NEWS & REVIEW 1. Editorial This edition has been edited by our first invited Editor, Don Clark and is dedicated to microscopic vibrational spectroscopy. With much pleasure I hand over to Don... This edition of IJVS is focused (sorry pun intended) on the use and applications of
vibrational microscopy, which is a collective term for FT-IR microscopy, NIR microscopy
and Raman microscopy. The feature that links these and a number of other emerging
techniques, is that spectral information is obtained via a modified light or atomic force
(AFM) microscope. The ability to view a sample before, after, and sometimes even during a
spectral data acquisition is very important in the analysis of microscopic samples and
areas. In terms of the IR and Raman spectroscopy being performed, these microscope systems
offer a convenient method of obtaining a spectrum from a small sample or area. It can be
easy to overlook the visual data that is also available. This includes information such as
sample size, shape, colour, morphology, crystallinity, and number of phases present. The
combination of these physical properties along with the specificity of the spectroscopic
data provides a more complete analysis of the sample than by microscopy or spectroscopy
alone. To help set the scene, the following pictures show you what a typical FT-IR
microscope and a typical Raman microscope look like.
Nicolet Continum m FT-IR microscope Renishaw System 1000 Raman Microscope I have had an interest in the technique since the mid 1980’s when I purchased one of the first commercially available FT-IR microscopes in the UK. To date, the purchase justification for that instrument was the easiest I’ve ever had to justify to management. The benefits in non-destructive microscopical sample analysis were obvious to all concerned. My perception since then is that for many of the laboratories involved in investigative analysis today, the FT-IR microscope is now a standard technique in the spectroscopist tool box. And in recent years Raman microscopy is becoming more widely used. Having given an external presentation earlier this year on developments in microspectroscopy, I was flattered when Patrick Hendra asked me to get involved # with this microscopy issue. Needless to say I now have an appreciation of the effort which goes into producing each edition of this journal. # Wrong word - lumbered! - Patrick Hendra The subjects covered in this edition are the design of FT-IR and Raman microscopes, a personal view on the use and trends in the applications of vibrational microscopy, use of the techniques in combinatorial chemistry and the study of semiconductor materials. It closes with features on FT-IR and Raman chemical imaging, which for me, represents the major application development area in microspectroscopy. The data from these experiments are usually presented in a pictorial manner and provide chemical information about the sample. In very simple terms, these data can be considered as a chemical photograph. The ability to perform routinely non-destructive analysis on solid phase mixtures, (e.g. polymers, biological samples and pharmaceutical products to name but three), and to visually show the location of the components present marks a significant technological advance in vibrational spectroscopy. Through both academic groups and instrument manufacturers, the boundaries of what is possible with microspectroscopy systems is continually being extended. In my opinion this is what makes vibrational microscopy an exciting science to be involved in. My only regret is that these techniques have not made a bigger impact within the microscopy community. Perhaps it’s instrument cost that cause this inertia? However, speaking from experience, I know that when spectroscopists and microscopist share their skills and knowledge there is a significant and positive impact in their studies of microscopical heterogeneous samples and systems. This IJVS edition presents information on what is currently possible in vibrational microscopy. So what about the future? A consequence of imaging experiments is the vast amount of data they produce. Everybody's experiments will be different, based on sample response, instrument availability, sampling area and spatial resolution. However, one common feature of all these experiments is the vast amount of spectral and image data they produce. I would estimate that most images contain between 1,000 to 25,000 discrete pieces of information. This can equate to file sizes of 40+Mb, so large PCs with hard disc capacity and fast processing speed are essential for handling these large data sets. For many applications the creation of images is relatively easy provided each of the (known) components in the samples has a unique characteristic spectral band. Data analysis becomes more challenging when their are unknowns in the sample, as it is impossible to predict where their unique bands are. Other difficulties arise when the sample components have similar spectra or complex spectra with many overlapping bands. To create meaningful images from these data sets more sophisticated software is required. This software may already exist, but if it does, it is either not readily available or has not yet been applied to these types of problems. (If you know of anything suitable, I’d like to hear from you - don_clark@sandwich.pfizer.com). At the time of writing, the applications of atomic force microscopes and probes to obtain images with spatial resolutions of below the defraction limit are becoming more widely published. Anecdotally, I have heard of the use of future systems to identify each amino residue in order along a single protein strand. If this becomes reality it will open up many new areas and applications of chemical imaging. I hope you find this edition interesting, and I’m sure Patrick would like some feedback and any discussion items that arise from the following articles. If you are interested in learning more about the these and emerging microspectrometry techniques I recommend making contact with the following groups/organisations. Europe The Microspectrometry Applications Group ( MAG) - Affiliated to the Association of British Spectroscopists and the Royal Microscopical Society. The MAG is a UK based discussion group with a goal to promote small sample analysis by spectroscopy through microscopes. It meets twice a year at different locations around the UK as a forum to discuss different areas of applications and new techniques. The group has a triennial award for the best oral presentation at its meeting for young microspectroscopists. To keep all the membership aware of group activities the MAG produces abstracts of all its meetings and appropriate newsletters.. For further details contact Don Clark, Physical Sciences - D265, Pfizer Central Research, Ramsgate Road, Sandwich, Kent, CT2 8EP. Tel. +0044 01304 646036 e-mail: don_ clark@ sandwich.pfizer.com North America The Molecular Microscopy Laboratory is based at Miami University, Oxford, Ohio under the direction of André Sommer. This group’s work is dedicated to microspectroscopy research, development and education whilst providing a molecular microscopy service to both scientific and business communities. Each year they run an annual course for any one who wants to learn more about these techniques. The next is in June 1999. More information can be found at http://www.muohio.edu/~ sommeraj In addition, the annual Federation of Analytical Chemistry and Spectroscopy Societies (FACSS)conference has sessions covering the FT-IR and Raman microscopy and images. Details on next years conference in Vancouver can be found at http://facss.org/info.html Don Clark The views expressed here are personal, and are not those of Pfizer Central Research. Assistant Editorial
Introduction Microscopes A microscope is a precise optical instrument . It has been developed over centuries in order to reveal the small, and fractions of larger samples that are beyond the normal sight. The microscope allows a sample to be held in a stable manner without vibration so that small areas of a sample may be illuminated and seen at high magnification without distortion. Many years of research and development has resulted in lenses that are aberration free, these produce an image that is in focus across the full field of view. They are used on an infinite variety of samples, some are transparent and some are opaque. The microscope is used to characterise and identify many samples on its own, and for this a number of different illumination methods are used. It is not sufficient to just shine a white light onto or through the sample to reveal all of the components of the sample, many details can only be revealed if the appropriate illumination method is used from the following:- bright field, dark field, polarised, phase contrast and fluorescence. If you do not reveal all of the components then when you try to identify what you see you can only be partially successful. The ability to reveal components of a sample is also governed by the methods of sample preparation and how the sample is presented and the magnification used. A microscope usually has a number of lenses mounted on a turret that have different magnifying powers. These lenses should be parfocal. This means that once one of the objectives in the nosepiece set has been focused on the specimen, all of the other lenses may be used and the focus will be approximately correct for each. In addition the lenses should be co-linear. This enables you to move the lens and select higher magnifications without varying the centre point of the examination. The hyphenated techniques and their widespread use is a result of the development of more sensitive detectors for both infrared and Raman spectroscopies. FTIR - microscopy FTIR is a technique that enables us to identify compounds from their infrared absorption spectrum. Microscopy is a method of sample handling that is suitable for infrared examination . In order to combine these two methods a number of criteria need to be met .
