Principles of Raman Spectroscopy (5) Raman spectroscopy FAQ

October 28, 2019

Equipment related

Q1. I heard that a Raman spectrophotometer is difficult to operate. Can I use it?

One of the most time-consuming aspects of Raman microscopy is identifying the measurement location in the sample. This can be done quickly for samples that require distribution analysis by using 2D or 3D matrices, with measurements made at fixed intervals. However, for randomly arranged samples the user has to select each position by searching the microscopic image and individually selecting the points of interest. The Sample Search function in Spectra ManagerTM can be used for particulate or powder samples to automatically analyze the microscopic image and identify the position based on size, contrast and/or color of the target defined by the user. Simply click the start button to execute spectral acquisition and the stage is automatically positioned for optimized spectral measurement of all identified sample points. Measurement points can be recognized in real time, allowing simultaneous spectral acquisition and qualitative analysis. This function is recommended for foreign materials contaminating Si wafers or polymer films, powder components in pharmaceutical samples and micro-plastics.

Fig. 17 Automated sample search for particles and powder samples

Q2. What is Class 1 of the Laser Safety Standards? What are the precautions for handling?

In order to ensure safe use of lasers, they are classified according to specific standards specified in the “Safety standards for laser products”. Class 1 corresponds to a laser power of approximately 0.39 mW or less, and it is required to “prevent continuous viewing of the beam”. JASCO employs an automatic opening and closing mechanism for the sample chamber and an interlock mechanism so that the beam cannot be directly viewed, so complying with Class 1. Being able to close and measure the sample chamber prevents stray fluorescence light and helps detect weak Raman light. In addition, sample room lighting makes it easy to set up samples.

Fig. 18 Automatic opening and closing of the Raman spectrophotometer

Q3. How long does it take to measure a Raman spectrum?

If the sample produces a sufficient amount of Raman scattering, it can be set and measured within 1 minute. After turning on the power to the spectrometer, set the sample on the stage, focus while watching the sample image, and close the sample chamber door. Focus the sample so that the laser spot is small, set the measurement conditions while checking the spectrum preview, and start the measurement.

Q4. Does Raman spectroscopy destroy the sample?

Basically it is nondestructive. However, if you keep the high power laser on a small point, you may damage the sample. In general, it is recommended to use a attenuator to reduce the amount of laser irradiation to the sample and extend the exposure time.

Q5. What is the spatial resolution of Raman measurements?

The spatial resolution depends on the laser, objective lens, and the confocal aperture size. The beam spot size at 532 nm laser is as follows:

x100 objective lens: approx. 1 μm

x 50 objective lens: approx. 2 μm

x 20 objective lens: approx. 5 μm

Further details are described in “spatial resolution” in Section 2.

Q6. What does confocal optics refer to?

Laser Raman microscopes use confocal optics. A pinhole is placed at a position conjugate to the focal point of the objective, lens and light from other regions is cut off. This makes it possible to extract the signal at the focal position of the laser beam spot, enabling measurement in the depth direction.

Q7. I heard that no pre-treatment is required. Can I measure embedded samples?

The laser Raman microscope uses confocal optics and can change the focus position in the depth direction, as explained in Q6. Therefore, if you can focus on an embedded sample and perform measurements on it. Additionally, the JASCO Raman system uses dual spatial filtration (DSF) and has a high resolution of 1 μm in the depth direction.

Q8. To what depth can measurements be performed?

This depends on the working distance for the objective lens, which is shown in the table below for different lenses. Measurements can be performed at a depth of up to 10.6 mm with a 50× long objective lens.

Table 2 working distances for objective lenses

Q9. What is the benefit of being able to mount multiple lasers?

There are three purposes to changing the type of laser:

1. Reducing damage to the sample.

2. Avoiding fluorescence. Fluorescence emitted by a 532 nm laser can be eliminated by using the 785 nm laser (Fig. 19).

3. Increasing the sensitivity using the resonance Raman effect. Figure 20 shows Raman spectra of the yolk of a boiled egg. Peaks due to amide III, which are difficult to detect at 532 nm and 633 nm, are clearly seen using the resonance Raman effect.

