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Raman

Raman Spectroscopy Ocean Optics

 

Raman spectroscopy is like panning for gold. A wealth of information is there, if you can just sift through the rock, dirt, and sand obscuring it. The art to finding the gold in Raman spectra is the instrumentation, which must collect as many photons as possible while rejecting scattered laser light efficiently.

 

Raman spectroscopy examines materials not through direct absorption, but by scattering of high intensity light in the hopes that one in a million photons scattered will commune with the vibrational and rotational states of a sample molecule and emit light of a slightly different wavelength.

 

 

Though Raman spectra are very low in intensity and coded in the most mysterious of energy units (wavenumbers, or cm-1), they probe molecular structure as effectively as IR spectroscopy, but with greater ease of use, more versatility, and lower cost. Raman spectral signatures can play a role in fundamental research, or be matched to a known database for instant identification and quantification of materials.

 

It’s amazing that C.V. Raman ever discovered this effect, but we’re glad he did. His belief that “success can come to you by courageous devotion to the task lying in front of you” is essential in every new project you undertake, and in every product we develop to make it happen. So come on… let’s dive in together and find the gold.

 

Advantages

  • Chemical identification: signature can be matched to a known library; no interference from water, so samples can be aqueous or have high moisture content; distinguishes between isomers
  • Ease of use: no sample preparation required, rapid, non-contact, non-destructive, no hazardous byproducts
  • Versatile: applicable to solids, liquids, or powders in bags or vials by altering depth of focus; can test samples in the microliter range or large objects

Applications

Other Common Applications

  • Pharmaceutical QA/QC: incoming raw materials, polymorph analysis, final product testing, through packaging analysis
  • Forensics: currency, art and archeology authentication, counterfeit detection, identification of chemicals, fibers, hairs & inks
  • Law enforcement/ Homeland security: pre-cursor andexplosives identification, illegal drug identification
  • Process control: contaminant analysis, reaction monitoring, product validation, purity analysis
  • Fuel analysis: octane content and density, counterfeit fuel detection, biofuel production quality & process monitoring, identification and characterization
  • Life sciences: clinical diagnostics, tissue biopsy, photosensitive biological samples
  • Materials science: plastic and polymer characterization, study of graphene and carbon nanostructures like fullerenes, organic and inorganic chemistry research, composition of semiconductors

Application Note:

Application Blog Post: Ensuring Food Safety Using SERS

 

Technical Note

What Is Raman Spectroscopy and How Does It Work?  

When light is incident on a sample, it can be absorbed, transmitted, or scattered. Almost all of the scattered light is elastically scattered. That is, the photons change direction but keep the same energy and momentum. This process is also called Rayleigh scattering. But something different happens to about one in every million photons. These photons are inelastically scattered by the molecule, resulting in a photon that is redirected at a slightly different energy and therefore wavelength.

What happens to this inelastically scattered photon? There are different ways to describe it. One is that the photon is simultaneously absorbed and re-emitted in a single quantum event, with an energy difference that is equivalent to the difference between two vibrational modes of the molecule. Others say that the light scatters off a virtual state that is temporarily created to facilitate absorption and re-emission. For all intents and purposes, both processes can be considered to be instantaneous. However it is described, this process of energy exchange between scattering molecules and the incident light is known as the Raman effect.

A good way to visualize the Raman effect is to imagine a ball bearing being dropped onto a drum. The drum starts to vibrate at its own frequency, and the ball bearing bounces off with slightly less energy (analogous to Stokes radiation). If the drum is already vibrating and the ball bearing hits at just the right time, the drum acts like a catapult to give energy to the ball bearing and it bounces off with even more energy (analogous to anti-Stokes radiation). The energy difference before and after the ball bearing strikes the drum provides information about the vibrational mode of the drum. [Fundamentals of Molecular Spectroscopy, Banwell and McCash, John Wiley & Sons, Inc., New York, 1988]

Raman Effect

Rayleigh Energy

Now that we know what’s happening with individual molecules and photons, let’s take a step backward to view the big picture. Raman scattering is very weak, so to collect enough photons to make a meaningful measurement, a very strong light source must be used. Lasers serve this purpose well, as they are both intense and monochromatic.

The sample is literally bombarded by so many photons at once that even with a one-in-a-million success rate, Raman scattering occurs often enough to be detected. Since molecules have many different vibrational states, there can be as many as a hundred different Raman scatter wavelengths, all resulting from the same laser excitation wavelength.