A small aperture that allows sufficient energy to pass to give a reasonable spectrum is
selected. This is moved in a regular manner pausing after each step in the horizontal
direction to collect a spectrum. At the end of each row the stage moves rapidly back to
the start of the next row in the directions shown. After all of the data has been
collected an absorption band is selected that is characteristic of one portion and by
plotting the absorbance value against step distance a map of the distribution may be
obtained.
An example of a full research microscope that has all of the microscope techniques
available and will interface to Raman and/or any FTIR spectrometers with external beam
facilities, the AIRE ENT IR 800R, is shown below.
Conclusion These hyphenated techniques are excellent ways of doing most solid and liquid samples. Raman microscopy is probably the quickest way of examining most samples as there is no sample preparation and you know that when the laser spot is in focus that you will have the best Raman signal. Fluorescence is not as damaging in preventing you from seeing the spectrum in Raman microscopy as it is in conventional 90° collections. This is due mainly to the smaller focal volume of the laser focus burning out the fluorescence more quickly than is possible in 90° work. The CCD detectors and computer techniques help enormously in improving the use of Raman spectroscopy. Infrared spectroscopy has always required sample preparation, and infrared microscopy is no exception. Good sample preparation and the correct use of the microscope greatly improves the quality of the information that you may obtain from the technique. The more you use the microscope the more familiar you will become with the controls. Problem solving will be easier if you can combine the information you gather from microscopy with the spectroscopic results. Microscopy when used in a combined technique with a spectroscopy gives an added value to the analytical results that is not possible when they are used separately.
Don Clark Physical Sciences, Introduction The term vibrational microscopy is one that is becoming more frequently used to describe collectively the techniques of FT-IR microscopy, NIR microscopy and Raman microscopy. All allow vibrational spectra (i.e. IR or Raman) to be obtained from small samples via adapted light microscopes. This paper is an introduction to the practical aspects of vibrational microscopy, the types of experiments that are currently possible, and some interesting applications of these versatile methods. For more information, reviews,[1, 2, 3] reference books,[4, 5] and other journals should be consulted. It should be pointed out that the nomenclature used to describe these techniques is not globally consistent. European publications tend to use the terms FT-IR microscopy and Raman microscopy. This is consistent with the nomenclature used by the Royal Microscopical Society (e.g. Light microscopy, Scanning electron microscopy etc.). American publications use the terms IR microspectroscopy and Raman microspectroscopy. The term microspectroscopy is also appropriate for microsampling techniques which include beam condensers, diamond anvil cells, and the new generation of single reflection ATR accessories, as well as being used to describe microscope based systems. My personal belief is that the European nomenclature is scientifically specific and correct. However, with the dominance of the US publications and market forces it is unlikely that there will be a unification of terminology in the foreseeable future. Historically, it was the Raman microprobe [6] which first made an impact on small sample analysis. However, this type of system was typically only found in multi-national companies and a few academic institutions. The applications afforded by FT-IR microscopy, and introduced to the scientific community in the early 1980’s, proved to be much more applicable and accessible to many more analytical laboratories. Raman microscopy is currently going through a renaissance due to the emergence of bench top systems using CCD (charge coupled device) detectors and notch filters (rather than double or triple monochromators) for laser rejection. NIR microscopy is an emerging technique which has effectively extended the spectral range of the FT-IR microscope into the near IR region. Its applications have yet to be established. It should be stressed that vibrational microscopy is only one of a number of spectroscopic and microscopical tools the analyst can call on to solve problems. There are many forms of microscopy that can give information on the chemical composition and/or sample structure. Many of these techniques are included in a review paper by Peter Cooke on chemical microscopy and this is a good reference point for identifying modern microscopy techniques and their applications. The bulk of this article concentrates on how to carry out vibrational spectroscopy and its current applications. With the wide scope of information, it has been divided into the following sections. Practical aspects of vibrational microscopy i. Sample preparation for FT-IR microscopy Recent applications and technological advances i. Instrumentation and techniques Practical aspects of vibrational microscopy When or how should an FT-IR or Raman microscope be used ? This question will have a wide variety of answers depending on what information is required. Vibrational microscopy is used in its most simple form as an expensive beam condenser coupled with a high performance viewing system. This allows the analyst to survey the sample and position the area of interest in the IR or laser beam. At the other extreme it may be used to determine the number and physical shape and size of the different phases present. By observing samples under white light and through crossed (plane) polarisers, areas of birefringence will be observed enabling identification of crystalline materials to be made. If the system is fitted with photomicrography or image capture equipment, a permanent white light image of the sample can be obtained. With respect to size, transmission FT-IR microscopy measurements can be made on samples having XY dimensions of ca. 10-250 m m with a thickness of <30 m m. Typically samples have dimensions of several 10’s of microns. Recent developments using synchrotron sources enable sub 10 m m samples to be studied [8]. Raman microscopy systems allow significantly smaller samples to be studied routinely. Depending on whether a dispersive or FT instrument is used the sample size is typically 1-30 m m. Before performing any vibrational microscopy, I would recommend observing the sample using a low power stereomicroscope. This has two benefits. Firstly, it allows the whole sample to be surveyed quickly, and as these microscopes are binocular instruments, a full perception of sample depth is provided. This simple sample observation may give many clues as to the samples identity, or to what has happened to the sample prior to analysis. From my own experience dark coloured features that appear to be on the surface of a sample when viewed by the naked eye, may in fact be close to the surface, but covered by clear or less coloured matrix. Secondly, if required, the stereomicroscope can be used as an aid in microsampling preparation. Its low power magnification (10-60X) and large working distance* make, for example, the extraction of a specific particle or fibre from a solid or liquid matrix with a fine needle considerably easier than performing the same operation whilst viewing the sample by the naked eye alone. (* working distance = distance between objective lens and sample stage) i. Sample preparation for FT-IR microscopy
In some cases the sample will have to be studied without preparation. This may be
because the sample could change during preparation (e.g. undergo a polymorphic
transformation), or is required in its intact state for further testing or archiving.
