Spectrophotometer Cuvette - Features, Types, Materials, Precautions, Advantages

Spectrophotometer cuvette is a small transparent tube like laboratory vessel that is used to hold liquid, gas or solid sample during spectroscopic analysis. It is derived from French word cuvette, meaning little vessel. It may be square or circular in cross-section and has straight sides.

It has at least two parallel and optically clear windows. Through these windows, the light of definite wavelength enters from the front side, passes through the sample and comes out from the back side in a straight line. Then the detector measures how much light is absorbed, transmitted or emitted by the sample.

The cuvette is not only a simple container. It is a precision optical component of the spectrophotometer. It fixes the exact path length through which light travels in the sample. It also reduces stray light and helps in getting correct optical reading.

Most cuvettes are made of glass, quartz or plastic depending on the type of light and sample used. Quartz cuvette is generally used for ultraviolet light because it can transmit UV radiation. Glass and plastic cuvettes are mostly used for visible light range.

In the mid-1950s, thermal bonding and heat fusion techniques were developed for cuvette manufacturing. In this process, optically polished quartz or glass parts are fused together by heat. This removed the use of adhesives and improved optical accuracy, leakage resistance and chemical stability of the cuvette.

Thus, spectrophotometer cuvette acts as an important part of spectrophotometric measurement. It holds the sample, allows proper light passage and maintains fixed optical path for accurate absorbance or transmittance reading.

Characteristics Features of Spectrophotometer Cuvette

Spectrophotometer cuvette is a small transparent tube like vessel that is used to hold the sample in the optical path of a spectrophotometer.

It is used to measure the absorbance or transmittance of light passing through the sample.
The standard absorbance cuvette has two parallel clear windows. These windows are optically polished and allow the light beam to pass in a straight line.
The other two sides of the cuvette are generally frosted. These sides are used for handling the cuvette and prevent fingerprints on the clear optical surface.

Cuvettes are made up of different materials depending upon the wavelength of light used. The common materials are quartz, optical glass, polystyrene and PMMA.
Quartz cuvette is used for both ultraviolet and visible light range. Glass cuvette is used mostly for visible light range. Plastic cuvettes are disposable and used for common visible range work.
The optical path length is the inner distance between two transparent windows. It is the distance through which light travels inside the sample.

The standard path length of a cuvette is 10 mm. But it may vary from 0.01 mm to 100 mm according to the concentration of the sample.
The standard outer size of a spectrophotometer cuvette is about 12.5 mm × 12.5 mm and height is about 45 mm. This helps the cuvette to fit into normal instrument holder.
The volume capacity of cuvette varies according to its type. Macro cuvette holds more than 3.5 mL, standard cuvette holds about 3.5 mL, while micro and sub-micro cuvette hold very small volume of sample.

The cuvette is usually filled up to about 80% of its capacity. This prevents spilling of the sample during handling.
Z-dimension is the distance from the base of the cuvette to the center of the light aperture. It is important in micro-volume cuvettes because it must match with the light beam height of the instrument.
High quality cuvettes have very accurate path length. They also have flat and parallel optical windows which reduces light scattering and distortion of signal.

Reusable glass and quartz cuvettes are made by thermal bonding or heat fusion. In this method, adhesives are not used and the cuvette becomes more resistant to heat and organic solvents.
Some cuvettes are provided with caps or closures. These may be PTFE lids, silicone lids, stoppers or screw caps with septa. These are used to prevent evaporation, spilling and oxygen exposure of sample.

Which Material is used to build Spectrophotometer Cuvette?

Quartz is one of the most important material used to make spectrophotometer cuvette. It is highly transparent and used for ultraviolet (UV) and visible range measurement.

UV Quartz or fused silica cuvette is used for UV-visible spectroscopy. It allows the passage of ultraviolet light without much absorption.
Synthetic quartz is also used for making high quality cuvettes. It gives good optical clarity and is suitable for accurate absorbance reading.
IR Quartz is used when measurement is done in UV, visible and infrared (IR) range. It has broad spectral transparency.

Optical glass is used for visible light colorimetric measurement. It is not suitable for deep UV range because glass absorbs ultraviolet light.
Special optical glass or crown glass is used for some near-UV measurement. It gives better transmission than normal optical glass.
Borosilicate glass such as Pyrex is also used to make cuvettes. It is used when chemical resistance and low thermal expansion is needed.

Plastic cuvette is commonly used as disposable cuvette. It is mainly used for routine visible range analysis.
Polystyrene (PS) is a low cost plastic material. It is used for simple visible light measurement and is thrown away after use.
Polymethyl methacrylate (PMMA) or acrylic is a clear and rigid plastic. It is used for visible and some near-UV measurement.

UV-polymer cuvettes are made from special plastic materials such as polycyclical olefins. These are designed to transmit ultraviolet light and give better chemical resistance than normal plastics.
Sapphire is also used for special spectrophotometer cuvette. It is very hard and durable material and used in high temperature and high pressure conditions.
The selection of cuvette material depends on the wavelength range, chemical nature of sample and whether the cuvette is reusable or disposable.

