Understanding Cuvette Volume, Material, Path Length etc.

Understanding Cuvette Volume, Material, Path Length etc.

A spectrophotometer is an ideal choice for measuring materials of interest light absorbance or transmittance to a specific spectrum wavelength, in this way to get data on substance concentration and purity. A cuvette is a small vessel containing samples used for spectroscopic measurements which are placed in the spectrophotometers. Cuvettes are made of various sizes, volumes, and materials, and should be transparent for the targeted wavelength range.

For the photometric measurements of liquid solutions, the samples must be positioned into the optical light path of a photometer (spectrophotometer, fluorometer, or colorimeter) in a predefined format. The standard option for this application is cuvettes, sample containers containing 2 or 4 optical clear windows.

Figure 1. Standard Optical Path Length of Cuvettes

There are so many types of cuvettes available, (we have over 260 skus of cuvettes listed on our sites), even when narrowing down the range to only those used for absorbance measurements in the field of UV-vis Spectrophotometry.

Different Cuvettes Schematics

Different Cuvettes Schematics, a – e are Standard Size Cells

Sample solution is accommodated inside the red lines of the cuvettes above. Cuvette cells a – e are drawn to be 12.5 x 12.5 x 45 mm external sizes. 

  • a: 10 x 10 mm inner size fluorescence cuvette; 
  • b: 2 x 10 mm semi micro fluorescence cuvette; 
  • c: 2 x 10 mm semi micro absorption cuvette; 
  • d: 2 x 10 mm sub micro volume cuvette; 
  • e: 2 x 2 mm inner size fluorescence cuvette. 

Fluorescence cuvettes are cuvettes that have 4 clear walls (some specialized types of cuvettes have three transparent walls) while absorption cuvettes are usually 2 walls clear. Cuvettes f and g are examples of not standard dimension cuvettes, which are also called short path length cuvettes. The path length and external size are smaller than standard cuvettes.

The path length of the cuvette is the length of light that traverses the cuvette. 

Cuvettes a and c have 10 mm path lengths. Cuvette cells b, c, and f have the same inner dimensions (2 x 10 mm) and cuvettes e and g have the same inner dimensions (2 x 2 mm) but different outer dimensions. 

NOTE: Cuvettes f and g outer dimensions are not standard and are not applicable to in this discussion unless specifically referred to. Nonetheless, those who are comfortable with non – standard cuvettes can always use them, by using suitable adaptors or sample holders specially designed.

Cuvette c is a semi micro volume absorbance cuvette ( 2 clear walls ). It has two dark (black) walls that no light transmits. This is useful because a 10 mm path length cuvette may be used with a much smaller volume and any light not passing through the solution will be masked from reaching out to the light detector. 

It is very important not to use cuvette b instead of cuvette c in an absorbance experiment. By using b in the absorbance measurement will lead to an incorrect reading even when the background of the cuvette b has been measured. Cuvette c, on the other hand, is not suitable for an emission measurement, which is usually performed by a different geometry.

The factors including sample characteristics, volume availability, levels of concentration and types of measurements to be made should affect the decision when choosing the appropriate cuvette for your applications.

Figure 2. Different Types of Cuvettes

Cuvettes by Volume

The most widely used cuvette is square type with an external dimension of 12.5 x 12.5 mm and a height of 45 mm and an internal size of 10 x 10 mm. This is a standard dimension for which most spectrophotometers and fluorometers holders are designed. Many types of larger and smaller cuvettes are of course also available. Adaptors are also available which allow smaller short path length cuvettes into the standard holder that accepts 12.5 mm x 12.5 mm square cuvettes.

With this external size, the cuvettes are available from <100 microliters (sub-micro cuvettes) to a few hundred microliters (semi-micro cuvettes) to standard volume cuvettes of 3.5 milliliters. Of course, a bigger volume of cuvettes (macro type) larger than 3.5 mL is also available, and the external size will be larger.

Figure 3. Cuvettes Differ in Volume with Same Footprint

How is Cuvette Volume Determined?

The volume of the cuvette also refers to the sample liquid volume they are designed to hold.

