What factors will affect absorbance?

absorption measurement

1. What are absorbance and transmittance?


Absorbance (A), also known as optical density (OD), is the quantity of light absorbed by elements.
When light hits an object, the molecule or atom of analyte can absorb the light, usually because the wavelength of the absorbed light corresponds to an electronic excitation in the object.

The rest of the light is transmitted, in other words, it passes through the object, which is called transmittance (T).
The more analyte is found in the solution, the lower transmittance will be due to the more light is absorbed by it.

2. Why measure absorbance?

In biochemistry, biology or chemistry, when analyte absorbs the light at a specific wavelength, a unique relationship exists between the individual atom/molecule and its UV Vis spectrum. This relationship can be used for:

  • Qualitative analysis – determining the presence of certain substances.
    For example, determining pesticide residues in food, identification of contamination such as COD in water, identification of nucleic acid such as COVID-19 testing.
  • Quantitative analysis – determining the amounts of certain substances.
    For example, determining the concentration of substances in air such as PM2.5, determining the concentration of harmful substance such as mercury and asbestos in makeup, measuring calcium and protein content in dairy products.

3. How is absorbance detected?

  • Light source
    The all-important thing for light transmission or absorption measurement is the light source.
    Different light sources can be used for absorbance measurements. They differ in the spectral wavelength range, in their optical intensity and in the light stability.
  • Sample
    The solution with analytes of interest in proper volume usually needs to be placed into a cuvette, in order to measure the absorbance of the interested substance.
  • Cuvette Material
    The third important thing is choosing the right cuvette material. The material of cuvette is always clear and smooth, to ensure maximum light transmission and less light scattered, on account of researcher’s interest is in the absorbance of the solution rather than the material.
  • The appropriate blank
    In order to correct undesired absorbance of absorbance measurements, such as light scattering, a blank is measured in parallel to samples. The appropriate blank includes all components except the analyte of the assay.
    Side Note: Particles in the solutions scatter the light, it will increase the measured absorbance value since they block the light path, as a result, less light reaches the detector.
  • Detector of absorbance measurements
    A UV-VIS spectrophotometer is an instrument designed to measure absorbance in the UV-VIS region using the Beer-Lambert law. Measures the intensity of light passing through a sample solution in a cuvette and compares it to the intensity of the light before passing through the sample.

4.Theoretical background of absorbance measurements

  • Detection path in cuvettes
    A solution contains interested analytes with known absorbance characteristics is placed into a cuvette. Then insert the cuvette into the instrument chamber, the absorbance reader will determine the light absorbance by calculating the optical density difference before and after passing the sample.
    (Light that does not pass through the detector is either absorbed or scattered. The scattered part can be corrected by measuring appropriate blanks, and is subtracted from this value to obtain real absorbance of the interested substance.)
absorption measurement
  • Absorbance in chemistry and life sciences
    After the absorbance measurement, the result is a value given in either transmission or optical density. Whereas, quantification of a substance in solution is the goal of the measurement, then the obvious question is how to convert the known signal into the concentration value. Generally, we can employ the Beer-Lambert law to get it.
  • Beer-Lambert Law
    The Beer-Lambert law is very helpful as it allows quantification of absorbing substances without the need to add any other reagents, it describes the relation of absorbance, path length and concentration of an absorbing substance:
    A – absorbance
    ε – molar attenuation coefficient or absorptivity of the attenuating species – M‾¹cm‾¹
    l – optical path length – cm
    c – concentration of the attenuating species- M

It says that there is a linear relationship between the concentration, absorbance, path length and molar optical coefficient, which enables the concentration of solution to be calculated by measuring its absorbance.
For instance, in a standard cuvette the path length is 1 cm. For an absorbing substance and a specific wavelength, the extinction coefficient (ε) is a constant specific, usually the absorbance maximum of the substance, which provides information on how strongly the specific substance absorbs light at the specific wavelength.
As an example, the molar extinction coefficient for oligonucleotides is 32.4 µl*cm-1*µg-1. Therefore, a solution of 1 µg/µl oligonucleotides with a path length of 1 cm has an absorbance of 32.4 OD.

5.What will affect your absorbance measurements?

