Color Spectrophotometer - Principle, Parts, Procedure, Applications

Color Spectrophotometer is a precise analytical instrument that is used to measure and define the colour of a material. It measures colour by studying the interaction of light with the sample. It gives the colour in numerical form and reduces the error of human eye observation.

It works by passing or reflecting a beam of light on the sample. The light may cover visible region and sometimes ultraviolet (UV) and infrared (IR) region also. The instrument then measures how much light is absorbed, transmitted or reflected at different wavelengths.

The colour obtained from the sample gives a special spectral pattern. This is known as the spectral fingerprint of the material. By this method, the colour can be measured accurately and compared with standard colour.

Color spectrophotometer is used in different industries for colour matching and quality control. It is used to maintain same colour in paints, textiles, plastics, food products, cosmetics and printing materials.

The spectrophotometer was invented in 1940 by Arnold O. Beckman and his colleagues at National Technical Laboratories. This company later became Beckman Coulter. Beckman developed this instrument to measure the absorption of ultraviolet light, which earlier instruments could not measure properly.

The first instrument was known as Model A. It used a glass prism. Later, Model B was developed, which used a quartz prism and gave better absorbance result.

After more improvement, Model D was introduced. It was also known as the DU spectrophotometer. It was produced from 1941 to 1976 and became an important instrument in bioscience studies.

In 1979, Hewlett-Packard introduced the HP 8450A. It was the first commercially available diode-array spectrophotometer. It was controlled by microprocessor and could scan many wavelengths within few seconds.

This development helped in making modern spectrophotometers more rapid, accurate and useful for colour measurement. The same basic principle is now used in advanced color spectrophotometer for exact colour analysis.

Principle of Color Spectrophotometer

Principle of Color Spectrophotometer is based on the measurement of light absorbed, transmitted and reflected by the coloured sample. When a beam of light is passed on the sample, some part of light is absorbed by the material. The remaining part is passed through the sample or reflected from the surface.

In this instrument, the light is produced from a built-in light source. The white light then enters into the monochromator. The monochromator has prism or diffraction grating, which separates the light into different wavelengths.

These wavelengths are allowed to fall on the sample one by one. During this process, the molecules of the sample absorb some selected wavelengths. Other wavelengths are not absorbed and they are transmitted or reflected.

The transmitted or reflected light then reaches to the photodetector. The detector measures the intensity of this light. It also compares this light with the original beam of light.

The difference in light intensity gives the amount of absorption, transmission or reflection. The instrument then calculates these values at each wavelength. A spectral curve is formed, which gives the exact numerical record of the colour. This is referred to as the colour fingerprint of the sample.

Parts of Color Spectrophotometer

Light source – It is used to produce the first beam of light in the instrument. The light source gives stable light which falls on the sample. Tungsten-halogen lamp, deuterium lamp, xenon flash lamp or LED are commonly used as light source.

Monochromator – It is used for selecting the required wavelength of light. The white light from the source enters into the monochromator and gets separated into different wavelengths. It may contain a prism, diffraction grating or optical filters.
Collimator – It is used to make the light beam straight and parallel. The collimator directs the beam in proper path through the instrument. It helps the light to pass correctly towards the sample.
Sample holder – It is the part where the sample is placed for measurement. The sample may be kept in glass cuvette, quartz cuvette, petri dish or clamp. It keeps the sample in fixed position, so that light can pass through or reflect from the sample properly.

Optical system – It consists of lenses, mirrors and other optical parts. These parts are used to guide the light beam from one part to another part of the instrument. It also helps in focusing the light on the sample and detector.
Filters – These are used to allow only selected wavelength of light to pass. Sometimes filters are used instead of monochromator. They also reduce unwanted light and make the measurement more stable.
Detector – It is used to receive the light that is transmitted through the sample or reflected from the sample surface. The detector converts the light energy into electrical signal. Photodiode, photomultiplier tube (PMT) and charge-coupled device (CCD) are used as detector.

Reference cell – It contains blank or standard solution. It is used for baseline correction before measuring the actual sample. This helps to reduce error due to lamp variation and other optical changes.
Digital processor – It receives the electrical signal from the detector. The raw signal is processed and converted into useful data. It calculates absorbance, transmittance or reflectance of the sample.
Display unit – It shows the final result of the measurement. The result may be shown as wavelength, intensity value, absorbance value or spectral curve. It may be present as digital screen or computer software window.

Software – It is used to control the instrument and adjust different measurement conditions. Scanning range, wavelength interval and data storage are controlled by the software. It also helps in comparing the sample colour with standard colour.