Where these samples are too thick or opaque for transmission measurements, the objective
Cassegrainian lens can be used in reflectance mode. It can be considered to be a micro
diffuse reflectance accessory; one half of the lens delivering the IR beam to the sample
and the other half collecting the resulting reflected energy. This (transflection)
approach provides ca. 50% of the incident energy available in transmission experiments, so
in practical terms reflectance measurements require extended acquisition times in order to
obtain the same quality spectra that are obtained in transmission mode.
There are two other approaches to reflectance FT-IR microscopy. These
are through the use of attenuated total reflectance (ATR) - See Figure 1, and grazing
angle objectives. ATR FT-IR microscopy uses the same principles as traditional ATR methods
and allows spectra of microscopic areas to be obtained through direct contact with the ATR
objective - See Figure 2. This method is so easy to use it can also be used to obtain
information from sample surfaces that would normally be obtained using macro ATR
accessories. (N.B. this approach has been modified in the design of the latest single
reflection ATR units that are proving extremely useful in providing rapid analysis of bulk
solids, liquids, and pastes). Grazing angle accessories have been used for many years to
study thin coatings on substrates, and thin film lubricants. The availability of grazing
angle objectives means that these types of studies can now be performed routinely at a
microscopic scale.
Another approach to obtaining spectral information from thick or opaque
samples is to use NIR microscopy. NIR bands originate from overtone and combination bands
of IR fundamental bands and as a consequence are significantly weaker than those in the
mid IR region. This is advantageous in NIR microscopy as much thicker samples ii. Sample preparation for Raman microscopy The major problem associated with any Raman measurement, including microscopy methods, is sample fluorescence. Fluorescence can often be avoided by using FT-Raman systems using 1064 or ca.780 nm lasers to irradiate the sample. Unfortunately, sensitivity at these wavelengths is poor. Sensitivity is improved by using an excitation wavelength of 633 or 514 nm, but here fluorescence may be the dominant spectral feature. A new approach to avoiding fluorescence is to use a deep UV laser, but by this method the sample may be damaged due to the much higher energy of this excitation source. In my experience, a suitable laser wavelength with which to obtain Raman spectra can be found for the majority of samples submitted for analysis. Recent applications and technological advances i. Instrumentation and techniques Several classical light microscopy techniques have been successfully adapted for use in vibrational microscopy. A technique which is becoming increasingly more used is confocal Raman microscopy. This exploits a feature found in some optical microscopes. Using an aperture at one of the microscopes focal planes, it is possible to study non-invasively a layer within a sample rather than just at its surface. This allows a sample to be depth profiled, and if a XY stage is available, provides an opportunity to image a sample volume rather than just a surface. Crystallinity and molecular orientations in individual particles and fibres can be probed by using polarised FT-IR microscopy. Here an infrared polariser is placed in the IR beam and spectra acquired from the sample placed alternatively perpendicular and parallel to the polarised beam. Differences in the spectra can be used to determine specific orientations and interactions of functional groups within the sample. In the case of fibres, dichroic ratios (Iparallel/Iperpendicular) can be used in the non-destructive prediction of their physical properties [4]. FT-IR and Raman thermomicroscopy use a heated / cooled stage to enable samples to be studied at non-ambient temperatures. The main applications of vibrational thermomicroscopy are to study spectroscopically (and visually, though not necessarily mutual) the changes in polymorphism and crystallinity of single sample particles/crystals. In these experiments the transformation from one form or phase to another is temperature dependent. Another approach is to use a modified differential scanning calorimeter to heat and cool the sample. This combination of techniques allows structural information to be correlated to the thermogram obtained from the same sample. ii. Areas of Application An exciting application has used Raman imaging techniques to study fingerprints for traces of explosives such as TNT and Semtex. [17] The imaging identifies which components are present, and by making confocal measurements an estimate of the amount of each compound present across the image can be made. Vibrational microscopy has many applications in biomedical studies (extensively reviewed by Victor Kalinsky [18]), including an often quoted example of the imaging and identification of silicone in breast tissue biopsies following the failure of implants. FT-IR can be used to differentiate between the a -helix and b -sheet secondary structures in proteins. This has been used successfully to show that the anticancer drug adamantyl maleimide ( AMI) produces a conformational change in both the membrane and intracellular proteins of the human gastric carcinoma cell line SC-M1 [19]. This gross change in secondary structure has been linked to the cell viability, and can be used to monitor the effect of different drug concentrations on the carcinoma. Principle component analysis and reflectance FT-IR microscopy data has been used to differentiate between the properties of bacterial colonies [20]. By observing the frequency of the asymmetric phosphate band, it was possible to determine which colonies were Gram positive and which were Gram negative. The advantage of this technique is that it is non destructive, requires no sample preparation, and is better at discriminating between bacteria types than other spectroscopic methods. The use of synchrotron sources has allowed IR spectra to be obtained from single living cells [21]. With spatial resolution of a few microns now possible, the distribution of functional groups of proteins, lipids and nucleic acids in a cell have been mapped. In addition, the changes in lipid and protein distributions during cell division and necrosis have been determined using this method. Within the pharmaceutical industry, vibrational microscopy has a special role in the analysis of samples in their solid state. This is important as the polymorphic form of the drug and/or excipients is often vital to the performance of the product. For example [22], a capsule formulation of SCH 48461 showed a slowing of dissolution with respect to time. Using ATR FT-IR microscopy this was shown to be due to a conversion in the formulation of the drug from an amorphous state to crystalline form; the latter having a lower solubility in the water. Similarly, Raman chemical imaging has been used to determine the size, form, and distribution of the drug and excipients in tablet formulations. Figure 1 shows the chemical images from two batches of a tablet formulation which have good and poor dissolution profiles. These images show that the particle size and distribution of the drug and excipient A are different in the two batches. With this information it has been possible to optimise formulation manufacture to produce tablets with good dissolution properties [23].