A 1 cm square cuvette accommodates 1 mL of liquid per 1 cm of height. Therefore, a 43.75 mm height (45 mm – 1.25mm base thickness) cuvette may hold up to 4.375 mL of liquid. If the cuvette is 80% full, then the total volume will be 3.5 mL, which is the so-called standard volume.

4.375 mL x 80% = 3.5 mL

The reason 80% is that you should never fill the cuvette more than 80%. When the liquids are too close to the edge of the cell (> 80%), they might easily spill and will cause a lot of issues during measurements.

What’s the Cuvette Volume Options?

Four types of volume options are available when choosing a cuvette:

  • The measuring volume of a macro cuvette is greater than 3.5 mL (7 – 35mL).
  • A standard volume cuvette holds a measuring volume of 3.5 mL.
  • A semi micro cuvette volume holds samples of 0.35 mL – 1.7 mL. Among these types of short path length cuvettes may require cuvette mounts or spacers.
  • A sub micro volume cuvette holds samples between 20 uL to 350 uL.

Figure 4. Macro Cuvettes

Figure 5. Standard 3.5mL Cuvettes

What’s the Choice of Micro Volume Cuvettes for Limited Samples?

For many biological measurements, the samples are so valuable and a few milliliters of the volume are difficult to target. The required volume can be decreased by making two or four sides of the cuvette inside walls thicker.

For instance, a 4 mm cuvette is a cuvette with an external size of 12.5 mm x 12.5 mm and an inner size of 4 mm x 10 mm. Such a 4 mm cuvette will require a liquid volume of 1.4 mL per – 45 mm of height; similarly, a 1 mm cuvette corresponds to a liquid volume of 0.35 mL per – 45 mm of cuvette height. 

See the examples below:

Figure 6. Semi Micro Cuvettes with 12.5*12.5 mm Square Bases

Figure 7. Mount and Spacer Enabling Short Path Length Cuvettes into Standard Holder

Spectrophotometers and fluorometers take measurements at one of three standard optical-beam Z dimensions (the distance from the bottom of the cuvette to the center of measuring aperture)—either 8.5, 15 or 20 mm—depending on the specific instrument.

Standard cuvettes are usually straight walled and will fit into most spectrophotometers. On the other hand, semi micro cuvettes have the same exterior footprint, but their interior is typically tapered to a restricted sample amount. Regardless, the sub micro cuvette is designed to measure through the sample at a specific Z dimension in the chamber. It is important to make sure that the cuvette you select for your sample measurements is compatible with the Z dimension height of your instrument.

Figure 8. Sub Micro Volume Cuvettes Differ in Z Dimensions

Cuvette by Path Length

In a typical research laboratory, the spectrophotometer has a standard chamber size in which a cuvette is accommodated to allow a specific light wavelength passing through the sample solution. This distance between the cuvette parallel optical windows is accurately manufactured and pre-known, which is known as the cuvette path length.

Cuvette Path Length

Figure 9. Cuvette Path Length

The standard path length of a cuvette is 10 mm, however, a shorter path length and longer path length is also available listed in stock. Shorter path lengths cuvettes are usually smaller in volume and longer cuvette path length makes the cuvette larger volume. 

Here are some important cuvettes features to consider:

  • When taking samples measurements that are found at low concentrations — for example, RNA, single-stranded DNA, and oligonucleotides — it’s recommended a long enough path length for the data readings to be within the instrument linear measuring range. The standard size is 10 mm path length, the good news is that cuvettes with an optional path length are now available with us (dual path length).

Figure 10. 10mm Cuvette that can be Rotated 90 Degrees to Allow for a Reading at 2 / 5 mm Path Length

  • Measuring a small volume sample over a short path length is easier and quicker than taking multiple steps to dilute a sample. Also, because there are fewer transferring pipetting steps, the readings are most likely to be more reliable and accurate.
  • Semi micro volume cuvettes that have a shorter path length can work with cuvette spacers or mounts. These testing results with short path cuvettes are more accurate and trustworthy when measuring concentrated samples such as nucleic acids or proteins compared with diluting sample preparations.

Figure 11. Short Path Length Cuvettes and Mount

Cuvette by Materials

Cuvettes made of different materials can fit into different spectral ranges. It’s important to ensure that the selected cuvette is transparent to the specific wavelengths when measuring the samples of interest. An ideal cuvette material choice would also only accommodate samples (mostly liquid solutions) and does not interact with the samples used in the measurement. 

