COLOUR QUANTIFICATION

As published in LabPlus international - October 2006

Principles and applications of reflectance spectrophotometry

The precise measurement and comparison of the colour of various objects is frequently required in a wide range of industrial and laboratory applications. The most objective way of measuring colour is by use of reflectance spectrophotometry, a technique which can be carried out with the use of appropriate accessories in a modern spectrophotometer. This article describes the principles and applications of reflectance spectrophotometry.
fluorescence or phosphorescence of the sample. stray light (i.e. light other than light of the selected wavelength reaching the detector). non-monochromatic radiation. changes in refractive index at high analyte concentration. shifts in chemical equilibria as a function of concentration. very large and complex molecules. However in practice, provided that steps are taken to ensure that the concentration is measured in the linear part of the calibration function, the Beer-Lambert law applies.

Basic principles of UV/Vis spectrophotometry

Most analytical scientists are familiar with UV/Visible spectrophotometers for the measurements of concentrations and wavelength scanning of UV/Visible absorbing analytes in liquids. There are also occasional analyses where the measurement of either transmittance in solids or absorption of gases is needed. The basic principles of UV/Visible spectrophotometry are well known: Light is generated by a source lamp which is normally a tungsten lamp for the visible region of the spectrum and deuterium for the ultra-violet range. The light is dispersed into its constituent wavelengths in a monochromator which results in a narrow band of the dispersed spectrum passing from the exit slit of the monochromator. Suitable optics are used to lead this light, of a narrow wavelength band, to the sample to be measured. A sample with a UV/Visible chromophore sample absorbs a certain amount of light and the remaining light is detected by a suitable detector in the spectrophotometer. The Beer-Lambert law is then applied to determine the concentration of a specific analyte in the sample at a specific wavelength: A=xlxc where, at a specific wavelength, A is the measured absorbance, is the molar absorptivity or extinction coefficient (M-1 cm-1), l is the path length (cm),

Reflectance spectrophotometry
In addition to the familiar absorbance measurements described above, there are other types of measurements which can be carried out using UV/Visible spectrophotometers. Among such additional measurements are those which are based on the ability of a spectrophotometer to measure the reflectance of materials. Reflectance measurements are of great value in providing a reference standard for the comparison of the colour of different samples. A reflectance spectrophotometer is similar to a standard UV/Visible spectrophotometer. It should have a bandwidth narrow enough to provide well resolved visible spectra yet wide enough to provide a good energy level for diffuse reflectance measurements. The reflectance spectrophotometer must also have optics and electronics systems of high sensitivity, and should be able to physically accommodate reflectance and transmission accessories. The adapted spectrophotometer must be able to make measurements both at selected fixed wavelengths or perform scans over the complete wavelength range.

Figure 1. The Cecil Instruments ReflectaScan spectrophotometer. c is the analyte concentration (M). The relationship between Absorbance and Transmittance is: A = log T The Beer-Lambert law describes the linear relationship between absorbance and concentration. However, there are some restrictions to the law, and the linearity of the Beer-Lambert law is limited by chemical and instrumental factors. Causes of non-linearity include: deviations in molar absorptivity coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity. scattering of light due to particulates in the sample.

COLOUR QUANTIFICATION
An example of a modern high quality reflectance spectrophotometer is the Cecil Instruments' ReflectaScan Reflectance system which uses a 50 mm sphere [Figure 1]. Reflectance measurements are made using both diffuse and specularly reflected light. In diffuse reflectance, light is scattered in all directions from the sample. Provided that this scattered light can be collected onto an optical detector, the surface reflectance may be measured either at a given wavelength, or by performing a scan over a range of wavelengths. Such a wavelength scan can then be used to characterise colour. In specular reflectance, the light reflected from a sample has an angle of reflectance equal to the angle of incidence of the illuminating light. Specular reflectance is normally used for the measurement of samples whose surface reflects light but shows very little scatter. For example, the reflecting properties of a coated optical mirror may be measured using specular reflectance. In the past, mirror systems were used to measure diffuse reflectance, but nowadays accurate diffuse measurements are carried out by means of an integrating sphere [Figure 2]. sphere to minimise the attenuation of the light reaching the detector. For this reason the inner surface must be constructed from material of the highest reflectivity available. For accurate measurements, the surface area of the sphere that is used for the inlet, outlet and sample ports should not exceed 8% of the total surface area. The design of the Cecil Instruments' sphere satisfies this condition; provision is also made for the easy insertion of the sphere into the spectrophotometer [Figure 3].