Operating Procedure of Color Spectrophotometer

The color spectrophotometer is first placed in a stable working area. The temperature and humidity should be controlled. The instrument should not be kept in direct sunlight, smoke, dust or chemical vapours.
The instrument is switched on and allowed to become stable for some time. The measuring conditions are then selected. The standard illuminant, observer angle, colour space and reference colour are selected according to the type of sample.

The aperture and calibration tiles are cleaned before calibration. Dust or stain on the tile can give wrong reading. So the surface of the tile and measuring port should be clean and dry.
For solid or powder sample, black calibration is first done by using a light trap. It gives 0% reflectance. Then white calibration is done by using a certified white tile, which gives 100% reflectance. Sometimes green tile is used for checking the proper working of the instrument.
For liquid sample, a clean and scratch free cuvette is taken. It is filled with pure solvent or distilled water. This is placed in the cell compartment for zero adjustment or blank measurement.

The liquid sample is prepared before measuring. Air bubbles are removed by degassing or by using ultrasound bath. If the sample is turbid, it is filtered or centrifuged. If the sample is wax like or solid, it is melted gently in water bath.
The powder sample is prepared by pressing it into a tablet form or by placing it in a glass sample cell. The cell is tapped on a hard surface, so that the powder settles evenly. This reduces shadow formation and light trapping.
The prepared sample or cuvette is placed firmly in the specimen holder or cell compartment. The sample should be placed in proper position. The light path should not be blocked or disturbed.

The reading is started after placing the sample. The instrument produces a beam of light. This light is separated into different wavelengths by the monochromator and then directed on the sample or through the sample.
The light transmitted through the sample or reflected from the sample surface is received by the detector. The detector changes the light signal into electrical signal. This signal is then sent to the internal processing system.
The instrument calculates the absorbance, transmittance or reflectance of the sample. It may also calculate colour values such as CIE Lab* and Delta E value. These values are shown on the digital display or in the software.

In case of irregular sample like loose powder, the sample is removed and refilled again. The reading is taken at least three times. The average value is taken as the final result, because it reduces variation due to uneven texture.
After measurement, the cuvette, sample holder and calibration tiles are cleaned properly. They are dried and stored in proper place. This prevents contamination and helps to maintain the accuracy of the instrument for long time.

Types of Color Spectrophotometer

Visible (VIS) spectrophotometer – It is used to measure light only in the visible region. The range is generally 400-700 nm. This type is mainly used for colour analysis of different materials.

Ultraviolet (UV) spectrophotometer – It measures light in the ultraviolet region. It is used for studying proteins, nucleic acids and other chemical compounds. It is mostly used in laboratory analysis.
UV-Vis spectrophotometer – It measures both ultraviolet and visible light region. This type gives more flexible measurement. It is used in research work, chemical analysis and industrial colour testing.
Infrared spectrophotometer – It is used to measure absorbance in the infrared (IR) region. It helps to identify chemical bonds and functional groups present in the sample. It is more related with chemical structure study.

Fluorescence spectrophotometer – It measures the intensity of fluorescent light emitted from the sample. In this type, the sample absorbs light and then emits light at another wavelength. The emitted light is measured by detector.
Atomic absorption spectrophotometer (AAS) – It is used for analysis of metal ions. In this type, vaporized atoms absorb radiation. The amount of absorption gives the quantity of metal present in the sample.
45/0 spectrophotometer – It is a directional type color spectrophotometer. The light falls on the sample at 45° angle and the reflected light is measured at 0° angle. It is used for smooth and matte surface.

d/8 spectrophotometer – It is a diffuse sphere type spectrophotometer. It uses a spherical inner surface to spread light evenly on the sample. This type reduces the effect of gloss and texture, so it is used for textiles, plastics and rough surface materials.
Multi-angle spectrophotometer – It measures reflected light from many different angles. It is used for materials whose colour changes with viewing angle. Metallic paint, pearlescent paint and automotive coating are measured by this type.
Single-beam spectrophotometer – It has only one path of light for measurement. First blank is measured and then the sample is measured. It is simple, less costly and used for routine work.

Double-beam spectrophotometer – It divides the light beam into two paths. One beam passes through the sample and another beam passes through the reference. It gives stable reading and reduces error due to lamp fluctuation.
Scanning spectrophotometer – It measures light intensity at one wavelength at a time. A movable diffraction grating is used for selecting wavelength. The wavelengths are scanned step by step and spectral curve is formed.
Array spectrophotometer – It measures many wavelengths at the same time. The complete light spectrum passes through the sample and a sensor array like CCD detects the wavelengths. This type gives faster reading.

