resolution case. High pixel count, or 'large format', cameras for infrared use have lower read-out speeds than their smaller counterparts. Therefore, the apparent speed advantage of large cameras over smaller ones is not as great as may first be perceived. 

Figure 2.  Area of photoresist target shown as (a) a video micrograph, (b) an infrared image at standard resolution and (c) an oversampled high-definition infrared image.			

In all three cases, file sizes increase and acquisition times get longer as resolution increases. As there is a significant trade-off between resolution and data collection time, many imaging systems include options for altering resolution. 

A fourth technique for resolution enhancement in infrared imaging does not involve either additional optics or large format cameras. Introduced in OMNIC™ Atlμs™ 7.2 software, oversampled high-definition infrared imaging offers the ability to vary image fidelity via software-controlled data acquisition. As with most image acquisition schemes, the system builds an image by collecting a number of contiguous frames from a sample that is moved step-wise relative to the camera and stitches them together to produce a single large image. In oversampled mode, the software moves the sample a fraction of the apparent pixel size. In the example above, in order to obtain the high resolution image, the first frame would be acquired and then two subsequent frames would be collected with an off-set of 1/4 frame (or 1/2 pixel) in the X and Y direction respectively. Each pixel is then registered to its correct spatial coordinates in order to build the final image. Images collected with and without oversampling are shown in Figure 2. The data is presented in 3-D in Figure 3. 

Figure 3.  Three-dimensional renderings of (a) standard image and (b) oversampled hgh-definition image.			

Both infrared images were collected with the same optics on the Nicolet™ Continuμm™ XL FT-IR imaging microscope. This software-based approach to high-definition imaging provides two significant advantages over the optical techniques described above. 

1. As the dimensions of the sample observed by each detector element are constant, the light striking the detector, and therefore the signal-to-noise is largely independent of resolution. 

2. As the resolution is software controlled, a range of resolutions may be provided that are not limited by the availability of optics or cameras. 

As image resolution increases so does acquisition time with any infrared imaging system. Therefore, it is beneficial to have the ability to acquire a low-resolution image for survey work and then to obtain high-resolution images for more detailed investigations. Oversampled, high-definition infrared image acquisition provides a range of resolutions without the need for additional optics or cameras and does not sacrifice signal-to-noise for resolution. 

High-Definition Infrared Imaging

The widespread proliferation of "electrotechnical" products has drawn increased attention to their impact on the environment. Many parts of the world, including the European Community, China, and California have current or proposed regulations regarding the use of certain substances such as lead, mercury, cadmium, hexavalent chromium, and some types of brominated flame retardants. For example, the European RoHS directive prohibits these materials from being used in electronic and electrical equipment after 1 July 2006. 

Therefore, the industries involved are looking for testing protocols for these regulated substances and while there are in most cases-approved analytical methods available, one major issue concerns that of sample preparation. These electrotechnical products come in many forms from relatively simple devices such as toys and fitness equipment all the way through to complex instruments and computers each of which contains many different sub assemblies falling under this same category. The challenge is then producing a representative neutral-to-analysis sample from a heterogeneous mixture of plastic, metal, ceramics, and composites. 

In order for the required analyses to be performed, the materials need to be reduced in size to 200 microns or less for analysis. Even after initial size reduction using cutters and similar devices, it is necessary to go through a two stage size-reduction process in order to reach this required size. Following the recommendations of a recent draft document from IEC, the materials should be initially processed in a Cutting Mill, such as a Retsch SM 2000 with 1 and 4 mm sieves. This mill is particularly suitable since it can handle hard and ductile as well as soft and elastic materials. Up to 5 L of material can be processed to a fineness of less than 6 mm. With the help of a sample divider (Retsch PT 100 or equivalent), a representative subsample is obtained for further size reduction to analytical fineness. 

 Figure 1.  From power cord to analytical sample.			

The IEC document recommends that fine grinding be carried out in a Centrifugal Mill (Retsch ZM 200 or similar) equipped with tungsten reinforced grinding tools to help minimize wear and avoid the risks of introducing impurities during milling. This very efficient mill is characterized by very short grinding times and also by its ability to handle material that has been prechilled with liquid nitrogen or dry ice. This not only improves the breaking properties of the material but avoids frictional heat and ensures that volatile fractions are not adversely affected. 

In the USA, there is some debate amongst the standards community as to whether or not compliance should be based on the homogenous approach which is one that looks only at the individual components and materials that are used within a finished product, or the whole product approach which requires the final product to be tested in its entirety. 

Figure 2.  Preliminary size reduction of Eproms (with Cutting Mill SM 2000).			