Another advantage of chemical imaging is that it can be applied to complex multiphase systems, which by light microscopy are of inherently low contrast. Emulsions are good examples of such systems, and Raman imaging offers a unique method for identifying the components present in aqueous, droplet and structural phases present in these formulations [24]. A review of this type would be incomplete without a few recent examples from the polymer industry. A paper by John Chalmers and colleagues [25] demonstrates the applications of several of the techniques in the characterisation of industrial materials. The use of a synchrotron as a light source now allows the IR spectra of sub 10 micron thick samples to be obtained, which has applications in studying thin layers in polymer laminates. One example demonstrates how IR imaging shows not only the location of known polymeric material in a sample, but also locates and allows identification of contaminants which were invisible by light microscopy. Imaging is not confined to static systems. Time domain FT-IR microscopy utilising a focal plane array ( FPA) detector has allowed the observation of low molecular weight liquid crystals diffusion into poly( butylmethacrylate) film in real time [26]. The performance of a polymer is dependant on a number of factors, a number of which can be probed at the microscopically level by vibrational microscopy. FT-IR microscopy in conjunction with scanning electron microscopy (SEM) and light microscopy, has been used to assess the crystallinity and molecular orientation and by implication, the performance of nylon 6,6 vibrational welds [27]. There are many examples of the Raman spectroscopy/microscopy to determine the stress/strain present in a sample. A recent example demonstrates how this microscopical method can be used to measure these properties at localised spots in polymer based composites [28]. Mention of other applications should be made which include quality and microstructure measurements of CVD (chemical vapour deposition) diamond films used as coating for computer hard discs, analysis of combinatorial chemistry products whilst attached to their solid phase substrates, and the investigative analysis of unknown fibres, particles, crystals and contaminants. Although, not an exhaustive review of vibrational microscopy applications, this article has given an overview of the type of studies that can be tackled using FT-IR and Raman microscopies. The continued development of new instrumentation and on-going research projects clearly demonstrates that vibrational microscopy has established itself as a valuable tool in solving microscopical problems in analytical laboratories around the world. References
Robert Alexander This application note will give a brief overview of the Combinatorial Technique and outline how FT-IR spectroscopy can be used to monitor these synthetic sequences and also consider the relative merits of various sampling methods available for this type of analysis. Why use Combinatorial Chemistry? Traditionally, drug discoverers have looked to natural product extracts or existing company libraries of compounds as a source of such materials. Unfortunately, many of these libraries can lack structural diversity and finding active products from natural sources can be difficult and very time consuming. This has lead to a change in emphasis away from a structured, rational approach to new compound discovery to a more empirical approach where the emphasis is on the synthesis of a very large numbers of new structures and using the rapid screening methods now available to test for activity within these groups. Once activity has been established a more step-wise approach to the synthesis is employed to isolate the active structure. What is Combinatorial Chemistry? Combinatorial Chemistry is the synthesis of a large number of different molecules in a quick and convenient manner. The starting substrate(s) is linked to a solid support material and subsequent chemical modifications to the molecules take place on the solid surface. At the end of the reaction sequence the final products are cleaved from the solid support using an appropriate chemical reaction. The difference between a conventional synthesis and a Combinatorial synthesis is shown in Figure.1.
Figure 1. The advantages of this type of synthesis are that the many different products exist as easily separable "packages" (as opposed to a homogeneous solution) and that purification between stages in the synthesis is minimal. The ease with which large numbers of different products can be separated and re-reacted lends itself to automation of the total synthetic sequence. The Solid Support materials are normally polymers that are
designed to be inert and have a large surface area. These are commonly
in the form of beads (50-100 microns) or crowns (pins) although other
forms are being developed. The earliest form of resin was the Merrifield
Resin (Figure 2), based on polystyrene beads derivatised with chloromethyl
groups and used in peptide synthesis.
Other polymers are used in order to generate differing reaction environments; polystyrene giving a hydrophobic environment whereas a polyamide would generate a hydrophilic environment. Tentagel, which is 80% polyethylene glycol grafted to polystyrene has become increasingly popular since it is believed to generate a more "ether-like" environment which lends itself to a greater range of chemical reactions and hence a greater range of possible structures. The Linker Groups are the chemical groups that link the substrate to the polymer resin. Since it is this group that that must be broken at the end of the chemical sequence, their structure determines the method of cleavage from the resin. It is important that this cleavage reaction does not destroy the new products that the chemist has synthesised. The real importance, however, of these Linker groups is that they determine the terminal functionality of the product ; thus, by using the same Linker group for an entire chemical library all the resultant products will have the same end group functionality. Examples of these Linker groups are Wang Resin Linkers (Figure 3) which can be used in the synthesis of carboxylic acids.
Figure 3.Wang resin linkers Other Linker groups include Rink Amide Linker (Figure 4) for primary amide synthesis and REM (Figure 5), HMB and Oxime Resins (Figure 6) which are for tertiary amine, C-terminally amidated peptide, and alkylamide synthesis respectively.
Figure 6. HMB and Oxime resins There have been several different techniques used in COMBINATORIAL SYNTHESIS to generate large libraries of new structures quickly and efficiently. One such method is described as "Mix and Split" (Figure 7) and was developed by Furka. This is where, for example, three initial materials X,Y, and Z are bound to a resin and mixed together. The resultant mixture is split into three separate reaction vessels and each is coupled with a further set on reagents. Dimer samples from each of the three reaction vessels are saved for subsequent reactions. The remainder of the dimers are mixed, split and put into three separate reaction vessels again. These dimers are reacted again with a further set of reagents resulting in twenty-seven different structures being formed, nine in each of the three vessels.
