The following details are important and supplied by the manufacturer. A practical step-wise procedure has been written.
Getting Started The Advantage 200A requires two connections, a power supply and a link to the computer. First, plug the 12 volt DC power supply into the back of the instrument and into a wall receptacle. When initially plugged in, the instrument performs a self-check. Next, insert the telephone-type jack located at one end of the serial port connector into the back of the instrument. The opposite end of the connector, the standard 9-pin serial adaptor, plugs into the computers serial port.The system is now ready to take Raman spectra.
The software necessary to take Raman spectra is opened by clicking on the DeltaNu icon located on the Windows Desktop. As soon as the NuSpec data acquisition program initializes, a red indicator in the upper right hand corner will be visible. The indicator remains red until the system reaches the proper operating temperature, at which point the indicator turns blue. This process takes approximately 4 to 5 minutes.
Place a sample in the sample holder; the system is shipped with a vial of polymethylmethacrylate as a standard. Make sure the integration time is set to 1 second. Press the Reference button. A green indicator in the upper left hand corner signifies that a spectrum is being acquired. Next press the Acquire button and again note the green indicator during the actual acquisition. After 1 second a Raman spectrum of polymethylmethacrylate will be seen on the monitor.
Taking a Raman Spectrum The Advantage 200A is specifically designed for ease of use. Spectra of liquids or solids in standard sample vials are obtained by placing the vial in the sample holder. It is recommended that the sample vial contain approximately ½ “ sample material to ensure detection by the laser beam in the first few weeks of use. As the instrument becomes more familiar to the user, it is possible to use very small amounts of sample material. Simply adjust the sample vial vertically within the sample holder to ensure alignment of the sample and the laser beam. Once the sample is in place, ensure that the integration time is on 1 second and press the Continuous button. The system will acquire a reference spectrum (with a shutter blocking the laser beam) and then continually acquire spectra (with the reference subtracted out) every second. This real-time mode allows the user to observe changes in the spectrum while manipulating the sample holder.
Focus for Laser Now that the system is displaying real-time spectra, properly focus the laser beam onto the sample material by adjusting the silver knob on the front of the sample holder. For liquids the focus is not critical, and there is a large range over which the adjustment makes very little change in the spectrum. When the sample is far away from the instrument, there is a decrease in signal and perhaps an increase in background as the glass sample vial itself moves into focus. Similarly, if the sample is too close, the sample vial will dominate the spectrum. Once the laser is focused for optimal signal, the user should not have to make adjustments. However, the distance is dependent on the index of refraction of the sample and may differ slightly with different samples. Solids are optimized the same way. If the solid is opaque, the optimization is more critical than with liquids. The ideal distance will be that at which the laser is focused just on the sample material, and not the glass vial. The Advantage 200A has an interlock that shuts the laser off if the sample holder is removed. If the laser turns off while focusing because the interlock has been tripped, focus back in until the laser turns on again.
Cyclohexane or toluene provides an excellent liquid sample to practice adjusting the sample holder. Acetaminophen (Tylenol) or benzoic acid are good solid materials to use for practice in focusing. Once the intensity is optimized and the background noise is minimized, press OFF next to the Continuous button in order to stop the real-time acquisition. Now, adjust the integration time to the desired time and press Reference followed by Acquire. The integration time required to obtain a spectrum depends on the sample and the application. A new reference should be taken every time the integration time is changed. Reference spectra need not be taken before each acquisition.
Detector Saturation The Advantage 200A detector begins to saturate around a signal greater than 70,000 counts per second. To obtain the best signal to noise ratio, keep the largest peak in the spectrum close to, but under, 70,000 counts per second.
Improving Raman Spectra Obtaining a high quality Raman spectrum requires following a few simple rules and a little practice. Initially, practice with a strong Raman scatterer such as toluene (shown at the right) or cyclohexane. As optimizing the sample distance and choosing the correct integration time becomes clearer, move on to more challenging samples. The spectra at the right is for toluene.
Water has a weak Raman feature around 1650 cm-1. The simplest way to improve spectra is to increase the integration time. The noise level will decrease by 1/(integration time)1/2 while the signal increases linearly with the integration time. With clean samples and good Raman scatterers, this is the best way to improve spectra.