In both approaches, sample preparation is a critical since without a good representative sample, there can never be a precise and meaningful analysis result. Meanwhile as of 1 July 2006, any electrical or electronic product exported to the European community must show that the whole device complies with these regulations.  

U. S. manufacturers are starting to realize the importance of these regulations and are looking to equip themselves in order to be in compliance. Some companies are already prepared for this and others rely on the expertise and testing facilities offered by the environmental laboratories such as Environmental Monitoring and Technologies (EMT, Morton Grove, Illinois). In this well equipped facility, laboratory manager Vince Donndelinger uses both the Retsch SM 2000 and ZM 100 mills to process various electronic devices including cell phones. Marketing manager Kris Erikson comments that the presence of the Retsch equipment has allowed them to stay fully abreast and in fact ahead of the current regulations, and they see an increasing demand for their services in the future as the regulations take effect. 

Figure 3.  Retsch Cutting Mill SM 2000 a primary grinder and Ultra Centrifugal Mill ZM 200 for the fine grinding stage.			

As EMT have found, having these two Retsch mills makes it relatively easy to be in compliance with the existing and future regulations which are expected to become more stringent as the world realizes the importance of not continuing to dump toxic materials into the environment. 

Sample Preparation of Electrical and Electronic Scrap

Monomolecular layer samples have traditionally been a challenge for spectroscopic measurements. Specular reflectance at high "grazing" angles (around 80 degrees from normal to the surface) has been recognized as one of the best spectroscopic techniques to monitor monomolecular layers on reflective surfaces due to the enhancement of the infrared signal (1, 2). The versatility of this sampling technique is further optimized using different specialized grazing angle accessories. 

An FTIR spectrometer was equipped with an MCT detector and with three different grazing angle accessories; an 80Spec™ - fixed grazing angle, an AGA™ - variable illumination area and with a VeeMAX™ - variable angle reflectance accessory. The fixed angle 80Spec is a dedicated single reflection accessory with simple input and output optics channeling the infrared light with two gold flat mirrors. While this is a powerful tool, its use is limited to larger area samples. When the light from an FTIR instrument is directed to the sample at a high angle, the illuminated area is not as well controlled, its shape is elongated and the IR image size becomes relatively large. When the sample is smaller than the illuminated spot, apertures could be used to limit the size of the infrared beam. In this case, however, the contrast and repeatability of the measurement usually suffers considering the sensitivity required to detect monomolecular quantities of materials. The controlled projected image size therefore is a very important advantage of the AGA accessory, which has a special imaging system with a set of variable diameter mirrors. In this unique optical design, the illuminated spot on the sample is the same as the size and shape of the gold mirror at the base of the optical layout (Figure 1). The selector mirrors in this design are round and are variable in five steps from 3 mm to 12.5 mm diameter. 

 Figure 1. In this unique optical design, the illuminated spot on the sample is the same as the size and shape of the gold mirror at the base of the optical layout.			

The variable angle VeeMAX accessory (Figure 2) on the other hand allows other reflectance and reflectance-absorbance measurements in addition to grazing angle measurements. 

All three optical arrangements can be used with additional polarizers to enhance the monomolecular surface species. In this work, a ZnSe wire grid polarizer was used for all samples. All measurements were done using the MCT detector as the IR throughput is relatively low at grazing angle of incidence in combination with an IR polarizer. All spectra were collected at 8 cm-1 resolution with a 1 min data collection time. 

Figure 2. The variable angle VeeMAX accessory.			

For the comparison of the performance of different optical geometries, monolayer systems used in various biotechnologies were selected. Monolayers are considered initial surface coverages with reactive groups to be modified further for various biospecific and ligand interactions, fabrication of protein, DNA and carbohydrate chips, and biosensors. Gold is known to have high affinity to sulfur containing groups, such as thiols, and generate more or less well-ordered monolayers (4). One of the systems, a self assembled hydroxyl-terminated alkanethiolate monolayer that was formed on a freshly evaporated gold surface using the method and materials from Platypus Technologies, Inc (Madison, Wisconsin, http://

). The monomolecular layer is formed over a timeframe of several h in a dilute solution of HS(CH

X, where X can represent different functional groups. The ω-functionalized alkanethiolate layer (5) was stable throughout the experiment and was easily measured on all of the accessories. 

Figure 3. (3A) shows the NH-terminated monolayer measured on the 80Spec with a ZnSe polarizer. Figure 3B shows the example using the AGA is an OH-terminated thiol.			

