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Introduction to UV/Visible Spectrophotometers
This specific wavelength is often the Lambda max i.e. the wavelength which gives rise to the highest clearly resolved Absorbance value.
The scan itself may also be useful in aiding in the identification of the analyte.
Single Wavelength Quantitative Measurements
Once a specific wavelength has been chosen, the spectrophotometer is set at that chosen wavelength. A calibration or standard curve of Absorbance or Transmittance versus concentration needs to be plotted, (where various levels of concentration of a known standard and the resultant Absorbance/Transmittance readings are plotted). The amount of the unknown analyte in the sample is read off against the curve, or as a factor is derived from a calibration curve.
In general, Concentration = Factor x Absorbance.
The factor is generally the gradient of a calibration curve and its intercept on the y axis. A factor is specific to each individual spectrophotometer, for a particular analyte, at a specific wavelength, using a specific blank, after a specific type of sample pre-
Before a standard or sample reading is taken, a blank/reference (this material should be similar to the standard/sample, but does not contain the analyte of interest) should be inserted in the spectrophotometer and the instrument zeroed for that blank at that the chosen wavelength.
The measured Absorbance/Transmittance of the standards and samples should not be beyond the spectrophotometer’s measuring range. Depending on the stray light of the spectrophotometer, the best Absorbance’s to aim for are 0.6 to 0.7 Absorbance units.
Stray light is the detected light of any wavelength that is outside the bandwidth of the
selected wavelength. The greater the stray light, the higher the degree of inaccuracy of Absorbance readings which are more than around 1.5 Absorbance units.
Many compounds produce the types of calibration curves shown below.
An analyst cannot therefore use the whole of the calibration curves shown below. They should only use the linear portions, where a clear single gradient (p to q) is seen. Hence standard and sample concentrations are valid in the concentration range R.
Some commonly required measurements, such as time plots, thermal melts, kinetics, concentrations of oligonucleotides, levels of chemical elements, amounts of proteins, observance of cell growth, Transmittance of light through lenses etc., may then be produced using UV/Visible spectrophotometers.
There are occasions when the reflectance of light, on normally solid samples needs to be determined, measurements may then be expressed as internationally recognised colour values, such as CIE L*u*v*. UV/Visible spectrophotometers may also be used for these measurements.
Most UV/Visible spectrophotometer samples are in the liquid form, but sometimes solid samples are used, especially in the determination of Transmittance and for reflected colour analyses. Occasionally samples in the gaseous form are used in a UV/Visible spectrophotometer.
The cuvette/cell, sample container, should be appropriate to the type of sample used and measurement to be determined.
If an analyte does not naturally contain a UV/Visible chromophore, a UV/Visible chromophore may be chemically added to it, so that the resulting chemically modified or derivatised analyte, can be detected in a UV/Visible spectrophotometer.
In addition, samples may need pre-
Single Beam Spectrophotometers
Here a single beam of light is passed through a single sample container and the resulting light is detected by a detector. In the case of single beam spectrophotometers, most detectors are silicon diodes.
A filter and a monochromator with a slit, split the light to the chosen wavelength.
The bandwidth (bandpass) of the spectrophotometer, relates to the size of the monochromator’ slit for example 4 nm.
A narrow bandwidth will produce wavelength scans of higher resolution than a wider bandwidth. However, there will be less light energy to reach the detector, consequently, there can be a loss of sensitivity at narrow bandwidths.
If a filter alone is used to split the light, a very wide bandwidth results. In addition, it is not possible to perform wavelength scanning, as discrete filters need to be physically inserted for each wavelength range used.
Single Beam Spectrophotometers are of the simplest in design hence have lower capital and maintenance prices than other spectrophotometer types.
Double Beam Spectrophotometers
Here the light leaving the monochromator is split, using a beam splitter, into a sample beam and a reference beam. After each beam of light is passed through its respective sample/reference (blank) container, each beam is then detected by its own detector. The sample and reference are simultaneous /measured/scanned, saving time and providing for optimum accuracy.
Hence the double beam spectrophotometer can have two detectors. Most detectors are silicon diodes. Double beam spectrophotometers ensure that any fluctuations in the light emitted from the lamp are applied equally to both the sample and the reference beams, hence double beam spectrophotometers offer around ten times better stability and 2 to 5 times lower baseline noise, than single beam spectrophotometers of the same brand.