The frequency difference or shift between each peak in a Raman spectrum and the laser excitation corresponds to the vibrational frequency of a specific molecular bond, and therefore gives us a clue to the molecule’s structure. These transitions could be probed more easily via direct absorption, but to do so would require mid-infrared light, which is highly susceptible to interference from any water in the sample.

Thanks to the availability of low-cost, portable lasers and relatively sensitive CCD array-based spectrometers, Raman spectroscopy can easily and economically be performed in the lab or in the field. The strength of the Raman signal is directly proportional to the average laser power, so the greatest amount of Raman signal will be generated at the laser’s focus. This can be used to good advantage, and allows a sample inside a clear plastic bag or vial to be probed without opening the packaging. In pharmaceutical samples it allows the system to “look” below the external coating of a tablet to study the medicine within.

Raman spectra consist of distinct peaks based on the chemical formula and structure of the compound, the functional groups attached, and skeletal vibrations and modes. It is ideal for identifying closely related chemical samples, and including isomers and polymorphs. This spectral fingerprint can be compared statistically to a library of known compounds for positive identification using chemometric software. It can also be used for quantitative determinations of mixtures, from 90 – 100% concentrations down to ppt levels (ppb with SERS detection).

Compared to IR or FTIR, Raman spectra typically have fewer and narrower peaks, varying by up to 1000x in intensity within the spectrum, which makes it easier to resolve the compounds in a mixture. IR spectra are easily swamped by water absorption lines, but Raman spectra are not. Water peaks still occur in Raman spectra, but are similar in intensity those of other components in the sample. Raman spectroscopy can thus be used to capture data on aqueous samples or samples with high moisture content, making it particularly useful for biological, pharmaceutical and homeland security applications.

For these reasons, Raman spectroscopy has become the preferred technique for chemical identification. It enables reliable, non-destructive chemical analysis of aqueous solutions, powders, tablets, gels, and surfaces, making it extremely versatile and applicable to a wide range of fields.

Technical Note

What Is the Difference Between Modular and Turnkey Systems?

Modular systems can be lower in total cost, and offer more flexibility for reconfiguration. The excitation laser, fiber optic Raman probe, spectrometer and software are configured by the user, freeing each component for use in other applications.

Turnkey systems are convenient, and are often optimized for a specific application or sample type. Use of free-space optics instead of fiber optic Raman probes improves sensitivity, and enables unique sampling modes like raster orbital scanning (ROS). Some turnkey systems may come with special software for chemometric analysis and libraries customized to the target application.

Turnkey systems for Raman spectroscopy include:

  • Compact handheld units
  • Portable/benchtop systems
  • Raman microscopes

 

Featured Products for Raman:

Raman - Biofuels Analysis

 

QEPRO-RAMAN QE series spectrometer preconfigured for 785 nm Raman analysis; modular options for 532 nm and other wavelengths are also available
LASER-785-LAB-ADJ 785 nm diode laser for Raman excitation; 532 nm and other options are available
RIP-RPB-785-SMA-FC Raman coupled fiber probe for 785 nm with FC Excitation -SMA Collection; 7.5 mm working distance
RIP-PA-SH Raman sample holder
LASER-GL-ML1 Laser safety glasses block 785 nm/808 nm/1064 nm lasers; OD 7+, VLT 45% Green
OceanView Spectrometer operating software
RAM-ANIQ-LAB Single-license chemometrics package from Analyze IQ

What light source can I use for excitation?

Raman scattering requires a very high intensity light source or long spectral acquisition times in order to generate enough photons for detection. The source should also be monochromatic to interrogate the narrowest Raman features and with the greatest resolution. Since the amount of Raman scattering is proportional to the intensity of the light source, lasers are a natural choice, particularly as they can be focused very precisely into a small sample or through a window.

That leaves the question of wavelength, λ. Raman scattering is proportional to 1/λ4, but excitation at shorter visible wavelengths can generate a lot of autofluorescence, particularly in organic samples. Autofluorescence appears as a broad background that is strongest close to the laser line, and can make it difficult to resolve weaker Raman peaks in this area. Blue and green lasers work best for inorganic materials, studies of carbon materials like nanotubes and fullerenes, and surface enhanced Raman scattering (SERS). Organic materials are best studied using red or near-infrared wavelengths (660 – 830 nm), as fluorescence is significantly lower.

