UV/Vis Spectroscopy

UV/Vis spectroscopy

Whether measuring chromatic properties of foodstuffs, analyzing wastewater quality according to standard procedures, or verifying the concentration and purity of a drug's active ingredient: UV/Vis spectroscopy is the right method for these applications. This spectroscopic method plays a role in every industrial sector, but especially in pharmaceuticals, environmental engineering, and the chemicals and foodstuffs industry. Here, spectrophotometers are an integral part of special applications and routine analyses, where they are used to ascertain the properties and concentration of individual atoms, molecules, or ions.

Principles of this method

Spectroscopy refers to a number of physical methods that use electromagnetic radiation to investigate material properties such as electron transfers or vibrational states in samples. To this end, UV/Vis spectroscopy exploits the interactions of molecules with electromagnetic energy (molecular spectroscopy). This spectroscopic method is thus one of many types with a number of sub-methods:

• Atomic absorption spectroscopy (AAS)
• Molecular spectroscopy (UV/Vis, NIR, fluorescence, NMR)
• Atomic emission spectroscopy (OES)
• X-ray spectroscopy (XRS, XRF)
   

What is UV/Vis spectroscopy?

UV/Vis spectroscopy relies on the way that radiation interacts with matter. When electromagnetic radiation hits solid objects, fluids or gases, various effects can occur. One can observe absorption, reflection or scattering. In the field of optics, these phenomena fall under the label of "extinction". All are processes involving the weakening, or attenuation, of radiation. UV/Vis spectroscopy is only concerned with radiation within a certain range of the spectrum: In addition to UV radiation and the visible electromagnetic range (Vis), high-performance machines like the SPECORD Plus extend coverage into the near-infrared range (NIR). Other terms for this method are spectrophotometry and electron absorption spectroscopy.

UV/Vis spectroscopy is used to analyze the absorption of radiation. When photons of UV or visible light are absorbed, the result can be the excitation of valence electrons of atoms or molecules. Here, the electrons go into a higher-energy state.

The wavelength of the radiation reveals the energy that was absorbed. A shorter wavelength means a higher energy. UV/Vis spectrophotometers can measure the location of the absorption sites and the magnitude of the absorption. The result are transmittance spectrums that describe the relationship between the incident light and the transmitted light. This enables UV/Vis spectrophotometry to tackle the following tasks, to name just a few:

• Wavelength
  • Identification of substances
  • Chromatic measurement of substances
• Absorption
  • Measurement of the quantity of absorbed light
  • Quantitative substance measurements
• Relative absorption of two signals
  • Verification of substance purity
  • Measurement of reaction kinetics

Structure of the method

Measuring a sample's absorption of electromagnetic radiation as a function of wavelength: this is the principle behind UV/Vis spectroscopy. A modern UV/Vis spectrophotometer is constructed from essentially the following components:

• Radiation source: Continuum emitter (deuterium or halogen lamp, for example)
• Light decomposition unit: Interference filters, prisms or holographic grids
• Sample chamber: mainly for fluids or solutions in cuvettes
• Detector: Photodiodes or photomultipliers (PMT)
• Control computer

In addition, measuring instruments also have optical components that direct the light through the various components.

Principle of operation behind UV/Vis

Different variants of UV/Vis spectrophotometers differ mainly in their radiation source, but perhaps more importantly, in the element that breaks down spectral light and directs the radiation. This becomes apparent when considering their principle of operation.

UV/Vis with monochromator system

In a monochromator system, such as the one used by Analytik Jena in the SPECORD PLUS Series, spectral decomposition of the light occurs before the light hits the sample. Thus, only monochromatic light illuminates the sample. This has a crucial advantage in that measurements only deliver precise results if the sample absorbs no more than a small amount of radiation.

The optical path in a monochromator system is structured as follows:

1. Radiation source
2. Inlet slit
3. Filter wheel
4. Outlet slit
5. Sample chamber with cuvettes
6. Detector

As it leaves the light source, polychromatic light hits the inlet slit of the light decomposition unit. It houses a moving filter element that breaks apart the light. The subsequent outlet slit only lets through a narrow spectral range of light, which then hits the sample. From that point, the light beam goes to the detector. The absorption of the sample under analysis is obtained by comparing the light intensities between when the detector has a sample and when it does not (instead a blank). This type of UV/Vis spectrophotometer makes it possible to scan spectra with continuous measurement of the absorption value at each defined wavelength (scanning UV/Vis spectrophotometer).
The few moving parts also help give the SPECORD spectrophotometer a long service life. In addition, SPECORD 210 PLUS and SPECORD 250 PLUS machines feature variable slit widths in the outlet slit. This adjustable spectral resolution makes it possible to optimize the machines for use with accessories for specific sample types and varying concentrations.