Figure 7. At this stage each of the three reaction vessels are tested for activity (Figure 8). On the basis that one of the vessels shows activity this limits the active structure to be one of nine.[This description is simplistic because the yield is likely to be less than 100% and impurities and bi-products may be generated tending to confuse the process involved.] In the example given it is clear that the active structure must have Y as its terminal group. At this stage the retained dimer structures are reacted only with reagent Y to give nine final products, three in each of the reaction vessels. A further activity test reveals which of these vessels contains the active structure and each of the three compounds in this vessel can be synthesised independently
Analysis of combinatorial intermediate Product analysis can be carried out by cleaving the product from the resin and carrying out a normal classical chemical analysis by some form of spectroscopy. The chemist, however, would like some means of determining the success of his reactions whilst the substrate is still attached to the resin. This would allow him to proceed with the next stage in the sequence without the inconvenience of the cleavage step. For single products this is normally done using IR or NMR whilst for multi-component mixtures HPLC-MS is becoming the method of choice. The remainder of this note will consider the advantages and disadvantages of FT-IR (and Raman) spectroscopy as an analytical technique and also compare the various IR sampling techniques. It is worth noting at this point that in most cases the synthetic chemist wants to follow a reaction sequence rather than obtain a definitive chemical structure. This means that the monitoring by IR of the appearance or disappearance of specific functional groups is the required objective. This is fortuitous since the resultant IR spectrum from the resin-substrate combination, although dominated by the contribution of the resin, does indicate the presence of the required substrate functionality. It is also worth noting that it is preferable for the analysis to be carried out in the same laboratory as the synthesis and by a non-specialist and therefore the analysis should be as straight forward as possible. Once again it is fortuitous that most synthetic chemists are used to following reaction sequences by following the progress of various functional groups by IR spectroscopy. The sampling techniques studied were:
Although photo-acoustic spectroscopy has been used by other investigators, it has not been considered here since the technique requires some degree of expertise to be fully successful. Results for Bead Samples Figure 9 shows the spectra obtained from a Wang resin bead with substrate
attached. The three spectra were obtained using Diffuse reflectance
(Spectrum 1), Single Reflection Diamond ATR (Spectrum 2), and Reflectance
Microscopy (Spectrum 3).
Although the spectra are dominated by contributions from the polystyrene Wang resin, the important functional group in this synthesis was the carbonyl group whose absorption band is clearly shown at about 1770cm in all three spectra. The same bead sample was analysed by micro-ATR on the IR microscope and Figure 10 compares the normal reflectance spectrum (Spectrum 6) with that using the ATR objective (Spectrum 7).
A similar set of spectra was run for a second substrate
on a Wang Resin. The diffuse reflectance (Spectrum 4), single reflection
diamond ATR (Spectrum 5), and reflectance microscopy (Spectrum 6)
spectra are shown in Figure 11.
Similarly, the beads were run using micro-ATR (Spectrum9) and normal
reflectance (Spectrum 10) on the IR microscope (Figure 12).
In this particular example the detection of the hydroxyl group (absorption band at ~3300cm) is paramount in the structure identification. It is clear from the above data that this it is extremely difficult to detect this band using single reflection ATR methods either on the Diamond ATR accessory or on the microscope. This may be because these techniques sample such a small area of the sample and hence small amount of sample or it could be due to the wavelength dependence of such ATR techniques; absorption bands at higher wavenumbers do appear less intense than lower wavenumber absorptions. In an attempt to eliminate this later effect an ATR correction was carried out on one of the above ATR spectra and the results are shown in Figure 13.
The top spectrum shows the original data and the bottom shows the
result of the ATR correction. Although the hydroxyl absorption band
is slightly more distinct it is still a weak feature and could be
overlooked. Also the fact that such post-run manipulation is required
would tend to compromise the ease-of-use criteria.
Since it is clear that transmission mode spectra do show the required
features without being totally masked by the contribution of the polymer
resin, an alternative to KBr disc making was sought. One such option
is to use a Diamond Anvil Cell to crush a few beads between
the diamond windows and run this directly on the IR microscope (Figure
15).
The quality of this data is very good, the top spectrum showing clearly a strong carbonyl absorption band and the lower showing the hydroxyl grouping. To determine if similar data could be obtained from other types of
beads, the same techniques were used to obtain spectra from a substrate
on a Tentagel bead. Figure 16 shows the micro-ATR (top), single reflection
diamond ATR (middle), and diffuse reflectance spectra from such a
material.
Once again in this example the detection of the hydroxyl group at ~3300cm is important. As with the Wang resin, the diffuse reflectance spectrum shows this absorption clearly whereas in the ATR data this feature is barely detectable. Results for Crowns (Pins) These Crowns come in a variety of shapes and sizes with the most common shape being shown in Figure 17. They are normally about 3 to 4 cm in length. The initial starting material is bound to the surface of the polymer Crown. In use they are suspended from a tray into a reaction vessel into which differing reacting substrates are found in solution. Between each step in the synthesis the tray is raised from the reaction medium and the Crowns washed with an appropriate solvent. The IR analysis of these Crowns does present additional difficulties
over that for the beads. The shape and their structure rules out performing
straight transmission measurements on them and since the surface of
one Crown may contain the total yield of the synthesised material
the chemist is particularly concerned not to destroy or damage the
surface; he will require the Crown for the next stage in the reaction.
These criteria tend to rule out the use of Diamond ATR accessories
which would require that the Crown be compressed or that a part of
the Crown be cut off. The three techniques used for the analysis were
Diffuse Reflectance (using the abrasive rods supplied with the PE
accessory), Reflectance Microscopy and Micro-ATR on the microscope.
The results for a substrate on such a Crown are shown in Figure 18. Spectrum A shows the diffuse result, Spectrum B shows the reflectance result and Spectrum C the micro-ATR measurement. In this example the product contains a carboxylic acid group and whereas both the diffuse and reflectance spectra show the carbonyl and hydroxyl grouping, the ATR measurement shows only very weak features for both.
Raman Spectroscopy One of the main advantages of Raman Spectroscopy is its ease of sample
preparation. This would be an obvious benefit to the combinatorial
chemist wanting data quickly from the beads or crown in a non-destructive
manner such that he can easily move on to the next stage in the synthesis.
The results shown in Figure 19 are the Raman spectra from two different
crowns. The top spectrum is from the crown with the initial starting
material on the surface whereas the lower spectrum shows the result
after two reaction steps; the result of these reactions is that the
new material contains a carbonyl group. Although the spectra are of
very high quality and are very easy to obtain, they are totally dominated
by the contribution from the polymer crown itself and show no bands
to the reactions. Additional Raman data was obtained from other samples
with the same result, namely that the intense Raman spectra from the
polymer support is much stronger than that of the substrate of interest.