Excitation at 1064 nm results in even lower autofluorescence background, but the Raman signal is much lower due to the long wavelength. Detectors are also less sensitive at NIR wavelengths, but it can be well worth it to excite the sample below any electronic transitions, as many materials cease to fluorescence at this point.

Raman and Energy Levels

The other option to avoid autofluorescence is to work in the ultraviolet, as the full Raman spectrum can often be seen before autofluorescence starts at ~330 nm. This is particularly advantageous for biological molecules such as proteins, DNA, and RNA thanks to resonant enhancement.  In practice, however, UV Raman spectroscopy is challenging due to the cost and intensity of UV sources, lower-performance and higher-cost filters for blocking the laser, and potential for damaging the sample with high-energy light.

We offer two spectrum-stabilized turn-key laser systems for use in modular Raman systems. Our Laser-532 is a diode-pumped solid state (DPSS) laser, which allows it to maintain ± 0.1 nm stability and a spectral linewidth of < 0.05 nm (FWHM).  Its output power is > 50 mW, typically stable to within 1%.

Our Laser-785 is a diode laser; as such its output wavelength will vary more with temperature than a DPSS laser. It offers a peak wavelength of 785 ± 0.3 nm, with a typical spectral linewidth of 0.2 nm. Its output power is > 500 mW, with less than 3% power variation over an 8 hour period.

What is the best sampling optic?

The beauty of Raman spectroscopy is that it can be performed on a wide range of samples, from liquids and gels to powders, capsules, and large objects. A fiber optic probe assembly is the sampling optic most often used in a modular Raman spectroscopy system. It consists of a fiber bundle to route excitation light from the laser to the sample and collect Raman scattered light. Light at the laser wavelength (Rayleigh scattering) is rejected on the path to the spectrometer using a dichroic filter to avoid saturating the detector.

We offer a range of fiber optic probes for Raman spectroscopy with 532 and 785 nm excitation. The RIP-series probes include models for laboratory, industrial and environmental applications with SMA or FC connectors.  The process probes are rated for use at up to 200°C and 1500 psi, while the immersion probes go up to 500°C and 3000 psi. Options include stainless steel and Hastelloy C housings and sapphire windows.

These probes mate well with the OOA-RAMAN-SH sample holder, which is compatible with a variety of cuvette sizes and vials. It allows the user to adjust the distance between the probe and the sample simply and accurately to optimize the Raman signal while blocking ambient light with a cap. The OOA-HOLDER-RFA has many of the same features, but is designed as a multipurpose sampling fixture for Raman, fluorescence, absorbance and reflection measurements, with accessories for each technique.

What spectrometer should I use for detection?

A high-performance laboratory Raman system typically has resolution in the range of 2 to 4 cm-1. A system with resolution in the range of 6 to 11 cm-1, however, is only a fraction of the cost. In reality, you don’t really need resolution below about 4 cm-1 unless you’re performing gas analysis. Many applications of Raman in industry are based instead on fingerprint comparisons – using the essential, identifying, basic spectral features of a molecule to identify or quantify it. Moderate resolution systems, therefore, are more than adequate.

We offer multiple spectrometer options for detection in modular Raman systems, each offering its own unique benefits in terms of size, sensitivity, resolution, and cost. We’ll start with the most basic and work our way through to the most sensitive.

Preconfigured spectrometers for Raman

Almost any of the preconfigured spectrometers can be adapted for collection of Raman spectra (USB2000+, USB4000, HR2000+, HR4000), but the units offering the best performance for Raman are the Maya2000 Pro-NIR and the QE Pro series.

A Maya2000 Pro-NIR configured with 780 – 1180 nm range, a 50 µm slit, and gold mirrors for enhanced NIR reflectivity offers high quantum efficiency and high dynamic range for use with 785 nm lasers. It covers a 200 – 4200 cm-1 range with 8 – 14 cm-1 resolution. With a back-thinned 2D FFT-CCD detector and low noise electronics, this configuration bridges the gap where silicon detectors typically fall off and InGaAs detectors are needed for good sensitivity (~1050 nm). The Maya2000 Pro can also be customized for other range and resolution needs.

The QE Pro series of spectrometers use a cooled back-thinned detector to minimize dark noise at long integration times, while the enhanced optical bench design provides improved thermal stability for reduced wavelength drift, and yields more symmetrical peaks over a wide ambient temperature range. A low stray light bench design and gold mirrors enhance S:N, while user-changeable slits provide flexibility.