Types of Spectrophotometer Cuvette

A. On the basis of material

Quartz cuvette – It is used for UV, visible and near IR range. It is strong and chemically resistant. UV quartz and IR quartz cuvettes are used according to wavelength range.
Optical glass cuvette – It is used mainly for visible light range. It is cheaper than quartz cuvette. It is not suitable for UV measurement because glass absorb UV light.
Plastic cuvette – It is disposable type cuvette. It is used for routine visible light measurement. It is generally made of Polystyrene (PS) or Polymethyl Methacrylate (PMMA). Some special plastic cuvettes can also pass UV light.

Sapphire cuvette – It is very strong cuvette. It can tolerate high temperature and pressure. It is used in special type of spectrophotometric work.

B. On the basis of clear windows

Absorbance cuvette – It has usually two clear polished windows. Light enters from one side and comes out from opposite side. It is used for absorbance measurement.
Fluorescence cuvette – It has three or four clear polished windows. Light enters from one side and emitted light is measured at 90° angle. It is used in fluorescence measurement.

C. On the basis of volume

Macro cuvette – It holds more than 3.5 ml sample. It is used when sample is available in more amount.
Standard cuvette – It usually holds about 3.5 ml sample. It is commonly used in normal laboratory work.
Semi-micro cuvette – It holds less sample volume. The volume is about 0.35 ml to 1.75 ml. It is used when sample amount is limited.

Micro cuvette – It is used for small sample volume. It holds about 20 µl to 350 µl sample.
Ultra-micro cuvette – It is used when very little sample is present. It holds about 2 µl to 50 µl sample.
Nano cuvette – It is used for nanoliter level sample. The volume may be about 0.1 µl to 2 µl. It is used in very small sample analysis.

D. On the basis of path length

Standard path length cuvette – It has 10 mm path length. It is the common cuvette used in most spectrophotometer.
Short path length cuvette – It has 1 mm to 5 mm path length. It is used for highly concentrated sample so that absorbance does not become too high.
Long path length cuvette – It has 20 mm to 100 mm path length or more. It is used for very dilute sample. It increases the sensitivity.

Dual path length cuvette – It has two path lengths in same cuvette. When the cuvette is rotated, different path length is used. Example 10 mm and 2 mm.

E. On the basis of shape and structure

Rectangular or square cuvette – It is the common type of cuvette. It is used in normal laboratory spectrophotometer. It reduces light scattering.
Cylindrical cuvette – It is round tube like cuvette. It is used in some special optical work like circular dichroism.

Round cuvette – It is less commonly used. It may be used in fluorescence work where light is collected from different sides.
Flow-through cuvette – It is used for continuous flow of sample. The sample is pumped through the cuvette. It is useful for real time monitoring.
Divided or tandem cuvette – It has two separate chambers. Two solutions can be kept separately and then reaction can be studied. It is used for reaction kinetics.

Demountable cuvette – It can be opened and cleaned properly. It is used in special tests where cleaning or changing path arrangement is needed.

F. On the basis of closure type

Open top cuvette – It has simple lid. The lid may be made of PTFE or silicone. It does not make tight seal.
Stopper cap cuvette – It has PTFE stopper. It gives better sealing than simple lid.

Screw cap cuvette – It has threaded cap. Sometimes septum is also present for needle injection. It is used for volatile and oxygen sensitive sample.

Precautions Should be Taken when handelling a Spectrophotometer Cuvette

Gloves should be used before handling the cuvette. Nitrile gloves are generally used. It prevents oil and fingerprints from touching the cuvette surface.
The cuvette should be held only from frosted side or upper edge. The clear optical window should not be touched. Finger mark on this part scatter light and change reading.

Plastic pipette tip should be used for filling the cuvette. Glass or metal tip should not be used. These may scratch the inner wall of cuvette.
The cuvette should be filled slowly. The sample is allowed to flow along the inner wall. This prevents formation of air bubbles.
The cuvette is filled about 75% to 80% only. It should not be filled completely. Overfilling may spill the sample inside the spectrophotometer.

If mixing is needed, it is done gently. Vigorous shaking is avoided because bubbles may form. Bubbles give wrong absorbance value.
Before placing in instrument, outside wall is wiped with soft lint free tissue. Kimwipes or optical tissue may be used. Rough paper or cloth should not be used.
The cuvette should be inserted in same direction every time. The logo mark or arrow may be kept in same side. This gives consistent reading.

After use, the cuvette is emptied and rinsed immediately. The sample should not be allowed to dry inside. Dried residue can stain the optical surface.
Abrasive brush should not be used for cleaning. It can scratch the inside surface. Scratched cuvette gives poor light transmission.
Strong alkali, laboratory detergent and hydrofluoric acid (HF) should be avoided. These chemicals can make glass or quartz cloudy and damaged.

Cuvette should not be cleaned in ultrasonic bath. Vibration may produce small cracks. It may also damage fused joint or coating.
Disposable plastic cuvette should not be reused. It is made for single use only. Reuse may give wrong reading and contamination.
After washing, cuvette is dried in inverted position. It may be kept on lint free tissue or drying rack. Dust should not enter inside.