Fig. 19 Raman spectrum of pigment (red), avoiding fluorescence by use of 785 nm laser

Fig. 20 Raman spectrum of yolk of boiled egg

Q10. What if I need a non-standard grating?

This may be the case if you are using a standard 532 nm laser, or if you need to perform high-resolution measurements. In JASCO spectrophotometers, as shown in the figure, up to four gratings can be mounted, and it is possible to easily switch between them (Fig. 21). In Fig. 22, it can be seen that a 2400 gr/mm grating provides superior peak separation and higher wavenumber resolution when measuring a taurine spectrum.

Fig. 21 Switching diffraction gratings

Fig. 22 Taurine spectra obtained using different gratings

Q11. Why are you using 532 nm as the standard laser?

JASCO uses a 532 nm green laser as the standard in consideration of sample damage, ease of use (such as visibility on the sample), laser quality, ease of alignment and durability.

Q12. What kind of laser can be used other than 532 nm?

The wavelengths that are available are 244, 257, 325, 441, 488, 514.5, 632.8, 785 and 1064 nm. Wavelengths of 244 and 257 nm can be used for photometric measurement of resonance Raman and wide gap semiconductors (GaN and SiC). Wavelengths of 785 and 1064 nm can be used for measurement of fluorescent samples or biological samples.

Q13. How can I perform measurements using a 1064 nm laser?

An InGaAs array detector is used to detect Raman scattered light from the 1064 nm laser. The JASCO NRS-5000 / 7000 series can be used as a second detector. Automatic switching between lasers, gratings and detectors makes it easy to switch between 532 nm and 1064 nm measurements.

Q14. What kind of data can be acquired with 1064 nm laser?

If you use a 1064 nm laser for a sample that emits fluorescence with a 532, 633 or 785 nm laser, you may obtain better data by eliminating the fluorescence. Figure 23 show spectra obtained from red paint at different laser wavelengths. The baseline is low only when using a 1064 nm laser, and a clear Raman peak is observed at 1000 to 1500 cm-1.

Fig. 23 Measurement of red paint using a 1064 nm laser

Q15. What is the procedure for switching lasers?

The setup for the laser, diffraction grating, and objective lens is automatically performed using simple software commands.

Q16. What is the maximum size of the sample can be placed in the sample chamber?

Depending on the drive range of the automatic stage, the maximum dimensions are 75 mm (L) x 50 mm (W) x 30 mm (H). By removing the stage, the sample height can be increased to 80 mm (Fig. 24).

Fig. 24 Sample stage in microscopic Raman spectrophotometer

Q17. Can I heat or cool the sample?

Heating and cooling measurements can be performed using a thermo-control stage. Figure 25 shows a trehalose measurement at a constant temperature of 80 °C at one minute intervals. The mode below 500 cm-1 assigned to lattice vibrations changes over time, indicating a crystal phase transition. Detachment of bound water from the amorphous hydrated compound causes recrystallization to occur.

Fig. 25 Time-lapse change of Raman spectrum of trehalose

Q18. Can I carry the instrument to a place other than the laboratory and perform measurements?

It is possible. JASCO also offers a portable Raman spectrophotometer (RMP-500 series) . Fig. 26 shows an example of performing a nondestructive analysis of the composition of a fresco in a church.

Fig. 26 Analysis of church fresco produced in the 16th century

Sample related

Q19. Can I measure a sample in a container?

Liquid samples are usually measured in a sample bottle or glass capillary. If the container is transparent to the laser wavelength, the sample can be measured, and samples in plastic bags and containers can be measured. However, since fluorescence and Raman scattering from the container may be observed at the same time, it is necessary to perform the measurements using a low light intensity. You can also use the macro measurement unit to measure liquid samples in a vial or square cell (Fig. 27).

Fig. 27 Measurement of liquid sample in vial

Q20. What concentration is required for solution and powder samples?