A good spectrometer for 785 nm Raman is the QE Pro-Raman, preconfigured for 780 – 1000 nm (range can also be adjusted to higher starting wavelengths). When configured for 785 nm Raman, it covers a 200 – 2800 cm-1 range with 6 – 19 cm-1 resolution.

Raman-specific spectrometers and integrated lab systems

Our Ventana spectrometers are designed to minimize light loss and maximize throughput in a small footprint, making it an excellent OEM solution. They are configured with a high density volume phase grating for high efficiency & low stray light, a back thinned silicon CCD detector, and low-noise electronics. This enables the use of a low power laser and short integration times to acquire high S:N spectra.

The Ventana 785 spectrometer spans a range of 200 – 2000 cm-1 with 10 cm-1 resolution at 810 nm.  It is also comes integrated with a 120 mW cooled and wavelength-stabilized 785 nm laser module to create a free-space coupled system, the Ventana 785L. The Ventana 532 spectrometer covers 350 – 4300 cm-1 with 20 cm-1 resolution at 810 nm. The Ventana 785 kit comes with spectrometer, probe, 785 nm diode laser, sample holder, software.

What are my software and data processing options?

Raman spectroscopy software varies in complexity, depending on the application. For research and basic spectra, the Raman mode in our OceanView software may be sufficient, displaying in wavenumbers or the Raman shift relative to a specified excitation wavelength. It can be used with any of the spectrometers described above. For more sophisticated analysis, a chemometrics software package may be warranted. We carry two powerful options, both of which are used widely in the industry.

RSIQ software offers a user-friendly interface, routine analysis, and one-touch data acquisition. It outputs in two data formats: SPC and ASCII. It allows easy interpretation of spectral results, including an identification of the spectrum (if the material already exists in the database), display of peaks (location and intensity), display of measurement parameters, and a unique measurement ID.

An optional software add-on, RSIQ-QUAL, allows substance ID by a database search of thousands of reference spectra using commercially available spectral libraries, or the user can create their own. RSIQ-QUANT is a multivariate analysis tool, while RSIQ-CFR supports 21 CFR Part 11 compliance for cGMP electronic records and signatures.

AnalyzeIQ software products work with spectra collected by OceanView, and are excellent at identifying a target substance in a mixture without correcting for the presence of other components. Analyze IQ Lab is an analytical chemistry package ideal for commercial R&D, forensic labs and academic research labs for chemometric techniques such as PCR.  It provides advanced spectral analysis and a broad range of pre-processing options.

Spectra Manager is a spectral database and data management package for managing your own library of spectra. It stores CAS registry numbers and QA details, retrieves and lists spectra by IUPAC and common names, and tracks mixtures that use the same materials by lot numbers, making it extremely useful for process applications.

The Raman Spectra Library is an option for Spectra Manager containing 1,870 Raman spectra, including the exact composition of each material and all associated data. A wide range of additional data can be incorporated, including manufacturer, lot, appearance, purity, IUPAC name, common name and more.

Predictor is the embedded software module that allows the analytical models built using Analyze IQ Lab to be used in your own custom-developed software package.  Its lower CPU and storage requirements make it perfect for integration into portable systems.

How do I read a Raman spectrum?

Raman scatter can be observed at both longer wavelengths (Stokes radiation) and shorter wavelengths (anti-Stokes radiation) than the excitation laser wavelength. The intensity of Stokes radiation is generally much higher than anti-Stokes, so most people choose to work at longer wavelengths. This is further facilitated by the availability of high-quality steep long pass filters for rejection of the intensely scattered laser line (Rayleigh scattering).

Spectrometers typically yield spectra as a function of wavelength in nanometers, but for Raman spectra, those units don’t really make sense. Raman peaks correspond to transitions between vibrational energy states within the molecule, and therefore their spectra need to be in energy units. For this reason, Raman spectra are reported in terms of wavenumbers, or cm-1 (the inverse of the wavelength in cm) since this is proportional to energy (E=hc/λ). Furthermore, the Raman shift is expressed relative to the laser, so that the laser wavelength, λ0, becomes zero on the x-axis of the Raman shift spectrum.

Two Samples of Butanol

To calculate a Raman shift, Δν, in cm-1 using the laser wavelength and peak wavelength in nm:

Conversion to wavenumbers

The conversion from wavelength to Raman shift in cm-1 is typically done automatically by the software, but several apps exist for conversion into wavenumbers. We like Raman Gear.

