UV detectors

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An ultraviolet detector (also known as UV detector or UV-Vis detector) [1] [2] is a type of non-destructive chromatography detector which measures the amount of ultraviolet or visible light absorbed by components of the mixture being eluted off the chromatography column. They are often used as detectors for high-performance liquid chromatography. [3]

The vast majority of liquid chromatographic systems are equipped with ultraviolet (UV) absorption detectors. The most common UV-Vis detectors used are variable wavelength detectors (VWD), photo diode array detectors (PDA), and diode array detectors (DAD). [4] Variable wavelength detectors decide in advance which wavelength is needed for the detection. Its absorbance as function of time is collected in a graphic format called a chromatogram. [5]

As can be seen in Figure 1, these detectors have a light source, a dispersion element that is a diffraction grating or prism, a flow cell, to where the sample arrives directly from the chromatographic column, an optical bench of lenses and mirrors, and a diode that receives the light coming from the optical system and translates it into a signal proportional to light intensity. When the user selects a wavelength for the detector, the optical system rotates the grating or prism in the space, so that the desired wavelength passes through optical system, then the flow cell and reaches the diode. The UV/Vis detector then produces a chromatogram as a two-dimensional (2D) output. This output plots time on the x-axis and response in absorbance units (AU) on the y-axis.  The chromatogram is then analyzed by integrating the peaks curves to get their area, then getting their retention time (RT) from the peak maximum to identify them, and then perform quantitative analysis, by comparing their area to those of samples whose concentrations are known, i.e, standards.

Diode Array UV-VIS Detectors

In recent years, diode array UV-Vis detectors have been increasingly used to collect entire spectra at any given moment of data collection. Diode array detectors (DADs) collect entire UV spectra at every point of the eluting peaks while operating as a multi-wavelength UV-Vis detector. This way they give additional information, which help understand more about the nature of the substances appearing in the chromatogram and allow their identification. [6] DADs are the preferred detectors for HPLC method development because they facilitate better peak identification.

Figure 1: Simplified schemes of the Variable UV-Vis detector compared to PhotoDiode Array detector. In the Variable UV-Vis the entire optical bench is located before the flow cell whereas in the diode array the flow rate is positioned before the main optical bench. UV PDA Scheme2.gif
Figure 1: Simplified schemes of the Variable UV-Vis detector compared to PhotoDiode Array detector. In the Variable UV-Vis the entire optical bench is located before the flow cell whereas in the diode array the flow rate is positioned before the main optical bench.

A schematic of the optical systems is shown in Figure 1. The variable UV-Vis absorbance detector's optical bench is showing how the flow cell is positioned after the optical system, including the monochromator, which typically has a physical slit and a moving grating, so it is illuminated by a selected wavelength, reaching a photo-diode. The bench of the diode array detector, however, is configured so that the flow cell is positioned before the optical parts, so that the beam containing the entire spectrum is passing through it. The optical parts consist also with a monochromator and a slit, but with a fixed grating, which disperses the light onto a diode array imaging element.

Related Research Articles

<span class="mw-page-title-main">Ultraviolet–visible spectroscopy</span> Range of spectroscopic analysis

Ultraviolet (UV) spectroscopy or ultraviolet–visible (UV–VIS) spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum. Being relatively inexpensive and easily implemented, this methodology is widely used in diverse applied and fundamental applications. The only requirement is that the sample absorb in the UV-Vis region, i.e. be a chromophore. Absorption spectroscopy is complementary to fluorescence spectroscopy. Parameters of interest, besides the wavelength of measurement, are absorbance (A) or transmittance (%T) or reflectance (%R), and its change with time.

<span class="mw-page-title-main">High-performance liquid chromatography</span> Technique in analytical chemistry

High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify specific components in mixtures. The mixtures can originate from food, chemicals, pharmaceuticals, biological, environmental and agriculture, etc., which have been dissolved into liquid solutions.