The relative usefulness of Raman compared to IR can be clearly illustrated by taking the above sample containing the carbonyl grouping and comparing it’s Raman with it’s IR spectrum (Figure 20). The top spectrum is the Raman data whereas the lower spectrum shows the IR data in absorbance units for ease of comparison. Although it could be argued that the Raman spectrum is a better spectrum, it is the IR one that shows the required carbonyl absorption.
[It should be noted that no attempt was made to find a system 'ideal' for Raman study. The aim was to compare the usefulness of several analytical techniques applied to the same type of sample]. Conclusions The relative ease with which FT-IR spectroscopy can determine the success or failure of a combinatorial synthesis appeals to the synthetic chemist. The fact that IR is a do-it-yourself technique means that the results are available immediately to the chemist. It would appear that the best techniques of analysis are diffuse reflectance using the aluminised pads, KBr discs, transmission through a crushed bead, or reflectance microscopy. For crowns, diffuse reflectance or reflectance microscopy would appear to be the best options. Although ATR sampling techniques result in very good quality spectra, there is a real danger that the relevant absorption bands are too weak to detect easily. Raman spectra seem to suffer in the same way; the absorption bands of interest are very weak compared to the very intense bands due to the supporting polymer.
Norman A. Wright Email: Norman_Wright@bio-rad.com Keywords:
imaging, spectroscopy, chemical analysis, mid-infrared Introduction This paper provides an overview of an infrared spectroscopic imaging system along with some applications. The technique combines the emerging commercially available technology of focalplane array (FPA) detectors with a step-scan Fourier transform (FT) Michelson interferometer based spectrometer. This coupling provides an optimized method for infrared spectroscopic imaging by simultaneously realizing both a multiplex and multichannel advantage. Specifically, the multiple detector elements enable spectra from all pixels to be collected simultaneously, while the interferometer allows all the spectral frequencies to be measured concurrently. This technique can rapidly generate very high quality, chemically specific images from a wide variety of samples. Early systems were built for special purpose applications. Among these were airborne gas analysis [1,2] and astronomical observation [3]. The instrumentation described here is specifically oriented to chemical analysis. Infrared microscopy is a tool that lends itself to the examination of heterogeneous samples. Using microscopy a researcher can more easily investigate a sample for defect analysis, domain identification or boundary information. Mapping techniques have been developed for infrared microscopes to expand the capabilities of and ease of use when studying this spatial nature of samples. However, creating a map becomes a time consuming task if both high spatial resolution and high signal to noise is required. Even with the current level of sophistication available in infrared mapping systems, this fundamental limitation cannot be overcome. A mapping system cannot generate, in a reasonable time frame, sufficient data to reconstruct an image with high spatial content as well as good signal-to-noise. Infrared imaging is the logical replacement of infrared mapping when the need to spatially characterize a sample is required. With array detectors covering suitable spectral regions, this capability is now available. Samples can be viewed either in transmission or reflection and no staining or coating is required. The technique is non-destructive and works well for solids and liquids. The contrast that is seen in these images arises from the inherent infrared absorption of the sample, i.e.; every chemical compound has its own characteristic absorption pattern that creates an infrared signature. This paper describes the instrumental components of the Bio-Rad FTS
Stingray 6000 infrared imaging system configured for microanalysis
(shown in Figure1), and a sampling of applications of this emerging
powerful analytical tool.
Experimental Set Up
A review of focalplane array detectors and their potential for spectroscopic applications is discussed by Colarusso, et. al. [4]. While much of the current effort in developing focalplane technology is being carried out in the area of uncooled arrays, the LN2 cooled arrays are more sensitive and in general, better suited for spectroscopy. The initial laboratory description of this technique (5) used a cooled InSb 128 x 128 pixel array detector. The detector has a spectral response window between 3 and 5 micrometers. This allowed collection of data in the narrow spectral region between 4000 and 2000 cm-1. Because of the natural interest in mid-infrared spectral information, an MCT array detector was located for this application. The detector is a 64x64 pixel MCT array, with a spectral window of 2.3 to 11 microns (ca. 4000 - 900 cm-1), which was developed for the Javelin missile program for the U.S. Army. Repackaged, it represents commercial diversification of military technology. Both the InSb and MCT FPA detectors provide digitized data output. The data is transferred to a host Windows NT based PC that also controls the FT-IR spectrometer. This simplification of the array detector control electronics has created a system rugged and reliable enough to be considered as a commercial product. The spectrometer system described here is a Bio-Rad FTS 6000 Infrared spectrometer with a UMA 500 Infrared Microscope. The focalplane array detector is mounted on the microscope. The MCT array detector with its 64 x 64 element array views a sample size of approximately 7 mm x 7 mm per pixel or a 400 mm x 400 mm overall sample size using the standard Bio-Rad 15x microscope objective. The FTS 6000 spectrometer is operated in step-scan mode for the imaging application. Although the integration time for the FPA is less than a millisecond, due to the time required for readout of the entire array, the use of the step scan mode is necessary. The data from the array detector is transferred directly to memory, via a digital frame grabber card that is installed in the single Windows NT based PC, used to control both the spectrometer and the array detector. The fixed mirror position of the Michelson interferometer is piezo controlled. This allows the interferometer to maintain continuous alignment as well as establish and maintain discrete optical positions thereby allowing data collection from the focalplane array. The spectrometer has a range of step scans speeds, which allows the user to "sit" at a single optical retardation and co-add multiple frames to improve the signal-to-noise of the resulting spectra. The following diagram shows how the data acquisition is synchronized with the steps of the interferometer. At each step of the interferometer, defined by the summed motion of the fixed and moving mirrors, the array detector is triggered to collect image frames, one or more per step. As shown below, multiple frames are collected and co-added at each step.