Cuvettes are stored in original case or soft box. They should not be stacked one over another. Touching with hard surface may cause scratch or chipping.
The cuvette should be removed from spectrophotometer after measurement. It should not be left inside the machine for long time.

Advantages of Spectrophotometer Cuvette

Spectrophotometer cuvette gives fixed optical path length. Most commonly it is 10 mm. This helps in correct absorbance reading and concentration calculation by Beer-Lambert law.

It holds the sample in proper position during measurement. The light passes through the sample in straight path. So the reading becomes more uniform.
It is available in different volume. Macro cuvette holds more sample and micro cuvette holds very less sample. So small and precious sample can also be measured.
It has special designs for different work. Screw cap cuvette is used for volatile sample. Flow-through cuvette is used for continuous measurement. Fluorescence cuvette has more clear windows for fluorescence reading.

Quartz cuvette is very useful because it can pass UV, visible and near IR light. Its transmission range is broad, about 190 nm to 2500 nm. It is used in accurate scientific work.
Quartz cuvette is strong and reusable. It can tolerate many chemicals and high temperature. If handled carefully, it can be used for long time.
Optical glass cuvette is useful for visible and near IR measurement. It is cheaper than quartz cuvette. It is also reusable and suitable for normal laboratory work.

Plastic cuvette is low cost and disposable. It does not need washing after use. So it saves time in routine testing.
Disposable plastic cuvette reduce cross contamination. One cuvette is used for one sample only. It is useful in clinical and high number sample testing.
Some special plastic cuvette can also pass UV light up to nearly 230 nm. So they can be used when disposable UV measurement is needed.

Limitations of Spectrophotometer Cuvette

Glass cuvette and quartz cuvette are fragile. They can break easily if dropped or handled roughly. Quartz cuvette is also costly.
Normal glass and plastic cuvette are not used for UV range. They absorb UV light. So DNA and protein measurement in UV range cannot be done with them.
Plastic cuvette has low chemical resistance. Organic solvents like acetone and chloroform may damage or dissolve it. So it is not suitable for all chemicals.

Glass and quartz cuvette also have chemical limitation. Hydrofluoric acid (HF) can etch and destroy them. Hot strong alkali may also damage the surface slowly.
Cheap bonded cuvettes may leak. The adhesive used in them can dissolve by organic solvent, corrosive liquid or high temperature. So glued cuvette is not useful for harsh sample.
The optical window is very sensitive. Fingerprint, dust, bubble and dried sample residue can scatter the light. Due to this the absorbance value becomes wrong.

Cuvette can be scratched easily. Rough tissue, paper towel or brush should not be used. Scratch on clear window reduces light transmission.
Some plastic and glass cuvettes show autofluorescence. The cuvette material itself gives background glow. This can disturb weak fluorescence signal.
Cuvette must be placed in correct direction and height. If the optical window does not match with light beam, light may hit the wall or miss the sample. Then reading becomes incorrect.

Ultrasonic bath should not be used for cleaning cuvette. Vibration may make cracks or break the polished surface. In some case, cuvette may shatter.
Cuvette should not be filled more than about 80%. Overfilling may spill the sample inside the spectrophotometer. This can damage the instrument.
Very concentrated sample may give absorbance saturation. Then the sample should be diluted or short path length cuvette is needed.

Completely filled and tightly closed cuvette may burst during temperature increase. The liquid expands and pressure is produced inside. So some space should be left.

Uses of Spectrophotometer Cuvette

Spectrophotometer cuvette is used to hold liquid sample in the light path of spectrophotometer. It helps to measure how much light is absorbed or transmitted by the sample.
It is used for absorbance and transmittance measurement. The sample is placed inside the cuvette and light passes through it. The instrument then gives the reading at particular wavelength.

Fluorescence cuvette is used in fluorescence spectroscopy. It has clear windows on more sides. The emitted light is measured at 90° angle after the sample absorbs the incident light.
Quartz cuvette is used for DNA, RNA and protein measurement. It can pass UV light. The readings are generally taken at 260 nm and 280 nm.
It is used in pharmaceutical quality control. Flow-through cuvette is connected with pump. It helps in tablet dissolution study, drug stability test and automated batch analysis.

It is used in water and environmental analysis. Long path length cuvette is used for dilute sample. Trace pollutants like nitrate, phosphate and heavy metals can be detected better.
It is used in clinical diagnostic work. Blood and serum samples are kept in cuvette for different tests. Bilirubin, hemoglobin, lipids and enzyme activity can be measured.
It is used in colorimetric assay. Glass or plastic cuvette is used for Bradford assay, BCA protein assay and bacterial culture density measurement.

It is used for reaction kinetics study. Special temperature controlled cuvette may be used. Enzyme reaction and other reactions can be observed with time.
It is used in particle and cell analysis. Micro-channel flow cuvette allows single cells or particles to pass through the light path. It is useful in some medical diagnostic studies.
It is used in material science and laser testing. The cuvette acts as optical container for studying refractive index, light scattering, thin films and nanomaterials. Some cuvettes can also tolerate heat, pressure and strong light.

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Spectrophotometer Cuvette – Features, Types, Materials, Precautions, Advantages