Cuvettes have a light transmission of a limited wavelength range, and have refractive index dielectric mismatch (different refractive index of air and solutions), and can have damages such as scratches which can be very small and unnoticed. All of these factors might affect the outcome experiment measurement. 

The wavelengths of the cuvette to be used is determined by the cuvette material. A sufficient transmission is important to the cuvette so that light attenuation to the cell transparent walls will not have a negative effect on the measurement outcome.

The transmission for all wavelengths is not uniform for standard optical cuvettes, and most commonly the spectral transmission in the UV or IR is the limiting range. Visible range is commonly transmitted by nearly all the cuvette material types.

However, there is NO universal agreement of the minimum transmission required for a specific wavelength range. And different standards are applied by different manufacturers ( varies between 10% and 90% ).

Usable Transmission Range of Different Material

Material  Transmission Range
Far UV Quartz 170 – 2700 nm
Near IR Quartz 250 – 3500 nm
UV Quartz 220 – 2500 nm
Optical Glass 340 – 2500 nm
Plastic 380 – 850 nm
UV plastic 220 – 900 nm

This table should not be taken or used as a reference literally. It is only used to let the reader understand that the usable range of cuvettes will vary considerably. It’s important to make sure that the cuvette to be used will work under the wavelength range of interest.

Quartz material has the highest transmission and temperature resistance, most importantly is transparent in both the visible light and UV range and is an appropriate choice when measuring samples in the UV-light spectrum.

Glass and plastic materials are normally transparent to visible light ( 380-700 nm ) but absorb in the UV ( 190 – 340 nm ) wavelength ranges. Therefore, glass and plastic cuvettes are ideal for colorimetric protein assays or measurement of the bacterial culture density, which cannot be used for concentration and purity measurements of samples in the UV ranges. Plastic cuvettes are low cost and disposable.

Plastic cuvettes work under 380 nm are available but the rest of the majority transparent plastic cuvettes are not suitable for fluorescence or absorption experiments.

In fact, the manufacturing process ( surface quality and wall purity ) leads to light attenuation  ( the absorption rate ). Therefore, cuvette performance can differ across brands. In addition, many manufacturers utilize their own proprietary materials and surface coating to increase their transmission range and/or reduce their cuvet costs.

Note: A high transmission may be needed to produce meaningful and trustworthy results, which may cause issues at the edges of the cuvette transmission ranges.

There are advantages and disadvantages to the types of cuvettes. Here are some more tips to guide you on selecting the more appropriate cuvette for accurately and reliably sample measurements.

How to make the decision?

The choice of the type of cuvette depends on the instrument to be used, on the experiment nature and the sample itself. It is important that the cuvettes and cells have as high transmission as possible for certain measured wavelengths, so we don’t usually limit the material to just the linear range of the photometer. Quartz cuvettes have the highest transmission among all materials.

Figure 12. Quartz Cuvettes with Highest Transmission

The requirements of the equipment necessitates the cuvette to be compatible with the instrument. The external size of the cuvette, because it must fit into the cuvette holder, is of major importance, and also take note of the height of the measuring chamber. 

Another factor to consider is the light beam position (Z dimension): the light source must be able to pass through the optical window of the cuvettes. This is particularly important to sub-micro volume cuvettes that have very small apertures. The clear windows for light beams could be very small (i.e. 2 mm x 5 mm). If not properly chosen the Z dimension (light height), the micro cuvettes might well not be compatible and cannot be used. Common Z dimensions are 8.5 mm, 15 mm and 20 mm.

Small Window Cuvette with Z Dimension 8.5 15 20mm

Figure 13. Small Window Cuvettes Z dimension of 8.5 15 20mm

The next major factor is the measurement of spectral wavelengths involved in the applications. PMMA, polystyrene, or optical glass cuvettes are transparent only in the visible range. When measurements are applying UV wavelengths of less than 300 nm, quartz cuvettes or IR quartz cuvettes which have sufficient transmission should be used.