For greater sensitivity, the two silicon diode detectors may be substituted with a single photomultiplier detector. Photomultiplier detectors are used in the more expensive spectrophotometers and have a maximum wavelength range of 900 nm. In practice, most UV/Visible measurements occur within the 190 to 800 nm wavelength range.
End window photomultipliers can collect up to 100 times more light. Hence it is possible to measure highly light scattering samples. A single photomultiplier detector simultaneously measuring both sample and reference beams, is more precise than a two detector system. Good accuracy and performance occurs, even at narrow optical bandwidths
Split Beam Spectrophotometers
Here light is split into two beams, one beam passes through the sample and the other acts as a reference at the detector/s. Hence a smaller amount of lamp energy is available for use in measurement, consequently there is a lowering of sensitivity.
Diode Array Spectrophotometers
Some spectrophotometers have a ‘bank’ of a thousand or so mini detectors or diodes, which are arranged as an array. Each diode will detect the band of wavelength of light which has been focussed upon it.
With diode array spectrophotometers, light from the active lamp is passed through a polychromator after passing through the sample. The polychromator disperses the light onto the array of diodes, so that each diode will measure a discrete band of wavelengths.
These diode array spectrophotometers can therefore produce a wavelength scan almost instantly, however the sensitivity of measurements is compromised. No sample compartment lid is necessary and no moving parts are used.
Common Accessories and Consumables
autosamplers. These are useful when large numbers of samples require unattended measurement.
calibration standards. As part of the instrument validation process, these are used to check the wavelength accuracy, Absorbance accuracy, stray light and bandwidth of spectrophotometers. They can be solid filters or solutions.
cells (cuvettes). A wide variety of rectangular or circular cells are available. Typical pathlengths range from 1 to 100 mm. Working volumes can start from 1 µL, especially if nano cells are used. Flow cells are used if liquids are to be continuously passed through a single cell. Fibre optic cells may be used if the sample is some distance from the spectrophotometer.
cell stirring. There are occasions where the sample should be stirred whilst the measurements are taken, magnetic flea, cell stirrers are available.
deuterium lamps. These are used to create wavelengths within the 190 to 365 nm wavelength range. They are a relatively expensive consumable, so that their life is often guaranteed. Many lamps will deteriorate over time, even if they are not used.
dissolution accessories. Controlled release tablets/capsules need to be tested to ensure that they do actually release the active pharmaceutical ingredient/s or nutrient/s at the required rate. Accessories include dissolution bath systems which involve dissolution vessels, paddles, tester sinkers/baskets, eight channel peristaltic pumps,
eight position automatic cell changer and the appropriate flow cells and dissolution software.
integrating spheres. Diffuse reflectance measurements are performed by means of an integrating sphere. An integrating sphere consists of a completely spherical chamber. The inner wall of the chamber is made of a material that provides the maximum possible reflectance over the entire visible wavelength range.
multi cell changers. These can be useful, especially when more than one sample is to be measured within a short time space, or over a given period of time, and the process is to be repeated. These changers may be incorporated with temperature controller devices.
PC use and control. Programmes are available to control spectrophotometers and to export spectrophotometer data to a PC/laptop.
sipettes. These are systems where a sample is aspirated into a flow cell, a reading taken, and the sample returned to its original container or to waste. This is useful for hazardous samples, as less user contact with the sample is achieved. Sipettes are also useful for continuous monitoring of processes.
specular reflectance accessory. Specular reflectance is normally used for the measurement of samples which surface reflect light but show very little scatter. For example, the reflecting properties of a coated optical mirror may be measured.
temperature controllers. Sometimes samples need to measured at specific temperatures. Thermoelectric Peltier devices offer a fast and temperature programmable means. Circulating laboratory water baths may also be coupled to spectrophotometer cell holders to achieve a specific cell temperature.
tungsten halogen lamps. These are used to create wavelengths within the 320 to 1,100 nm wavelength range.
xenon lamps. These are used to create wavelengths within the 190 to 1,100 nm wavelength range. They are a relatively expensive consumable, so they are often incorporated in ‘press to read’ technology, so they are only switched on when a measurement needs to be made. However, they do not emit good energy levels within the whole of the 190 to 1,100 nm wavelength range, and they often emit an irritating high pitched noise.
