L A B O R A T O R Y C O M P E T E N C E Compact Knowledge: Absorbance Spectrophotometry Flexible. Reliable. Personal.
The interaction of light with molecules is an essential and well accepted technique that is used to identify or quantify specific molecular substances. The study of the interaction of light with molecules is called Spectrophotometry. Spectrophotometry is the measurement of visible (VIS) and ultra-violet (UV) electromagnetic radiation (light) that is reflected, absorbed by or transmitted through molecules. What can be measured by spectrophotometry? Or in other words, what absorbs light? The answer is everything. Measuring Principle Light is absorbed by molecules in a manner that is characteristic of their chemical structure. Many different types of laboratories use spectrophotometry to characterize their samples. For example, spectrophotometers can be used to: used to determine the purity of food measure the concentration of DNA measure the presence of contaminants in water measure the UV light transmittance of clothes and eye glasses UV / VIS spectrophotometers are instruments that are specifically designed to generate light in the visible or ultra-violet spectrum or from light with wavelengths from 200 to 1000 nm in the electromagnetic spectrum. Visible light ranges from 400 to 750 nm and comprises the visible light that we can see. Absorbance of light in the visible range gives substances their characteristic colour in a manner that is complimentary. There are multiple ways that samples can interact with light. The most common application is the absorbance of light by a sample but light waves can pass through the samples, light is reflected off the sample, light waves can be scattered off an object, light can be refracted through the object. The absorbance of light by a sample is a result of the specific electronic configuration of the molecule. The energy of a certain wavelength excited electrons within molecules from their ground energy state to an excited level. Many molecules have energy sub levels gives rise to rounded peaks that are characteristic of the UV / VIS spectrum (Fig. 1). Most absorption spectrophotometry is based on the electronic transitions of atoms that are chemically bonded to one another. For example, the ethanal bond in ethanol has an absorbance maximum of 210 nm. The functional group which results in a specific absorbance is known as a chromophore. Many molecules have multiple chromophores which give rise to a complex absorption spectrum which never the less is characteristic of a specific molecule. Many organic molecules with simple double bonds between non-metal atoms like carbon, oxygen and nitrogen and small conjugated ring structures like protein or nucleic acids absorb light in the UV region. Large aromatic structures often absorb light in the visible spectrum. An example is TMB which is a substrate that is used to detect the presence of antigens in an ELISA assay. Lambert-Beer Law How is the absorbance of light measured in a sample? To understand this principle we need to discuss the laws of light absorbance as described by Lambert and Beer. Lambert's Law Beer s Law Fig. 2. Lambert s law states that the amount of light absorbed by a transparent medium is not affected by the intensity of the light and that multiple layer of equal thickness will absorb the same amount of light. Light absorbed by a sample is measured as both transmittance and absorbance. The transmittance (T) of light through a sample is simply the % of light which leaves the sample (I) compared to the amount of light that was directed at the sample l T = l * 100% 0 As concentration of the molecule of interest in the solution increases the transmittance is reduced exponentially. A more convenient expression of the absorbance of light in the sample can be described by Beer s law which states that Log10 Transmittance is equal to absorbance (A). Fig. 1: Electron energy levels UV-spectrum Fig.3: Molar absorptivity
The relationship of absorbance to concentration of the absorbing molecule is directly proportional. Absorbance equals concentration multiplied by the pathlength of light (b) through the sample multiplied by the molar absorptivity (ε). The molar absorptivity of a molecule is dependent on fixed ph and wavelength. A plot of absorbance (A) versus concentration (C) is a straight line with the slope of the curve defined as (ε) thus Beer s law expresses a linear relationship between light transmittance and concentration. The result is called Lambert-Beer law (sometimes Bouger-Lambert-Beer law): lg l 0 = εcd l The intensity of the radiation (light) sent through an absorbing medium is decreased exponentially. Clearly there is only a very small amount of light that is transmitted from