Benchtop spectrophotometer – It is a fixed type instrument used in laboratory. It is strong and gives more precise result. It is mostly used in quality control and research work.
Portable or handheld spectrophotometer – It is small and easy to carry. It is used for quick colour checking in field, factory floor and production area. It is useful where sample cannot be brought easily to laboratory.
In-line spectrophotometer – It is fixed directly in the production line. It measures colour continuously without touching the product. It is used for real time colour control in fast moving industrial process.

Applications of Color Spectrophotometer

It is used to check colour consistency and quality of food and beverage products. Products like beer, wine, juices, edible oils and dairy products are checked by this instrument. It also helps to know ripeness, clarity and turbidity of liquid sample.
It is used to detect contamination in food and liquid products. Any change in colour may show spoilage, impurity or unwanted chemical change. This helps in maintaining quality of the final product.
It is used in textile industry for matching dyes in different fabric batches. The colour of cloth is checked so that same shade is maintained during production. It is also used to study fluorescent optical brighteners.

It is used to control metamerism in textile and apparel products. In this condition, two colours may look same under one light but different under another light. The instrument helps to compare the colour under different lighting condition.
It is used in pharmaceutical industry for checking the colour of tablets, capsules and liquid medicines. It helps to maintain same colour in every batch. It is also used for measuring concentration and purity of active pharmaceutical ingredients (APIs).
It is used in clinical laboratory for analysis of DNA, proteins and other biological samples. It is also used in skin colour analysis. The colour reading gives numerical value, which is more exact than visual checking.

It is used in paint and coating industry for preparing new colour formula. It helps to match paint colour on different surfaces like metal body parts, plastic bumpers and coated panels. Matte and glossy finish can also be checked by it.
It is used in plastic and polymer industry for maintaining uniform colour in finished plastic products. It also helps to check the concentration of colour additives and pigments. Recycled plastic materials are also tested for colour consistency.
It is used in printing and packaging industry to control colour quality of labels, graphics and consumer packages. The printed colour is compared with the standard colour. This helps the final product to appear as required.

It is used in water treatment for checking true colour and clarity of water. Drinking water and wastewater can be tested by this method. Pollutants like chlorine and dissolved organic matter are also measured.
It is used in environmental monitoring to study pollution level in water sample. Colour change of water may indicate chemical contamination or organic matter. It is used in testing industrial discharge, river water and wastewater.
It is used in chemical manufacturing for checking colour and composition of dyes, pigments and liquid chemicals. Any change in colour may show contamination, degradation or process variation. So it is useful in routine quality control.

It is used in battery manufacturing to monitor the colour and condition of battery electrolytes. Change in colour may indicate impurity or chemical breakdown. It helps to keep the production process stable.
It is used in cosmetic industry for making and matching exact shades. Products like foundation, lipstick and powder are checked by this instrument. It helps to repeat the same shade in different batches.
It is used in agriculture to study crop health and nutrient condition. Leaf colour and crop colour can be measured by spectrophotometric method. Sometimes hyperspectral imaging is used for checking plant growth and stress condition.

Advantages of Color Spectrophotometer

It gives accurate and precise colour measurement. The colour is measured by numerical value, so the result does not depend on eye observation. It gives detailed spectral data from the sample.
It helps to reduce human error in colour matching. Human eye may see same colour differently under different condition. But color spectrophotometer gives fixed and objective reading.
It can detect metamerism in coloured materials. In metamerism, two colours may look same under one light source but look different under another light source. The instrument measures full spectrum, so this difference can be identified.

It is useful for making complex colour formulation. New paint recipes, colourants and standard colour formula can be prepared by using its spectral data. This spectral data is also referred to as colour fingerprint.
It can measure many types of samples. Solid materials, liquid samples, loose powders and transparent materials can be analyzed by this instrument. Some instruments can also measure colour and turbidity of liquid at the same time.
It allows adjustment of light condition during measurement. Different illuminants and observer angle can be selected in the instrument. This helps to know how the colour will appear in daylight, store light or other lighting condition.

It helps to detect impurity and degradation in the sample. Small change in colour or spectral value may show contamination, chemical change or product breakdown. This is useful in quality control.
It gives rapid measurement of colour. Modern instruments can give result within few seconds. The sample is generally not destroyed during measurement, so it is a non-destructive method.
It can be connected with computer software. The software stores the reading and prepares colour data. It also helps in automatic comparison, analysis and record keeping.