The acquired data is an array of interferograms that following a Fourier transform generates a spectral hypercube. Additional processing includes library searches for unknown compositions and the ability to perform quantitative analysis after calibration or reference correction. Using this technique we acquire a four dimensional array of data. We have a grid of points over the sample (two spatial dimensions) and at each point on that grid is an infrared spectrum (two additional dimensions -- infrared frequency and intensity). We can think of this ‘spectroscopic data hypercube’ in the following ways. First, the data can be thought of as a stack of images of the sample, each obtained at a different infrared frequency. Any card can be pulled out of the stack to ‘see’ what the sample looks like at one particular frequency. Second, an infrared spectrum exists for every point on the sample, and that spectrum can be used to identify a material, or for quantitation (how much is present). This data is collected in a short time, just a few minutes, and is typically 16-64 MB in size. It can represent over 16,000 spectra, collected at typical analytical spectroscopic resolution, and the individual spectra can be from as small as a 5 mm square area. We obtain an infrared spectrum (a plot of infrared frequency versus energy absorbed) at each pixel, creating a total of 4096 spectra. All the spectra are collected at the same time. The data set can be thought of as a deck of cards. An individual card gives a picture of the sample at one particular infrared frequency (one ‘color’ of infrared radiation). If we drill down through the deck at one particular pixel location, we obtain the infrared spectrum of the sample at that position. Examples Two simple examples of this imaging chemical analysis follow. The
first example is of a nylon mesh sample. The image shown below is
generated from the 3350 cm-1 band (NH stretch). The 3-dimensional
image consists of 4096 resolution elements to create the infrared
image. The red colour shows the area of highest absorption and the
blue area represents no absorption, between the strands. This spectral
region is shown selected in the spectrum shown on the right side of
the figure. Because the sample consists of a single material, an image
at any selected wavelength will result in essentially the same image.
The second example of chemical specific imaging is of a three-layer laminate comprised of an epoxy and a urethane component. The differences between the two materials is shown in the following two figures. The laminate is first imaged using the 1414 cm-1 band. This band highlights the edge layers of the laminate. The last figure (Figure 5) shows an image at 1467 cm-1 which highlights the center layer of the laminate.
Other Applications The areas in which infrared imaging has been applied and reported are varied, including biological systems, polymer systems, material defect analysis, agricultural products, and art restoration investigations. Some specific applications and their references are, silicone leakage from artificial implants in breast tissue[6], primate brain tissue imaging [7,8], biomineralized tissue, distribution in bone sections [9], curing of polymer blends [10], diffusion rates in Liquid crystals [11], and discrimination of co-extruded polymers [12]. Summary Infrared imaging spectroscopic analysis is an emerging technique that provides chemical specific information as images. From these images, precise information about the chemical composition and material structure of a sample is obtained. This type of information is significant as it allows the investigator to understand more fully a chemically diverse sample, which is generally the rule rather than the exception when studying either made-made or naturally occurring materials. Due to the ease of infrared image generation, and the power of image analysis software tools, the ability to study complex systems is now readily available so that the analysis and applications become the focus rather than the experimental technique itself. References
T. Jawhari (a) and A. Pérez-Rodríguez (b) (a) Serveis Científico-Tčcnics,
(b) Dpt. d'Electrňnica, Introduction We all know the importance of microelectronics in our everyday life and the huge advances that occur continuously in small geometry devices and integrated circuits. This is, for example, well reflected by the astonishing fast changes that take place incessantly in the field of computers. The rapid evolution in semiconductor technology, including device fabrication, thin-film deposition, interface preparation and microelectronic processing, have increased the need for characterization techniques that can provide more precise information, especially on the structure of the materials. The quality of this information is one of the most critical factors in designing and manufacturing microelectronic devices since high performance integrated circuits and devices demand an excellent quality material, either in the form of bulk material, thin-film or device structure. Furthermore, in the last few years there has been a new interest in silicon (Si) technology, related to the development of Micro-Electro-Mechanical Systems (MEMS) in which sensor devices and micro-actuators are integrated together with the electronic circuits for signal processing and control. In such systems, the characterization of the electrical, optical, mechanical and thermal properties of the thin films and structures are also critical for the performance of the device. The study of both crystallographic and physical features of the structure is of high technological interest in order to improve the process steps in MEMS technologies. Raman scattering is an extremely powerful contactless tool which allows non destructive and quantitative microanalysis of structural and electrical properties. This technique is very useful since the Raman signal is very sensitive to the microstructural state of the sample and other local environments giving therefore information on the structure of the material on the scale of a few lattice constants. Raman signal is a function of the electron-phonon interaction, i.e. lattice vibration, and is a good complementary method to other techniques used in the characterization of the solid state such as transmission electron microscopy (TEM), X-rays diffraction, X-ray photoelectron spectroscopy (XPS), secondary ion mass spectroscopy (SIMS), Auger electron spectroscopy, etc. Most of these techniques require a sample preparation that often alters the material whereas, in principle, the Raman technique does not affect the sample at all which is a crucial point when characterizing processed devices. Another major advantage of Raman spectroscopy is that when combined to a microscope, the technique can probe smaller dimensional scale devices. This is important for the analysis of microstructures and devices, where the properties of the layers are strongly process dependent and may differ significantly from the bulk material properties. Therefore, the micro-Raman system is now one of the preferred methods when characterizing semiconducting materials. We shall here illustrate the possibilities that offer micro-Raman spectroscopy in semiconducting materials with examples obtained in the Raman laboratory of the Serveis Científico-Tčcnics in collaboration with the Department of Electronics of the University of Barcelona. These studies are mainly centred in the analysis of structures and processes in Si and group IV technologies (SiGe and Si based alloys, SiC...). In principle, Si constitutes the main material for electronic devices and integrated circuits. In this sense, the maturity reached in Si technology has allowed the development of high performance devices, which may include heterostructures with SiGex and, more recently, more complex SiGexCy and SiCy based alloys. This deep knowledge in the Si based technology has also been applied for a wide range of sensor and MEMS devices, constituting the basis of the Si micromachining technologies. Other group IV semiconductor, which now constitutes an emerging technology, is SiC due to its potential applications for high temperature, high power electronics and sensors working in harsh environments. This is related to the electronic and mechanical properties of this compound, such as wide energy band gap (2.4 to 3.2 eV, depending on the polytype), high thermal conductivity, mechanical strength, high breakdown electrical field (i.e., ability of the material to stand high electrical fields), and extreme chemical inertness even at high temperatures. The examples presented here are representative of the wide range of characterisation problems related to semiconductor based materials and structures which can be solved by the micro-Raman probe. Instrumentation The great advances made in Raman microscopy since
its discovery in the mid-seventies were fundamental in the applications
of Raman spectroscopy to semiconductor devices since it provides a
structural and molecular picture with a very high spatial resolution.