the sample at higher absorbances which is why these measurements are typically not within the linear range of the instrument. Measurements made outside of the linear range are not reliable predictors of the absorbing molecule concentration and thus a deviation from Beer s Law. In addition to linearity, stray light can also interfere with the measurement. Stray light is radiation that is transmitted to the sample outside of the selected wavelength. Other deviations include solution effects such as when a solution polymerizes or ionizes. All three of these factors can result in inaccurate measurements. Optical Structure We know that a spectrophotometer measures light but how is this accomplished? A spectrophotometer must have these basic features: light source means of selecting specific wavelengths sample holder detector read out Fig.4: Lambert-Beer law How does Absorbance relate to Transmittance: 1A absorbance is equivalent to 10 % of transmitted light 2A is equivalent to 1 % of transmitted light 3A is equivalent to 0.1 % of transmitted light 4A is equivalent to 0.01 % of transmitted light Fig. 5: Assembly of a photometer
The major types of light sources are tungsten halogen which generate light in the visible range, deuterium which generate light mostly in the UV range and xenon flash lamps. For a visible spectrophotometer, a tungsten halogen lamp is sufficient as the range of light emitted is from 400 to > 800 nm fully covering the visible range. For any spectrophotometer that measures UV light, either a deuterium arc lamp which generates UV light from 190 to 400 nm or a xenon lamp which generates light from 200 to 1100 nm. A deuterium lamp emits constant UV light once the instrument is on where as the xenon lamp emits pulses of light only when measurement is occurring. Deuterium lamps are considered superior because of the constancy of the light emission however they require replacement over time whereas the xenon lamp rarely requires replacement. The pulsed light of the xenon lamp requires integration of the absorbance over time therefore resulting in a slightly less precise result. The light generated from the lamp must then be filter or selected so that only specific wavelengths of light are emitted to the sample. A simple method of light select is an absorbance filter made of coloured glass which transmits some light and absorbs others. Absorbance filters are used in colorimeters. A more sophisticated filter is an interference filter which is designed to refract some light and transmit narrow bands of light. Cuvettes The sample holder in a spectrophoto meter is traditionally a 10 mm cuvette made from quartz. Quartz is used because it does not absorb any light in the UV or visible region unlike many plastics which absorb in the UV region (Exception: BRAND UV-cuvettes, usable from 220 nm up). Many other sample holders exist, such as 40 mm wide cuvettes for heavily diluted samples like what exists in water testing or very short pathlength cuvettes of less than 0.5 mm such as those used for DNA measurements (e.g. Hellma TrayCell). Many other types of sample holders exist as well. For example, measurements can be taken from a flask using a small pump called a sipper which uses plastic tubing to send the solution into a nearly sealed cuvette called a flow cell. The constant stream of sample going through the cell allows for representative results and observing kinetics (changes over time). Another common type of sample holder is a film holder which allows for larger, partially transmitting objects. A common use would be the transmittance of UVA and UVB light thought sunglass lenses. Cell changers allow for quick and easy measurements of larger amounts of samples. Fig. 6: Affect of bandwith on absorbance spectra The best spectrophotometers use a wavelength selection device called a monochromator which uses a prism or diffraction grating to produce even narrow bands of light. Monochromators emit a narrower band of light then filters and also have the additional advantage of producing a spectrum of light where the absorbance of the sample can be measured over a range of wavelengths. This feature can be used to give more specific information about the sample. For example during a DNA concentration measurement, a sample is measured from 230 to 320 nm. The absorbance maxima of DNA is 260 nm and this value is used to calculate the concentration of DNA; however absorbances < 260 nm or greater than 260 nm gives the scientist information regarding the purity of the preparation as absorbance or the sample at < 260 nm indicates the presence of sample preparation contaminants such as phenol and guanidine HCl whereas absorbance of the sample at > 260 nm indicates the present of protein contamination. For