Challenges come when the sample has an inherent background due to fluorescence. In this case, background will also increase with the integration time, and smaller improvements are gained by integrating longer. The Advantage 200A shows no improvement by integrating longer than 60 seconds. (Delta Nu software does not allow integration longer than 59 seconds.) Furthermore, if the sample is fluorescing, it may saturate the detector long before the 60-second integration period. The best way to improve a spectrum in this case is to average over several spectra. The Average button can be found under the Acquisition menu in the top left hand corner. When selected, the feature will first ask how many spectra are to be used in the final averaged spectrum. The resulting acquisition will require n x integration time (seconds), where n is the number of averages selected. The more spectra averaged, the better the signal to noise ratio. An Abort button appears when Average is selected to allow the user to stop the averaging process.
NuSpec offers a very unique feature that can also help with difficult samples. This is the Baseline feature. An automatic baseline correction can be selected using the Baseline menu found on the top menu bar. This feature performs a real-time baseline correction to remove spurious features such as fluorescence from the spectrum. If the sample is a poor Raman scatterer or has a fluorescence background, we suggest the following procedure. Select baseline ON and go into the continuous mode. Carefully adjust the sample position. In many cases, it is possible to find a position that maximizes the Raman signal, while minimizing fluorescence. The baseline ON is very useful as it often allows for observation of a Raman feature that would normally be hidden in a more spurious baseline. Once the position has been optimized, select an integration time that produces a good signal-to-noise ratio and, if needed, average over several spectra.
The Resolution feature on the top menu also can be used to improve spectra. As a rule of thumb, in any dispersive spectroscopy (spectroscopy where a grating or prism spreads the spectrum across the exit plane of the spectrograph) the intensity is inversely proportional to the resolution. The Advantage 200A allows the user to select three resolution levels to improve signal-to-noise. At low resolution the signal-to-noise is optimal, but the spectral resolution is lower. This can be readily observed with a sample such as toluene. At the low-resolution setting the two large peaks around 1000 cm-1 are not resolved. However, note the excellent signal-to-nose ratio even at one second. As the resolution is improved, the two peaks are resolved at the cost of adding noise to the background. As a rule of thumb, many samples do not require high resolution, and the best spectra are obtained in the low-resolution mode.
Special Features The Advantage 200A software contains special features that enhance the performance of the instrument. Timed spectra: A particularly useful form of data acquisition is the timed spectral acquisition. This is used to collect spectra over a period of time at known delays. For example, if a sample is undergoing a chemical reaction over time, the kinetics can be found from the Raman spectra as a function of time. The timed spectra feature is located in the acquisition menu and is called the Multiacquire feature.
When selected, the feature prompts the user to name the files to be acquired. The files will automatically be sequentially numbered corresponding to their order during the acquisition. After the filename has been selected, the user must select the delay time and the number of acquisitions. The total time per spectrum is the sum of the integration period + delay. The spectra are stored in the ASCII XY format and can be loaded into Excel or GRAMS for analysis.
Polarization: Raman spectra contain special information in the polarization of the scattered light. Bands that are highly polarized pertain to totally symmetric modes. This can be used to aid in the characterization of Raman features in a spectrum. The Advantage 200A system is able to record spectra of both the perpendicular and the parallel planes of polarized light and display a spectrum consisting of a subtraction of the two polarizations. This can be performed by pressing the Polarization button.
Resolution: Three possible resolution settings are available on the Advantage 200A. The benefit of the low-resolution setting is improved signal to noise. High resolution allows one to see spectral features that would not be separated at lower resolution. Range: The Advantage 200A collects data over the range 200 to 3400 cm-1. This covers most Raman spectral features. At the low end of the spectrum the intensity is attenuated due to filtering within the system. The user can select narrower ranges in order to enhance the appearance of the spectra. Saving data: Our software offers two methods of data storage. The more ubiquitous method is the ASCII XY file. This contains a two-dimensional array of frequency vs. intensity data. The second method is the .spc format. This format is used by GRAMS from Thermo-Galactic Industries. We provide a select directory feature to allow users to place data in a directory of their choice. Both the ASCII XY and .spc formats contain a memo line for later identification of the sample and experimental parameters.
Plotting data: Many plotting programs will accept ASCII XY data. For example, Excel will accept this data and has plotting features to produce a high-quality spectral plot. The .spc format is accepted by GRAMS, and within the GRAMS package one can manipulate data and plot the spectrum.
Background: There are many sources describing the theory and background of Raman Spectroscopy. The following may be useful:
1. A background from Delta Nu
2. More theoretical background
3. Virtual Raman Demonstration
4. Instrument manufacture tutorial
5. Encyclopedic knowledge and the Nobel prize in physics for Raman in 1930
6. Data base of Raman spectra for minerals
7. What is Raman Scattering?