In the case of a general solution, the standard detectable concentration is 1%. However, it depends on the sample. If there is a component that exhibits resonance Raman scattering or a component with high intensity, detection may be possible at a concentration of 0.1% or less.

Q21. Is it possible to measure a large sample that cannot fit into the sample chamber with a Raman spectrophotometer?

By connecting a fiber probe to a Raman spectrophotometer, you can measure large samples that do not fit into the sample chamber. A variety of applications are possible, such as measuring the reagents in equipment, chemical bottles, and flasks.

Fig. 28 Measurement of reagent (bottom) in a medicine bottle (upper left) and reaction vessel (upper right)

Q22. How can mapping measurement be applied?

Mapping measurements can be performed on samples such as medicine tablets, electrode surfaces, thin films, and crystals. As an example, Fig. 28 shows the results of measuring the distribution of medicinal ingredients in a tablet. The automatic stage imaging (QRI) installed in the NRS-5000 / 7000 series enables a wide range of measurements (such as measurement of the distribution of pharmaceutically active ingredients in analgesics) in a short time.

Fig. 29 Raman mapping measurement of tablet

Q23. What is the maximum film thickness that can be measured?

In the case of a general thin film, the standard thickness is 1 μm. However, it depends on the sample. In the case of graphite or a DLC film with a high Raman scattering intensity, detection is possible even at around 10 nm.

Q24. Can I measure foreign particles in a film?

If foreign matter can be observed with an optical microscope, it can be measured. The more foreign material is inside, the harder it is because it reduces the laser light intensity and that of the scattered light. Also, if the surface is rough and not clear like frosted glass, or if it is not uniform, it will be difficult to focus the laser in the sample.

The upper part of Fig. 30 shows an example of foreign matter buried in a multilayer substrate consisting of glass, adhesive, and a transparent film. Such foreign matter is difficult to analyze in cross-section due to the presence of glass, and the adhesive layer is also removed when removing the foreign matter, which makes it difficult to measure using micro-FTIR. On the other hand, in Raman measurements, the use of confocal optics enables selective acquisition of the spectrum at the position where the laser is focused. As a result, nondestructive and noncontact measurement of the inside of the sample can be easily performed without complicated pretreatment. Here, the location of foreign matter was measured in the depth direction (Z-axis direction), and information on each layer was also acquired. The representative spectra (lower figure) obtained in each layer are shown.

Fig. 30 Foreign matter in transparent film (top) and Raman spectrum (bottom)

Q25. I often hear the words G band and D band in Raman literature. What are they?

The G (Graphite) band (1580 cm-1) corresponds to an in-plane vibration of the graphite structure. The D (Disorder) band (1350 cm-1) is a band caused by defects in the graphite structure. The ratio of the G band to D band intensities is used to evaluate the crystallinity of carbon materials.

Fig. 31 Raman spectrum of carbon materials

Q26. What kind of samples easily emit fluorescence?

Typical materials that easily emit fluorescence include colored materials such as polyimide and epoxy resin. Many mixtures are often more likely to contain fluorescent samples. When fluorescence is emitted from a black sample, it is susceptible to thermal damage even by near infrared excitation, and it is difficult to measure even if the laser is changed. Generally, fluorescence is easily emitted from colored samples. If the sample is transparent, the influence of fluorescence is often small. However, even with a colored sample, the peak may be enhanced by the resonance Raman effect and it may be possible to measure it.

Measurement related

Q27. Does high speed imaging have any merit other than shortening the measurement time?

By scanning the stage in the X, Y, and Z directions using the height information of the omnifocal image, it is possible to accurately image even samples such as tablet imprints on a surface.

Q28. When measuring the cross section of the tablet, there is unevenness and I can not measure well. What should I do now?

Make the cross section as smooth as possible and use a low magnification or long working distance lens. In addition, by using surface scan (SSI) as the measurement method, the stage can be scanned in the XYZ directions, and samples with large surface irregularities (tablet imprinted parts) can be measured.