Figure 3A shows the NH-terminated monolayer measured on the 80Spec with a ZnSe polarizer. The example shown using the AGA is an OH-terminated thiol (Figure 3B). The accessory was adjusted to project a 12.5 mm diameter spot on the gold-coated slide. Due to the better definition of the illuminated area, smaller samples can be measured easily without apertures which in some cases can introduce artifacts. Grazing angle measurements with the VeeMAX is shown with a third kind of monolayer, a CH-terminated alkanethiol. -CH terminated alkanes are generally more difficult to identify, due to the lack of characteristic differentiating molecular groups in comparison to organic contamination from ambient air, solvents on the sample or the background slides. For the experiment the VeeMAX was set to 78 degrees and produced the characteristic pattern seen in Figure 4. 

Figure 4. For the experiment the VeeMAX was set to 78 degrees and produced this characteristic pattern.			

Infrared spectroscopic measurements of monomolecular layers can be performed using all of the different grazing angle reflectance accessories discussed in this work and are capable of producing excellent quality spectra. The intensity of the mono-molecular signal may be enhanced with polarized light, and the quality of the result is greatly enhanced by using an MCT detector. By controlling the illuminated area with a special optical design of the AGA, one can measure small samples or examine small areas of larger samples. When, in addition to grazing angle analysis, other reflectance measurements are needed; the variable angle VeeMAX is an excellent choice. 

Modern Techniques in Applied Molecular Spectroscopy

3. Single Adjustment Specular Reflection Accessory for Spectroscopy, U.S. Patent 5,106,196. 

4. C.M. Pradier, M. Salmain, Z. Liu, and C. Methivier, 

2901 Commerce Park Drive, Madison, WIÂ 53719 

Tel. (608) 274-2721, FaxÂ (608) 274-0103 

Measurement of Monomolecular Layers with Specialized FTIR Grazing Angle Accessories

Jordan Valley's RoHS Analyzer uses a powerful X-ray tube and a new generation high resolution Si PIN detector (see Table 1) to measure low levels of heavy metals, allowing electronics manufacturers to comply with new regulations designed to reduce environmental waste and adverse health effects. 

The Restriction of Hazardous Substances Directive (RoHS), designed to restrict toxic metals in new electrical and electronic equipment, profoundly impacts the electronics industry. Producers must now document the levels of restricted substances in their equipment. RoHS restricts six substances — lead (Pb), mercury (Hg), cadmium (Cd), hexavalent chromium (Cr6+), and certain compounds of bromine (Br), PBB and PBDE. These elements occur in compounds which are commonly used as fillers, stabilizers, pigments, and flame retardants.  

 Table I. Features of Si PIN Diode, Si(Li), and Silicon Drift Detectors.			

To be compliant, manufacturers must prove that these compounds fall below their maximum concentration values (MCV). 

Jordan Valley's new RoHS Analyzer was specifically designed to help electronics manufacturers determine the precise level of restricted substances and certify that they fall below the MCV. This high-powered instrument provides fast quantitative analysis of lead in solder for Pb-free electronics requirements, and sorts chlorinated and/or brominated plastics. Highly reliable, accurate analytical results can be obtained in PE, PVC, or other polymers. In one easy step, the analyzer measures extremely low levels of Cr, Hg, Pb, Br, Cd, and other heavy metals and identifies PVC, Br- or Sb- or P-based flame retardants. 

The nondestructive energy-dispersive technique means that samples are left unaffected to allow further testing by the same or additional techniques for quality control purposes. Additionally, every step is fully documented with an audit trail for self-certification and compliance verification. 

Jordan Valley is a leading designer, manufacturer, and marketer of energy-dispersive X-ray fluorescence (EDXRF) systems and components with more than 30 years' spectroscopic experience. We provide quality and cost-effective answers to real-world analytical challenges by melding the latest technological developments with innovative engineering. 

8601 Cross Park Dr., Suite 200, Austin, TX 78754 

Tel . (877) 777-1222, Fax (512) 973-9282 

RoHS Analyzer for Compliance with Environmental Regulations for Hazardous Substances

The long anticipated PX4 Digital Pulse Processor from Amptek has joined the successful XR-100 family of X-ray detectors (as shown in Figure 1). The system allows for XRF analysis in the field and satisfies QC requirements for monitoring detector performance. The PX4 is also a powerful research tool that can be used to optimize detector performance.

As shown in Figure 2, the detection of lead (Pb) is an easy task for this pair of instruments.  The L lines are easily resolved within 1 minute of unit power-on.  Since the PX4 is driven by software, only one switch exists on the box (power). Everything else is software selectable and can be set to automatically load on startup.  This ensures consistent, fast, and easy operation. 

 Figure 1.  PX4 Digital Pulse Processor and XR-100CR X-ray Detector. 			