Quartz and Glass Material Absorbance Curve

Figure 14. Absorbance of Cuvettes Made of Quartz or Glass Material Between 220 nm and 400 nm

Temperature Controlled of Cuvette

For those methods that rely on reactions at a specific temperature and absorbance measurement over time, heating and efficient temperature control of the sample during the process is essential. In this case, the contact area between the cuvette wall and the temperature controlled cuvette shaft should also be as big as possible in addition to adequate material strength. Therefore, in temperature controlled applications certain cuvettes such as macro cuvettes offer advantages.

Other factors to consider when choosing a cuvette include the chemical resistance, the sample volume and concentration at hand.

Cuvette Chemical Resistance

The material from which the cuvette is produced is relatively less important when the sample is an aqueous solution. Plastic or glass or quartz cuvettes will all work and you can even choose the most affordable NRC glued cuvettes.

If organic solvents, on the other hand, are involved, glass and quartz cuvettes are the preferred choice because these are more robust compared to plastic alternatives. And the NRC will not work with organic solvents, instead, you should go with CRF or HTR versions.

Types of Cuvettes Differ in Chemical Resistance

Figure 15. Types of Cuvettes Differ in Chemical Resistance

*HTR: High-Temperature Resistant. Highest quality for advanced experiments. These types of cuvettes are melt and manufactured as one piece, with extraordinary resistance to high temperature (<1200℃) and corrosive chemicals. Transmission is 83% over. Zero data reading variation is available on request (default <0.3%) for 2 or more pieces. HTR-5 means the cuvette has 5 clear walls.

  • Note: These HTR cuvettes can be used with most popular organic solvents, as well as acids and bases. They are compatible with chemicals such as acetone, butanone, DMF, and concentrated hydrochloric acid.

*CRF: Chemical Resistant Fused. Bestseller! This type of cuvettes is resistant to most organic solvents, acids, and bases. However, it has a chance to be stained by some chemicals to the bonding edges. It’s a cheaper alternative to the HTR type. CRF-H has a higher transmission (83% v.s. 80%) than CRF and the same transmission with HTR. 

  • Note: please don’t use the CRF cuvette to store chemicals for a long time. Clean it after use.

*NRC: Non-Resistant To Chemicals. This type of cuvette is assembled with glue.

  • Note: Please note that these NRC cuvettes should not be used with benzene, toluene, aqua regia, ethanol, corrosive solutions, or other similar substances, as they may degrade the bonds between the pieces and cause the cuvette to leak. DO NOT rinse the cuvette with ethanol or similar solutions for cleaning.

Further reading: Explanation of HTR, CRF, NRC and Chemical Resistance

Very Small Sample Volume

If only a few samples are available, the reuse of the sample for the following measurements may be considered. In this case, it is recommended to use disposable plastic cuvettes. The risk of contamination will be minimized if the plastic cuvettes are individually packaged, and have an appropriate purity quality. 

Alternatively, sub micro quartz cuvettes may be selected which were designed to use with extremely micro cuvette volumes.

  • Sub micro or microvolume cuvettes can be reused, eliminating the necessity to continually replenish the stock of consumable cuvettes. It is possible for us to recover precious undiluted samples for some downstream measurements, especially those in which sterility is not a must requirement.

Figure 16.  Sub Micro Cuvettes 

Sample Concentration and Cuvette Path Length

The sample concentration also influences the selection of the cuvette as each instrument has an upper detection limit. For instance, a double-stranded DNA can be accurately quantified up to maximum concentration 100 μg / ml, when using a spectrophotometer with a linear measuring range of up to 2 A with a 10 mm cuvette path length.

Higher concentration solutions must either be diluted, or a cuvette with a shorter path length can be used to simulate the dilution. Known from the Beer-Lambert law, A path length of 1 mm cuvettes will allow dsDNS concentration to be as high as 1,000 µg/mL.

Extended Reading: UV visible Spectrophotometer Setup and Beer-Lambert Law

Generally speaking, quartz and glass cuvettes have higher transmission and accuracy of spectroscopy measurements, and these cuvettes can be reused many more times. However, the plastic cuvette is cheap and easy to use, no need to clean and avoid cross-contamination, which makes it an excellent choice for protein, DNA and RNA, and aqueous solutions.

That’s all for today! Thank you for your time reading!

The Cuvet.Co Team

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