all wavelength selection mechanisms, it is important to understand the bandwidth. Depending on the application, narrower bandwidths may be required to identify sharper peaks within a sample (Fig. 6). Fig. 7: Automatic cell changer Detector Once the light is transmitted from the sample, the remaining light must be detected. There are several ways of detecting light. A silicon photodiode is used in many spectrophotometers. Light falls on the active area of the photodiode cause electrons to flow across a gap junction which is then measured as an electrical current. Another means of light detection is using a diode array also known as a CCD. The diode array is a silicon chip with a linear or matrix array of integrated optically responsive elements. The complete spectrum of light is spread across the array. A silicon photodiode is the most sensitive but the diode array is very robust dues to no moving parts. How are all of these elements used in a colorimeter or a UV / VIS spectrophotometer? A simple photometer uses a tungsten halogen light source, an absorbance filter and a 10 mm cell holder. These systems are useful for VIS-only routine measurements. A more sophisticated optical arrangement can be seen in single beam spectrophotometers. These systems use a deuterium lamp along with the tungsten lamp to measure in the VIS- and UV- range. A monochromator is used to select the light and a diode array to detect the transmitted light. These instruments generate a single beam of light which is transmitted through the sample. The sample can be held in standard 10 mm cuvette, a heated cell holder or a test tube.
Libra S12 Optical fibre can be used Fig. 10: Split Beam Photometer with Optical Fibre Fig. 8: Assembly Single Beam Photometer Another step up in sophistication is a split beam spectrophotometer. This instrument uses a Xenon lamp to generate UV and visible light as well as a monochromator for light selection. The light beam that is emitted to the sample is split. The part of the light beam that is not emitted through to the sample becomes the reference which is monitored and used to compensate the measurement beam. FoodALYT Photometer a modification of the bandwidth from 0.5 nm to 4 nm. Extremely narrow bandwidths like 0.5 nm are useful to precisely identify sharp peaks in complex samples. Reducing the bandwidth means reducing the amount of energy emitted through the sample. For this reasons, using the smallest bandwidth possible is not always the best choice. A double beam optical configuration is useful for experiments that require a high level of sensitivity or when the refence is frequently changed. Modern systems often include efficient internal software that allows for precise control via touchscreen. To comply with international standards like the 21 CFR Part 11, powerful external software is available to allow control and data storage via external PC. Fig. 9: Biochrom photometer with reference beam A single cell holder is available for this instrument as well as temperature controlled 5 and 8 cell changers which can be used to measure multiple samples in quick succession for product formation assays. Double beam UV / VIS spectrophotometers are used where anything but the highest precision is not an option (e.g. pharma industry quality control). These instruments use a combination of tungsten and deuterium lamp to cover both VIS- and UV- spectra. The sophisticated monochromator allows for Fig. 11: Assembly Double Beam Photometer Choosing the right colorimeter or UV / VIS spectrophotometer means understanding the limitations of the instrument within the context of the application.
Abstract If a measurement has comparably low requirements, like measuring a colored solution, simple single beam systems or colorimeters are an appropriate choice. A more sophisticated system would only provide the same results at a much higher cost in both aquisition and maintenance. Simple systems are also useful in demonstrating chemical and physical basics in schools and universities. Measuring concentration and purity of DNA on the other hand demands the more sophisticated systems, capable of measuring whole spectra and operate in the UV-range or be able to have special cell compartments to hold bigger objects like lenses. The top-end double beam spectrophotometers would be a good choice for a pharmaceutical laboratory which needs to perform highly sensitive measurements requiring continuous referencing and / or variable bandwidth. Libra S80 Our product specialists will gladly assist you in choosing the right system for your needs. Feel free to contact us anytime. Robert-Hooke-Str. 8 D-28359 Bremen / Germany Phone +49 (0)421 / 1 75 99-0 www.omnilab.de international@omnilab.de Flexible. Reliable. Personal.