Fig. 32 Measurement of engraved part of tablet

Q29. When the measurement time is the same, should the exposure time or number of accumulations be increased?

Basically, if you set a longer exposure time (by setting the number of accumulations to 2 for cosmic ray removal), you can measure the SN ratio well.

A Raman spectrophotometer uses a charge storage type CCD detector as a detector. The shape of the spectrum is first understood by converting the signal accumulated in the CCD for a certain period of time into charge, which is displayed on the preview screen (usually 1 second). There is a slight noise when converting this accumulated Raman scattering signal to charge, so if the measurement time is the same, increasing the exposure time will increase the signal-to-noise ratio.

However, if the CCD detector becomes saturated when the exposure time is extended, increase the number of accumulations and increase the SN ratio.

Q30. What is the merit of low wavenumber measurements?

In the low wavenumber region, it is possible to observe skeletal vibrations such as in monosubstituted benzene, lattice vibrations (vibrations of atoms in the crystal) and vibration modes between heavy atoms. In particular , with a low wavenumber of 500 cm-1, it is possible to determine, for example, differences in crystal structure between calcium carbonate and titanium oxide, and between quartz and fused silica.

In Fig. 33, it can be seen that quartz (crystalline) has a sharp peak and fused silica (amorphous) has a broad peak.

Q31. With the 532 nm laser, is there a technique to reduce the influence of fluorescence?

The following three methods can be considered.

1. Use a high-magnification lens, and reduce the slit and aperture width to increase the spatial resolution.

2. Use the automatic fluorescence correction software function.

3. Apply a photobleaching technique for fluorescence fading before Raman measurement.

Fig. 34 Increased spacial resolution(top), software correction (middle), and photobleaching (bottom).

Q32. Is there any way to measure polarization with Raman? What information can we get?

There are two methods. One is to rotate the sample, and the other is to rotate the polarization plane of the laser by 90 degrees using a 1/2λ plate. The following information can be obtained;

1. Orientation of molecules.

2. Depolarization of a randomly oriented system such as a liquid sample or a gas. Information on the symmetry of vibrations can be obtained and used as a reference for peak assignment.

3. Analysis of lattice vibration in crystals. The results can be used to evaluate the crystal orientation or plane direction.

Fig. 35 Orientation analysis of polyiodine ions in polarizing film

Q33. I would like to measure carbon powder. How should I install the sample?

The powder can be measured by dispersing it on a glass slide.

Q34. Can a quantitative analysis be performed using Raman spectroscopy?

It is possible to perform a semi-quantitative using Raman peaks. However, in the case of non-uniform samples such as emulsions, it is difficult to obtain accurate results.

Data processing related

Q35. What is the maximum sample area for high-speed imaging?

QRI is an imaging method based on stage scanning. The number of points depends on the measurement area of ​​the sample. For example, a 10 mm tablet can be measured at 785 nm excitation and about 32000 points in 16 minutes.

Fig. 36 High-speed imaging using QRI

Q36. What kind of data processing is available with Raman spectroscopy?

Automatic fluorescence correction, sensitivity correction, and digital filters can be used.

An example of automatic fluorescence correction is shown in Fig. 37. It can be seen that the uncorrected baseline is high due to the influence of fluorescence, and it is difficult to clearly distinguish peaks.

The use of a digital filter can eliminate the influence of cosmic rays and noise, as shown in the lower spectrum.

Fig. 37 Raman spectrum before and after automatic fluorescence correction

Q37. Can I obtain complementary FTIR and Raman measurement data?

By using the sharing holder and performing FTIR and Raman measurements, you can easily acquire data for the same sample at the same position.

Figure 38 and 39 shows imaging and spectral data for a multilayer film using IR and Raman measurements. In the IR spectrum, peaks due to cellulose are clearly observed. In the Raman spectrum, a peak due to TiO2 is identified, and the two components can be used to perform a more detailed analysis .

Fig. 38 Measurement of multilayer film using sharing holder

Fig. 39 Measurement of multilayer film by IR and Raman spectroscopy