Since the PX4 has 24 programmable peaking times between 0.8 and 102 µs, it is well suited for a wide variety of applications. In addition, the determination of Resolution vs. Peaking Time (finding the noise corner), as in Figure 3, can be performed automatically by a software routine. With this information, the user can easily select the appropriate peaking time to maximize performance for the particular application, whether it be the best resolution, higher throughput, or a trade-off between the two.

XRF measurements used to involve costly, liquid nitrogen dependent, instrumentation.  Amptek has broken another barrier in offering a low cost, easy to use system that not only performs the measurements, but also characterizes the system to satisfy QC requirements and to optimize performance across a wide range of applications. 

Figure 3.  Resolution vs. Peaking Time. 			

A Powerful Tool for XRF and Detector Performance Characterization

Coatings and films play an important role in many fields of application (drug delivery coatings, coatings on medical devices, thin films for food protection, etc.), which makes an efficient technique for the investigation of these materials essential. Confocal Raman Microscopy is a powerful tool for nondestructively imaging the chemical composition of heterogeneous components within a film or coating. The confocal setup not only provides the highest spatial resolution, but also allows the acquisition of depth profiles ideally suited to the three-dimensional characterization of polymer films and coatings without sample preparation. 

The Confocal Raman Microscope WITec CRM 200 combines a highly sensitive confocal microscope and a high-transmission Raman spectroscopy system. With a resolution down to 200 nm at each image pixel, a complete spectrum is typically acquired in only 50 ms to 100 ms. The number of image points (spectra) is limited only by the computer memory. A typical image consists of 10,000 (100 × 100) to 65,536 (256 × 256) spectra. From this multispectrum file, an image is generated by integrating over a specific Raman line or a region in all spectra. As all spectra are stored in memory, a variety of properties such as peak-width, center of mass, or peak position of certain Raman lines can be calculated from a single measurement. For depth profiling measurements, the focal plane can be moved in the z-direction when performing either x-z scans or generating x-y image stacks in the z direction. 

In this experiment, the inner polymer coating of an orange juice container was investigated with the CRM 200 by performing an x-z scan with a scan range of 50 μm × 100 μm at 200 × 120 pixels (= 24,000 spectra) using a 100 × objective (NA = 1.25). For excitation, a 532 frequency doubled Nd:Yag laser was used. The acquisition time for each spectrum was only 50 ms. 

 Figure 1. Raman spectra (left) and Raman image (right) of the inner coating of an orange juice container.			

Within the acquired multi-spectrum file, four distinct spectra can be observed in Figure 1 (left). Each spectrum represents a dedicated chemical compound within the polymer film. With the integrated software tools, the distribution of each compound can be made visible by analyzing all acquired spectra. Figure 1 (right) shows the resulting color-coded image, clearly revealing that the coating consists of five different layers although only four components are involved. The software also provides the ability to measure distances within the image, indicating that all layers vary in thickness.  

Confocal Raman Microscopy is an ideal tool for the analysis of heterogeneous samples on the submicrometer scale. As shown in the example, different polymer layers in the film can be clearly resolved and matched with their associated Raman Spectrum. Detailed information on the structure of multilayered polymer films or coatings can thus be attained using the depth profiling capabilities of the confocal Raman microscope.  

Confocal Raman Imaging: Depth Profiling of Polymer Films and Coatings

Raman spectroscopy has been limited from widespread use due to its high instrumentation cost, especially in its excitation light source. To answer this challenge, a low-cost miniature Raman spectrometer was developed. The MiniRam NIR Raman spectrometer (shown in Figure 1) utilizes B&W Tek proprietary CLEANLAZE™ laser technology and TE cooled 2048 pixel CCD array spectrometer. As a result, this low-cost instrument achieved a spectral resolution of 8 cm

The CLEANLAZE™ laser is a high-power broad-stripe laser diode with its output spectrum narrowed and stabilized using a novel external cavity technology. Unlike conventional external cavity lasers that require a long cavity length, the wavelength selective element in the CLEANLAZE™ laser is directly integrated onto the laser diode. As a result, the laser is both ultra compact in footprint (4 mm × 4 mm × 3 mm) and stable to environmental change. The utilization of a broad-stripe laser diode guarantees the low cost of the whole module and at the same time, it can provide much higher output power (up to 1.5 W) than its DFB and DBR counterparts. The spectral linewidth of the laser can reach 0.8 cm

 Figure 1.  B&W Tek Mini Ram Raman spectrometer. 			

Utilizing a 785 nm CLEANLAZE™ laser as the Raman excitation light source, the miniature Raman spectrometer achieved a high spectral resolution and exceptional sensitivity. Other features of the Raman spectrometer include light weight (< 5 lbs), compact size (8.9" × 6.4" × 3.25"), integrated fiber optic probe, and a user friendly computer interface. 