Please be aware that individual spectrophotometers may be used with optional software and optional accessory hardware configurations, defined by a user’s individual requirements.
UV/Visible spectrophotometry is a mature and established technique, with inbuilt flexibility to detect and measure millions of compounds (analytes) in a wide variety of sample matrices. This technique is used within a wide variety of analytical chemistry laboratories, such as within the following sectors:-
• Life Science commercial enterprises.
• Research and Teaching.
• University, Life sciences, Chemistry, Materials Sciences, Environmental, Medical departments.
• Hospitals and Clinics.
• Food and Drinks manufacturing.
• Food and Drinks testing/regulation.
• Water Suppliers.
• Veterinary chemicals.
• Veterinary clinics.
• Industrial Chemicals.
• Manufacturing and Production facilities.
• Security facilities.
Some molecules are able to absorb certain ideal wavelengths of ultra-
At those given wavelengths, the amount of UV/Visible chromophore in a sample may be measured. Generally, the higher the amount of UV/Visible chromophore detected, the higher the concentration of that compound.
Light generated by certain lamps, such as tungsten, deuterium or xenon, is separated into discrete bands of wavelengths of light by a monochromator, and then is passed through a sample via a slit. A sample with a UV/Visible chromophore absorbs a certain amount of light, the remaining light is detected by a detector.
Liquid samples are held in cuvettes (or cells) within a spectrophotometer. The width of the cell, in which light passes through the sample is the pathlength, a standard pathlength used by many users is 10 mm.
A = Є x l x c
Where, at a specific wavelength,
A is the measured Absorbance,
Є is the molar absorbtivity or extinction coefficient (M-
l is the pathlength (cm),
c is the analyte concentration (M).
Provided that the concentration is measured in the linear part of the calibration curve for each specific analyte, the law will apply in practice.
If Transmittance measurements are required, then there is the following relationship:-
A = -
Cuvettes are made of thin, uniform glass or plastic that is highly transparent to light at the wavelengths used. Some wavelengths such as those in the ultraviolet region (190 to 300 nm) require cuvettes made of other materials such as quartz or silica.
The surfaces of the cuvette through which light passes through should be clean, clear and free of fingerprints and grease.
For very low analyte concentrations, cells of pathlength larger than 10 mm may be used. For very high analyte concentrations, cells of pathlength smaller than 10 mm pathlength may be used.
Of course, it is possible to dilute solutions which have high analyte concentrations, so that their Absorbances are within the Absorbance range of the spectrophotometer. And vice versa for solutions which have low analyte concentrations.
UV/Visible spectrophotometers are mainly used to measure the Absorbance/Transmittance of analytes within liquids. These liquids are placed inside cuvettes, which are then positioned within the spectrophotometer. These cuvettes and the liquids themselves, have their own Absorbance/Transmittance values. The Absorbance/Transmittance values of the cuvettes and liquids must be accounted for. This is performed by the use of a blank (reference).
In single beam spectrophotometers, a cuvette filled with the liquid, but without analyte, is placed in the spectrophotometer, and at the required wavelength is 'zeroed'. When a cuvette filled with the liquid and the analyte, is subsequently placed in the spectrophotometer, the difference in Absorbance/Transmittance value is therefore attributed to the analyte itself.
In double beam spectrophotometers, there are two beams of light. So initially, two cuvettes filled with the liquid, but without analyte, are placed in each beam the spectrophotometer. At the required wavelength, the spectrophotometer is 'zeroed'. Then a cuvette filled with the liquid and the analyte, is subsequently placed in the sample beam of the spectrophotometer, so that the difference in Absorbance/Transmittance value is therefore attributed to the analyte itself. The advantage here, is that any variations of the liquid, such as slight precipitation, chemical reactions and temperature etc., apply to both the sample and the blank, at the same time.
In order to determine the specific wavelength, at which a user wishes to make a measurement, it is prudent to perform a wavelength scan.
A wavelength scan is a plot of Absorbance on the y axis and wavelength on the x axis, for a particular analyte.
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