Narrow slit: better resolution

• For substances with peaks that are close to each other
• For differentiating very similar substances

Wide slit: more energy

• For certain accessories (e.g. integrating spheres for solid samples)
• For measuring close to the detection limit

UV/Vis with polychromator system

The polychromator system chooses a different sequence of components. Here, the complete light spectrum hits the sample and is spectrally decomposed only afterwards.

After passing the sample, the radiation proceeds to the light decomposition unit. The light beam split by the polychromator hits a diode array that acts as the detector. Each of these diodes measures the intensity of a very narrow range in the spectrum. This type of UV/Vis machine is thus also known as a diode array spectrophotometer. In this way, polychromator systems allow for extremely fast spectrum measurements, although their precision does not match the results of a monochromator system.

Single- and dual-beam machines

Monochromator and polychromator UV/Vis systems can be designed as single-beam machines. They use a minimal number of optical components with good transmittance. This simple arrangement enables an economical setup with low intensity losses. The spectra of the sample and the blank must be measured sequentially, however.

Two-beam photometers have a different optical structure that enables simultaneous measurements of sample and blank. A split beam passes the two cuvettes and hits two dedicated detectors. This makes it possible to eliminate variations in radiation intensity over time (drift) as a source of error. With the SPECORD PLUS Series, Analytik Jena ensures maximally precise measurements with this arrangement. Users from the chemicals and materials sectors, as well as in environment applications, rely on the capabilities to measure low substance concentrations.

Analytik Jena knowingly breaks new ground with the SPECORD 50 PLUS to achieve an irresistible price-to-performance ratio. This spectrophotometer uses split-beam technology. The light beam is divided in a beam splitter, where about 10% of the total energy is diverted to an internal reference mirror and the remaining 90% to the sample. This ensures the sample is irradiated with a high amount of energy, allowing excellent measurements of opaque samples in particular.

Different light sources

In practice, there is no ideal light source that covers all wavelengths. For that reason, UV/Vis systems often use a composite of multiple light sources in order to achieve the best possible conditions. Here, the primary objective is to attain a constant radiation intensity with low noise. For the sake of precision and economic viability, the light source must also possess good long-term stability.

Deuterium lamps offer stable intensity in the UV range. In the visible spectral range, however, the intensity is no longer sufficient. A halogen lamp is therefore used for the visible range.

Xenon lamps represent an alternative to this popular combination. They are equally good at covering the UV and visible ranges. Their disadvantage is their relatively high noise, and so xenon lamps are used mainly for applications with a high radiation intensity.

Analytik Jena equips its SPECORD PLUS Series spectrophotometers with a deuterium and halogen lamp combination. This allows the machines to achieve a uniform high energy intensity over a very wide range of wavelengths.

Optimal sample handling with the Analytik Jena accessory portfolio

The UV/Vis system operates mainly with fluids and solutions. Depending on the spectrophotometer, these samples can be measured either manually or (semi) automatically in cuvettes in the spectrophotometer's sample chamber. Suitable accessories also make it possible to test gases and solids with UV/Vis.

Cuvettes are optimized with respect to their absorption characteristics so as to limit the usable measuring range as little as possible. Depending on the requirements for stability and the measuring range, various materials may be used. Cuvettes in many different designs are available, for instance for especially small sample volumes (often the case in pharma and life science) or as flow-through cuvettes (used mainly in environmental analysis).

Analytik Jena offers a wide range of accessories for the SPECORD PLUS Series for liquid, solid and gaseous samples. With the right cuvette holder, spectrophotometers can measure minimum sample volumes down to 5 µl. As the measurement sensitivity increases with the cuvette's layer thickness, cuvette holders up to 100 mm are also available. The cuvette receptacle in the SPECORD PLUS spectrophotometers can also be equipped with agitators or an integrated thermometer. Numerous automation accessories are also available. Multiple cuvette changes are available, as are sipper systems for in-flow measurements. These accessories allow for specific optimization of the sample throughput in the instruments.

ASpect UV software

The potential of the hardware can only be realized with the right software. This is why Analytik Jena equips all spectrophotometers in the SPECORD PLUS Series with the ASpect UV software. UV/Vis data can be recorded and analyzed in just a few clicks. The modules cover a wide range of applications, even in the default configuration.

• Photometry
• Spectral analysis
• Time-dependent analyses (kinetics)
• Temperature-dependent analyses
• Chromatic measurement

Together with the wide array of sample handling accessories, the ASpect UV software enables simple and efficient handling of the machines. It takes into consideration application-specific requirements: Powerful user management ensures compliance with stringent regulations for measurements in the pharma industry. Moreover, various validation modules meet the requirements of the European and US Pharmacopoeia.