It is useful for maintaining same colour in different production batches. The sample colour can be compared with standard colour. This helps to keep uniform colour in food, textile, paint, plastic, cosmetic and printing industries.

Limitations of Color Spectrophotometer

It is costly instrument than simple colour measuring instruments. The initial cost is high, so it may not be suitable for small laboratory or small industry. Maintenance cost also becomes more in some models.
It is complex in working and handling. The instrument has different settings like illuminant, observer angle, wavelength range and colour space. So trained person is needed for proper operation.

It needs regular calibration before measurement. Black tile, white tile and sometimes green tile are used for calibration checking. If calibration is not done properly, the reading may become inaccurate.
It is sensitive to instrument drift. Lamp ageing, detector change and optical part variation can affect the reading. For this reason, regular maintenance and lamp replacement is needed.
It can be affected by environmental condition. Temperature change, high humidity, dust, smoke and chemical vapours can disturb the measurement. Some samples may also show colour change with temperature.

It gives wrong result if sample preparation is not proper. Fingerprints on cuvette, scratches on glass, air bubbles in liquid and variation in path length can affect the reading. So sample should be handled carefully.
It may show error due to stray light. Stray light is the unwanted light that enters into the detector. It can cause non-linear reading and reduce the accuracy of measurement.
It can be affected by optical misalignment. Mechanical shock, vibration or routine wear can disturb the position of lenses, mirrors and other optical parts. This can change the light path and affect the result.

It is difficult to measure non-uniform and textured samples. Loose powders and rough surfaces may cause light trapping and shadow formation. For this, sample is prepared carefully and several readings are averaged.
In liquid analysis, it has some limitation due to Beer-Lambert law. If extinction coefficient or path length is not known, concentration cannot be measured properly. Suspended particles may also scatter light and distort the data.
Some buffer or reagent may absorb light at same wavelength as the sample. This overlap can interfere with the actual absorbance of analyte. So blank measurement and proper wavelength selection is needed.

Transparent, glossy or highly reflective sample may create measurement problem. Reflection and scattering from the surface may not be uniform. It can produce variation in the final colour value.

Precautions of Color Spectrophotometer

The cuvette should be handled only from the frosted or ridged side. The clear side should not be touched by fingers. Fingerprints can scatter the light and give wrong reading.
Clean and scratch free cuvette should be used for measurement. The optical window of cuvette should be wiped gently with lint free tissue before and after reading. Dust, stain or scratch can affect the light path.

The liquid sample should be free from air bubbles. If bubbles are present, the sample should be tapped gently or degassed by ultrasound bath. Air bubbles can block or scatter the light.
The sample should not contain dust and suspended particles. Turbid sample should be filtered or centrifuged before measurement. Suspended particles may scatter light and change the absorbance value.
For translucent material, the sample should be folded, stacked or wound properly. This is done so that background colour does not interfere with the reading. The sample thickness should also remain same as far as possible.

The calibration tiles should be kept in protective case after use. They should be protected from direct sunlight and ultraviolet (UV) radiation. Dirty or faded tiles can produce wrong calibration.
The white reference tile should be touched only from the edges. It should not be held for long time, because finger heat and dirt can affect the tile surface. The tile should not be rubbed, rotated or slid against the instrument port.
Dust from black calibration trap should be removed carefully. Compressed air may be used for removing dust. The trap should not be scratched or touched inside.

The instrument should be calibrated frequently before use. Calibration may be done daily, at the starting of work or after few hours of continuous work. This reduces drift error due to lamp ageing and environmental change.
The instrument should be used in a stable temperature room. Direct sunlight should not fall on the instrument. Temperature change can cause thermochromic colour change in sample and also thermal drift in the instrument.
The room humidity should be maintained properly. Very low humidity may produce static electricity and very high humidity may cause internal condensation. The humidity should be kept around 20% to 85%.

The working area should be free from smoke, dust and chemical vapours. These substances can damage internal optical parts and integrating sphere. They can also affect the accuracy of colour measurement.
The instrument should be given continuous and stable power supply. Frequent switching on and off may cause heating and cooling variation. The instrument should be allowed to become stable before taking the reading.
The sample holder, aperture and optical surface should be kept clean. Any stain or deposited material in these parts can disturb the light beam. Cleaning should be done carefully according to instrument instruction.

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Color Spectrophotometer – Principle, Parts, Procedure, Applications