Figure 2.  Raman spectrum of an amethyst sample measured with MiniRam.			

The miniature Raman spectrometer has been successfully applied to a variety of applications, including gemstone identification, hazardous materials detection, pharmaceutical analysis, and epoxy cure monitoring. Some of the results are shown in Figures 2-5, respectively. Figure 2 shows the measured Raman spectrum of an amethyst sample. The MiniRam provides a quick, non-destructive gemstone identification method from their characteristic Raman spectra. Figure 3 shows the measured Raman spectrum of a Benzenethiol sample (a hazardous material) that was captured on a Mesophotonics Klarite SERS substrate (shown in the upper right corner of Figure 1). 

Figure 3.  SERS spectrum of a Benzenethiol sample measured with MiniRam.

The surface enhancement provided by the substrate and the high sensitivity of the MiniRam allow us to detect Benzenethiol at low concentration levels. Figure 4 shows the measured Raman spectrum of a Tylenol™ sample. The high spectral resolution of the Raman spectrum provides a convenient non-contact way to analyze the content and active ingredient distribution of the medicine. Figure 5 shows the Raman spectra of an epoxy sample measured in real time with MiniRam during the cure process. The variation of the relative intensity of the 1260 cm

  epoxy band was used to accurately monitor the curing status of the epoxy sample. 

Figure 4.  Raman spectrum of a Tylenol sample measured with MiniRam.			

A miniature Raman spectrometer utilizing spectrum narrowed diode laser was developed and applied to a variety of applications. The measured Raman spectra show good spectral resolution and sensitivity for detection, identification, analysis and monitoring of a target material. 

Figure 5. Raman spectra of an epoxy sample measured in real time with MiniRam during the cure process.			

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Miniature Raman Spectrometer Utilizing CLEANLAZE™ Technology

A commemorative series of spectrophotometric calibration artifacts has been developed in tribute to Dr. Radu Mavrodineanu, the "founding father" of the contemporary NIST high-accuracy spectrophotometry program. 

Molecular absorption spectrophotometry is a vital tool in analytical research, quality control, and manufacturing-support laboratories. The assurance of accurate spectrophotometric data relies on various validation measures including science-based procedures to demonstrate that the spectrophoto-meter is functioning properly and within specification for its intended use. Primary spectrophotometric Standard Reference Material (SRM) artifacts from the National Institute of Standards and Technology (NIST) serve as the traditional benchmark for this purpose. Additionally, these SRM artifacts provide the primary means of traceability linkage for secondary spectrophotometric artifacts produced by commercial suppliers. 

The current array of primary spectrophotometric SRMs is an extension of the high-accuracy spectrophotometry program that originated in 1969 within the Center for Analytical Chemistry of NIST, known at that time as the National Bureau of Standards (NBS). For more than 25 years, the overall success of this program can be attributed to the technical vision, leadership, and pioneering accomplishments of Dr. Radu Mavrodineanu, a renowned analytical spectroscopist and scientist (1). Included among his many valuable contributions to this program are the original SRMs developed for wavelength accuracy, transmittance and absorbance accuracy, and stray radiant energy in the ultraviolet (UV) and visible (VIS) regions of the electromagnetic spectrum. 

To continue the legacy of Dr. Mavrodineanu, or Mavro, as he was known to many of his close friends and colleagues, Stranaska is proud to introduce its new Vintage MAVRO Series. This commemorative series is an integral part of the company's expanding product line of secondary calibration artifacts for analytical metrology (CA2M). Produced on demand and in limited quantities only, the Vintage MAVRO Series is comprised of the company's top ten CA2M products for the definitive calibration of UV/VIS absorption spectrophotometers. Included in the series are eight photometric artifacts, one wavelength artifact, and one stray radiant energy artifact; the eight photometric artifacts are subdivided into five solid CA2M artifacts and three liquid CA2M artifacts. Each of the ten secondary CA2M products comprising the Vintage MAVRO Series is meticulously patterned after or derived from a corresponding primary spectrophotometric NIST SRM artifact (see Table I). 

 Table I.  Vintage MAVRO Series of Spectrophotometric Calibration Artifacts			

(1) J. Messman, "Historical Review of Standard Reference Materials for Analytical Spectrophotometry – A Tribute to Dr. Radu Mavrodineanu,"  Poster Paper Number 1 presented at Federation of Analytical Chemistry & Spectroscopy Societies 30th Annual Meeting, Fort Lauderdale, Florida, 19 October 2003. 

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Application Notebook-10-02-2005