Figure 2. A typical integrating sphere accessory. 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. Previously, barium oxide coatings were used but much improved results are now obtained by the use of the extremely reflective material, Spectralon. Sphere diameters are typically in the range of 50 mm to 300 mm diameter. Results are possible even with a smaller 50 mm sphere, which is used in the ReflectaScan spectrophotometer. Light is scattered in all directions from a sample within a half spherical space. This light is multiply reflected at the surface of the sphere and eventually reaches the optical detector for detection and measurement. In addition to the normal spectrophotometer signal channel, the ReflectaScan has a second optical detector and signal channel for diffuse measurements; this provides for optimal performance. Measurements of diffuse reflectance may be made either with the specular component included or excluded. The multiple reflection of the light at the inner surface of the integrating sphere demands the highest possible reflectivity of the inner surface of the

Figure 3. A schematic view of the inside of an integrating sphere. Light of the selected wavelength enters the integrating sphere via the aperture and is reflected by the internal mirror on to the sample. All reflected light, no matter at which angle or in which plane, is collected by the highly reflective internal surface of the sphere so that the total amount of light reflected by the sample can be measured by the detector. Changing of the angle at which the incoming light strikes the sample enables measurement of both diffuse and specular reflectance. Generally reflectance measurements entail fitting an integrating sphere into a UV/Visible spectrophotometer and using the reflectance or transmittance scale. UV/Visible spectral scans of diffuse samples are obtained with reference to a white standard, normally Spectralon. Measurements of reflectances found at various wavelengths under specified light conditions may then be used to compute many absolute colour concept values. These include X Y Z tristimulus values, x y Y chromaticity co-ordinates, CIE L* A* B*, CIE L* U* V*, L*C*h colour space, whiteness etc. Such colour values are used to define particular colours, shades and intensities of colours without the need for comparison to standard solutions or colour chart. These computations are performed automatically in the ReflectaScan.

COLOUR QUANTIFICATION

Hence one or more of the absolute colour values can be used to define: The brilliance of a shade of white paint. The whiteness of a piece of limestone, marble etc. The colour of a paper, paint, fabric, cloth. The colour differences encountered between shades of paint. The matching of paint colours. UV cover and shade protection factors of shade cloth. Luminous transmittance of lenses for sunglasses and sunglare filters. Spectral reflectance of fabric. The effectiveness of an anti-reflective lens coating. Diffuse reflectance of powder samples. The concentrations of dyes in fabrics. Integrating spheres may also be used to determine the transparency and reflectance to light of curved surfaces, such as bottles, vials, contact lenses and spectacle lenses. An incidental advantage of an integrating sphere is that since all the forward scattered light that has passed through the sample is collected in the sphere, measurements of transmittance can be easily made in turbid samples, such as blood or brewing media. Specular Reflectance may be used to determine the thickness of thin films and is accurate, non-destructive, fast and requires little or no sample preparation. Typical films include semi-conductor films, coated lens, deposited coatings, anti-reflective lens coatings etc [Figure 4]. Figure 4. A typical specular reflectance accessory.

The minimum thickness which may be determined by this method is of the order of 0.1 microns; a sample area as small as 2 mm diameter may be measured. The amount of light reflected from a thin film over a range of wavelengths is measured, with the incident light at a known angle to the sample surface. The thickness of the thin film can then be automatically computed from the wavelength separation of the interference bands of the wavelength scan [Figure 5].

Figure 5. The thickness of a thin film can be automatically computed from the wavelength separation of the interference bands of the wavelength scan. Cecil Instruments manufactures the ReflectaScan Reflectance spectrophotometer, which contains powerful embedded software for the automatic calculation of a comprehensive range of colour and film thickness measurements. Cecil Instruments, Cambridge, UK. Tel + 44 1223 420821