One thing to note about plotting Raman shifts in cm-1 is that the resolution of a Raman system will vary with wavelength (resolution is only constant in wavelength space). The resolution of a Raman system will be higher at longer wavelengths, so it is important to specify either a range of resolution for a system, or a single resolution at a specific wavelength or Raman shift.

How do wavenumbers and nanometers (nm) relate?

To calculate a Raman shift, Δν, in cm-1 using the laser wavelength and peak wavelength in nm:

Conversion to wavenumbers

The conversion from wavelength to Raman shift in cm-1 is typically done automatically by the software, but several apps exist for conversion into wavenumbers. We like Raman Gear.

Should I worry about drift of my laser?

Thermal drift of the excitation wavelength may affect signal to noise ratio over time, which is why most systems require the user to perform a calibration or validation step prior to use. Variations in power stability over time will affect intensity but not the shape or positioning of the peak.

How does SERS enhance sensitivity?

Surface enhanced Raman spectroscopy (SERS) uses interactions with specialized surfaces to enhance Raman signal strength. The enhancing substrate may be gold, silver, or copper, often patterned to maximize the effect.  It requires that the sample be prepared in liquid or vapor form and placed in direct contact with the SERS substrate, so it does consume some of the sample.

This compromise is well worthwhile, as enhancement by a factor of up to 1011 can be possible under the right conditions. It occurs when the laser interacts with the SERS substrate and excites surface plasmons that resonate with the vibrational transitions in the sample molecules adsorbed to that surface, thereby magnifying the Raman effect. One side effect of adsorption to the surface is that the symmetry of the molecule can change, changing the rules for which vibrational transitions can be excited by Raman and yielding different peaks than would be seen with conventional Raman spectroscopy.

SERS substrate are usually silver (for 532 nm) or gold (for 785 nm) particles or modified surfaces, often in a structured array on a slide or colloids in suspension for microfluidics. SERS tags can be embedded in silica particles and functionalized to tag specific biological markers for medical applications such as immunodiagnostics, molecular diagnostics, and proteomics. This has also been used as a tag tracer to identify counterfeit fuel.

When analyzing SERS samples, be aware that the sample material is likely to be dispersed non-uniformly on the substrate, so measurements with a static laser may easily end up measuring substrate instead of sample. A static laser beam can also cause localized heating and damage to the SERS substrate, further increasing the background of the spectrum. The raster orbital scanning method of sampling that we use in our IDRaman reader and IDRaman mini products rapidly samples many points on the substrate, improving both signal strength and consistency of results with SERS.

SERS Substrates

SERS Substrates

Surface Enhanced Raman Spectroscopy
QE Pro (Custom)

QE Pro (Custom)

High-sensitivity Spectrometer for Low Light Level Applications
Maya2000 Pro (Custom)

Maya2000 Pro (Custom)

High Sensitivity Spectrometer
QE Pro-Raman

QE Pro-Raman

High-sensitivity Spectrometer for Raman
Ventana-785-Raman

Ventana-785-Raman

High-sensitivity Spectrometer for Raman Measurements
Maya2000 Pro-NIR

Maya2000 Pro-NIR

High-sensitivity Spectrometer for Raman and NIR Applications
Ventana-532-Raman

Ventana-532-Raman

High-sensitivity Spectrometer for Raman Measurements
Ventana-785L Raman

Ventana-785L Raman

High-sensitivity Spectrometer for Raman Measurements
Raman Laser Safety Glasses

Raman Laser Safety Glasses

Laser Light Protection without Comprising Visibility
Raman Sample Holders

Raman Sample Holders

Sampling of SERS, Liquids, Powders and More
General Purpose Raman Probes

General Purpose Raman Probes

High Signal Collection and Effective Filtering Design
Raman Immersion Probes

Raman Immersion Probes

Immersible Probes for Lab and Process Applications
Raman Process Probes

Raman Process Probes

Process-ready Probes for Industrial Environments
Turnkey Raman Lasers

Turnkey Raman Lasers

High-power, Spectrum-stabilized Lasers
Multimode Raman Laser Subsystems

Multimode Raman Laser Subsystems

Ideal for Integrating into OEM Packages
OceanView 1.6.7

OceanView 1.6.7

Real-time spectral acquisition and analysis
Analyze IQ Chemistry Software

Analyze IQ Chemistry Software

Chemometric Package is Useful for Raman Analysis