Gel permeation chromatography (GPC) is a type of size-exclusion chromatography (SEC), that separates high molecular weight or colloidal analytes on the basis of size or diameter, typically in organic solvents. The technique is often used for the analysis of polymers. As a technique, SEC was first developed in 1955 by Lathe and Ruthven. The term gel permeation chromatography can be traced back to J.C. Moore of the Dow Chemical Company who investigated the technique in 1964. The proprietary column technology was licensed to Waters Corporation, who subsequently commercialized this technology in 1964. GPC systems and consumables are now also available from a number of manufacturers. It is often necessary to separate polymers, both to analyze them as well as to purify the desired product.

<span class="mw-page-title-main">Spectrophotometry</span> Branch of spectroscopy

Spectrophotometry is a branch of electromagnetic spectroscopy concerned with the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. Spectrophotometry uses photometers, known as spectrophotometers, that can measure the intensity of a light beam at different wavelengths. Although spectrophotometry is most commonly applied to ultraviolet, visible, and infrared radiation, modern spectrophotometers can interrogate wide swaths of the electromagnetic spectrum, including x-ray, ultraviolet, visible, infrared, and/or microwave wavelengths.

<span class="mw-page-title-main">Fluorescence spectroscopy</span> Type of electromagnetic spectroscopy

Fluorescence spectroscopy is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs of fluorophores.

<span class="mw-page-title-main">Monochromator</span> Optical device which allows selection of a narrow band of wavelengths from a wider spectrum

A monochromator is an optical device that transmits a mechanically selectable narrow band of wavelengths of light or other radiation chosen from a wider range of wavelengths available at the input. The name is from Greek mono- 'single' chroma 'colour' and Latin -ator 'denoting an agent'.

<span class="mw-page-title-main">Gas chromatography</span> Type of chromatography

Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.

<span class="mw-page-title-main">Photometer</span> Instrument to measure light intensity

A photometer is an instrument that measures the strength of electromagnetic radiation in the range from ultraviolet to infrared and including the visible spectrum. Most photometers convert light into an electric current using a photoresistor, photodiode, or photomultiplier.

A spectroradiometer is a light measurement tool that is able to measure both the wavelength and amplitude of the light emitted from a light source. Spectrometers discriminate the wavelength based on the position the light hits at the detector array allowing the full spectrum to be obtained with a single acquisition. Most spectrometers have a base measurement of counts which is the un-calibrated reading and is thus impacted by the sensitivity of the detector to each wavelength. By applying a calibration, the spectrometer is then able to provide measurements of spectral irradiance, spectral radiance and/or spectral flux. This data is also then used with built in or PC software and numerous algorithms to provide readings or Irradiance (W/cm2), Illuminance, Radiance (W/sr), Luminance (cd), Flux, Chromaticity, Color Temperature, Peak and Dominant Wavelength. Some more complex spectrometer software packages also allow calculation of PAR μmol/m2/s, Metamerism, and candela calculations based on distance and include features like 2- and 20-degree observer, baseline overlay comparisons, transmission and reflectance.

<span class="mw-page-title-main">Column chromatography</span> Method to isolate a compound in a mixture

Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential absorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.

<span class="mw-page-title-main">Fast protein liquid chromatography</span>

Fast protein liquid chromatography (FPLC) is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid and a porous solid. In FPLC the mobile phase is an aqueous buffer solution. The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the application.

<span class="mw-page-title-main">Spectronic 20</span> Single beam spectrophotometer

The Spectronic 20 is a brand of single-beam spectrophotometer, designed to operate in the visible spectrum across a wavelength range of 340 nm to 950 nm, with a spectral bandpass of 20 nm. It is designed for quantitative absorption measurement at single wavelengths. Because it measures the transmittance or absorption of visible light through a solution, it is sometimes referred to as a colorimeter. The name of the instrument is a trademark of the manufacturer.

Reversed-phase liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds. The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed phase mode. In the reversed phase mode, the sample components are retained in the system the more hydrophobic they are.

Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses a supercritical fluid such as carbon dioxide as the mobile phase. It is used for the analysis and purification of low to moderate molecular weight, thermally labile molecules and can also be used for the separation of chiral compounds. Principles are similar to those of high performance liquid chromatography (HPLC); however, SFC typically utilizes carbon dioxide as the mobile phase. Therefore, the entire chromatographic flow path must be pressurized. Because the supercritical phase represents a state whereby bulk liquid and gas properties converge, supercritical fluid chromatography is sometimes called convergence chromatography. The idea of liquid and gas properties convergence was first envisioned by Giddings.

Agilent ChemStation is a software package to control Agilent liquid chromatography, gas chromatography, and ultraviolet-visible spectroscopy systems such as the 1050, 1100 and 1200 Series HPLC system and the 8453 and 8454 single-beam diode array detector spectrophotometers. It is an evolution of the Hewlett-Packard ChemStation System.

A chromatography detector is a device that detects and quantifies separated compounds as they elute from the chromatographic column. These detectors are integral to various chromatographic techniques, such as gas chromatography, liquid chromatography, and high-performance liquid chromatography, and supercritical fluid chromatography among others. The main function of a chromatography detector is to translate the physical or chemical properties of the analyte molecules into measurable signal, typically electrical signal, that can be displayed as a function of time in a graphical presentation, called a chromatograms. Chromatograms can provide valuable information about the composition and concentration of the components in the sample.

<span class="mw-page-title-main">Instrumental chemistry</span> Study of analytes using scientific instruments

Instrumental analysis is a field of analytical chemistry that investigates analytes using scientific instruments.

An evaporative light scattering detector (ELSD) is a destructive chromatography detector, used in conjunction with high-performance liquid chromatography (HPLC), ultra high-performance liquid chromatography (UHPLC), purification liquid chromatography such as flash or preparative chromatography, countercurrent or centrifugal partition chromatography and supercritical fluid chromatography (SFC). It is commonly used for analysis of compounds that do not absorb UV-VIS radiation significantly, such as sugars, antiviral drugs, antibiotics, fatty acids, lipids, oils, phospholipids, polymers, surfactants, terpenoids and triglycerides.

<span class="mw-page-title-main">Cary 14 Spectrophotometer</span> UV-Vis spectrophotometer, scientific instrument

The Cary Model 14 UV-VIS Spectrophotometer was a double beam recording spectrophotometer designed to operate over the wide spectral range of ultraviolet, visible and near infrared wavelengths (UV/Vis/NIR). This included wavelengths ranging from 185 nanometers to 870 nanometers.

Gas chromatography–vacuum ultraviolet spectroscopy (GC-VUV) is a universal detection technique for gas chromatography. VUV detection provides both qualitative and quantitative spectral information for most gas phase compounds.

References

  1. ↑ L.C. Passos, Marieta; M.F.S. Saraiva, M. Lúcia (2019). "Detection in UV-visible spectrophotometry: Detectors, detection systems, and detection strategies". Measurement. 135: 896–904. doi:10.1016/j.measurement.2018.12.045. ISSN   0263-2241. S2CID   117622937.
  2. ↑ Wysocki, Jedrzej; Dong, Michael (2019). "Ultraviolet Detectors: Perspectives, Principles, and Practices". LCGC North America. LCGC North America-10-01-2019. 37 (10): 750–759.
  3. ↑ Meyer, Veronika (2010). Practical high-performance liquid chromatography (5th ed.). Chichester, U.K.: Wiley. ISBN   9780470688427. OCLC   613324719.
  4. ↑ Swartz, Michael (2010-07-13). "HPLC DETECTORS: A BRIEF REVIEW". Journal of Liquid Chromatography & Related Technologies. 33 (9–12): 1130–1150. doi:10.1080/10826076.2010.484356. ISSN   1082-6076.
  5. ↑ Dolan, John W.; Berry, Vern V. (June 1, 1984). "Optical Detectors, Part III: Variable-Wavelength UV Detectors" (PDF). Liquid Chromatography Troubleshooting. 2 (6): 439–442 – via Liquid Chromatography Troubleshooting Bible.
  6. ↑ George, S. A.; Maute, A. (1982). "A photodiode array detection system: Design concept and implementation". Chromatographia. 15 (7): 419–425. doi:10.1007/bf02261601. ISSN   0009-5893. S2CID   93087073.
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