The schematic diagram of the Raman instrument Jobin Yvon T64000 used
at the University of Barcelona is shown in Figure 1. This spectrometer
can work either in the micro-mode utilizing an optical microscope
or in macro-mode with a conventional macro-chamber. For semiconducting
materials, excitation source is usually provided by a typical Argon
ion laser which emits discrete lines between 458 and 529 nm, with
its two main lines at 488 nm and 514.5 nm. Sometimes other lines than
the two main ones are used when analysis at different depth penetration
is required. For these materials, changing the laser line from the
green (514 nm) to the dark blue-violet at 458 nm will diminish the
penetration depth. In the case of crystalline silicon, the penetration
depth of the light in the back-scattering configuration has been estimated
to be approximately 300, 600 and 800 nm for laser excitation at 458,
488 and 514 nm, respectively[1]. Higher penetration depth can be achieved
by decreasing the excitation energy, as optical absorption decreases
for energies in the visible-near infrared (NIR) region. For this,
a titanium-sapphire laser can be coupled to the argon ion laser. This
provides radiation in the NIR in a continuous way between 700 and
900 nm. The use of a NIR laser can sometimes be worthwhile, especially
for Raman resonance measurements since many semiconductors have their
electronic gap in the near infrared.
This instrument comprises of a double monochromator
and a spectrograph that disperses the Raman scattered light onto a
CCD detector cooled with liquid nitrogen.
As can be seen in Figure 2, the incident laser beam is reflected by a beam splitter and a flip mirror onto the microscope objective. This focuses light onto the sample. The scattered light is recollected by the same microscope objective (back-scattering configuration), passes through the beam splitter and is focused onto the entrance slit of the spectrometer. The flip mirror situated in the microscope also permits us to visualize the microscopic region under analysis with the TV camera. In the case of materials opaque to the laser, such as most semiconductors, the visualization of the sample in the camera must be achieved with a bright field reflection illumination system. The advantage of using a back-scattering set-up, as shown in Figure 2, is that the laser spot on the sample can be directly observed on the TV monitor which allows precise identification of the microscopic region of the sample analyzed. When maximum lateral and axial resolution is required,
a spatial filter, which consists of a pinhole, is placed behind the
microscope at the image plane. This rejects scattered light originating
from outside of the laser focal volume.
As already mentioned, micro-Raman spectroscopy allows
analyses of many kinds of materials with no sample preparation. Solids
are easily studied by placing them on a standard glass microscope
slide. However, with a Raman instrument coupled to an optical microscope,
it is sometimes necessary to take some precautions due to the high
power density, typically between 104-105 W cm-2,
as a result of tight focusing of the laser beam that is obtained with
the microprobe. This may cause those samples with high optical absorption
in the spectral region close to the laser frequency to be physically
affected by local heating. Sometimes, a small black patch of the size
of the focused laser spot appears due to local annealing. This local
heating can also affect the shape and position of the Raman bands.
For example, in Figure 3 we can see that the bandwidth of the Si Raman
band at 520 cm-1 increases as the laser power augments.
This phenomenon is not due to local degradation of the crystal, but
only related to the local heating of the crystalline network which
causes anharmonic effects in the spectra. Accordingly, this effect
is totally reversible and is determined by the temperature gradient
achieved in the scattering volume. For highly damaged or amorphous
semiconductors, absorption of light increases and more intense thermal
effects are observed. Other laser power effects such as wavenumber
shift were sometimes observed. This is illustrated in Figure 4 which
shows how the low frequency mode of CuS increases as the laser power
is increased from 1.2 mW to 5.0 mW. This shift effect was also observed
to be entirely reversible. These series of measurements were carried
out in Cu-rich CuInS2 layers of a solar cell device, where
CuS was found to appear as a secondary phase. All these results clearly
imply the need to carefully test the possible presence of thermal
effects in the spectra when using a micro-Raman probe. Typically,
for a X100 microscope objective, the laser power at sample should
be preferentially between 1 and 2 mW whereas with a X50 objective
the laser power can be as high as
Spatial resolution
The lateral spatial resolution has been checked in
the conditions of routine micro-Raman experiment by recording successive
Raman spectra at different positions across a polyethylene (PE)-silver
layer interface. The experiment was achieved by preparing a neat interface
through vacuum-deposition of an extremely thin silver layer on a flat
polyethylene surface and using a microscope xy translation stage controlled
by a computer to probe the Raman signal across the interface (see
Figure. 5). The intensity of the Raman band of polyethylene at 2884
cm-1 was measured for each spectrum recorded and plotted
in function of the analyzed position as shown in Figure 6. The experiment
was first carried out without any diaphragm and then repeated with
a
As already indicated, most semiconductors have a high
optical absorption in the spectral range of the laser frequency. In
this case, the axial resolution of the micro-Raman spectrometer is
higher than the penetration depth of light, therefore the depth of
the scattering volume is determined by the optical absorption. However,
this is not the case for wide energy band gap semiconductors such
as SiC or GaN. These semiconductors have a low optical absorption
in the visible region, and are now receiving special interest in the
field of high temperature applications and optoelectronic devices
in the blue part of the spectrum. For these semiconductors, the minimum
depth of the scattering volume of collected photons can be estimated
from the theoretical nominal depth of field of the microscope objective,
given by Applications First of all, we shall examine the different parameters that can be obtained from the Raman spectrum of solid state materials and analyze the type of information on their structure and physical properties they can offer. a) Frequency On the other hand, the presence of crystalline disorder also gives rise to changes in the frequency of the band, usually towards lower wavenumbers. These are related to the breaking of translational symmetry in the crystal, which can be produced by structural defects such as dislocations or by grain boundaries in nanocrystalline materials. The position and shape of the Raman band can be simulated with a correlation length model[3] which allows to estimate the value of the correlation length L from the analysis of the Raman band. This correlation length is defined as the characteristic size of crystalline domains in the scattering volume where the translational symmetry of the crystal holds, and is related to the average distance between defects for damaged crystals or to the grain size for nanocrystalline materials. Other parameters affecting the frequency of the Raman band are the temperature which affects the contribution of anharmonic effects and the presence of chemical impurities in the crystalline network which leads to changes in the mass of the atoms in the lattice sites. For example, the presence of Ge atoms at substitutional positions in the Si network produces a decrease in the frequency of the vibrational modes, due to a higher mass. This is known as the chemical effect. Besides, the presence of impurities at substitutional positions also affects the lattice spacing due to the different size of the impurity. Hence, a stress effect will also occurs in addition of the chemical one. b) Raman bandwidth |
/N.A.). For instance, a typical x100
microscope objective used in the Raman microprobe experiment with
a N.A. of 0.95 and 