  • Does cuvette material affect absorbance?
    When measuring in the UV-range, as it is required for nucleic acids or NADH, the UV-transparent cuvette is requisite to finish the measurement. Otherwise, you will get the maximum absorbance in all samples in this absorbance measurement because the material of cuvette absorbs UV light.

We have various cuvette material, and there are 5 common material you can choose to use, Plastic, Glass, UV Quartz (JGS-1), VIS Quartz (JGS-2), IR Quartz (JGS-3) cuvette, the same material has the same spectrum transmitting, but different fabrication has different light transmittance.
As an example, for our JGS-1 quartz glass material cuvette, Glued cuvette has 80% light transmittance, but the All Fused quartz cuvette has 83% light transmittance, and the highest applicable temperature is not the same.






UV vis ( 200- 2500 nm)

Matched Pair

Transmission > 80%

Transmission > 83%

Resistant to Acids and Bases

Resistant to Organic Solvents

Usable upto 600°C (1112°F)

Usable upto 1200°C (2192°F)

0 Variations Reading

✔ (On Request)



Assembled with Quartz Powder

Assembled with Quartz Powder

Assembled with Glue


Long Term

Clean after Use

Clean after Use

Clean after Use

Available Material

Quartz Material

  • Does cuvette size affect absorbance?
    According to the Beer-lambert Law, when the extinction coefficient (ε) and the path length (l) are constant, the absorbance (A) is proportional to the concentration (c) of the sample. When the ε and the c are constant, the absorbance is directly proportional to the length of the light path, it is also equal to the inner width of the cuvette.
    The path length affects absorbance. With a longer optical path length, the light has to travel through more solution, and can hit more molecules or atoms, and be absorbed more.
    For a low concentration sample, the analytes may not absorb enough light with short path length cuvette, then the measurement is less effective.
  • Will sample volume affect absorbance?
    For a standard cuvette, fill the cuvette about 80% full of the solution you wish to test is enough.
    But for a micro quartz cell or flow cuvette, and the sample you use is small enough to 10-400ul, then it’s important to make sure there is enough sample in the cuvette for the light to pass through. As we said before, the measurement will be less effective if the analyte can’t absorb enough light.
  • Will Z-Dimension affect absorbance?
    8.5mm, 15mm, 20mm Z-Dimension are the common distance of cuvette center window to the cuvette bottom.
    If you use 15mm ZD black wall cuvette to a 8.5mm Z-height machine, the light beam will smission too low to tranmist the black wall of cell, which can’t reach the dector because it is blocked, then you will get 0% light transmission.

    We have varieties of absorption cuvettes with different ZD shown as below, such as standard pathlength, long pathlength, or flow cell.
    8.5/15/20mm8.5mm Z Height 50ul black wall cuvettesQM7910mm50ulQB41-10mm100ul-Micro-Black-Wall-Cuvettes05QB4110mm100ul
    8.5mm8.5mm Z Dim 32ul black wall flow CellQB3510mm32ul8.5mm Z Dim 64ul self masking flow through cellQF7520mm64ul

    There are more Self-masking cuvettes, please go to our web or contact us for quote if you want to buy cells.
  • How would the test results be affected if the fingerprints left on the sides of the cuvette?
    Fingerprints on the cuvette window will interfere with target light transmission, and will cause inaccurate measurements. It will lead a slightly higher absorbance reading and the measured concentration will be corresponding higher than the actual concentration, because fingerprints absorb the light.
  • How would the absorbance results be affected is there is disturbance of the light-path?
    The reading of measured absorbance will be increased if there is anything in the light path.
QF94 20mm 10ml Flow-Through Cell01

Usually, these are air bubbles in the sample, condensation on a lid, dust, scratches or fingerprints on the window of the cuvette. Therefore, checking the cuvette just before measurement is recommended.
We have bubble-free New-Arrival cuvette to reduce the air bubble influence in the measurement.

TypeItem CodePathlengthInner WidthWindowsVolumeLink
bubble free flow cellQF6110mm10mm4 Clear Windows3.5mlMore Details
QF62,-3.5ml,-Bubble-free-Flow-cell05QF6210mm10mm2 Clear Windows3.5mlMore Details
QF4703 Quartz Funnel Fluorometer Cuvettes, 10mm Pathlength06QF4710mm10mm4 Clear Windows3.5mlMore Details
  • How does pH affect light absorbance?
    When the pH of the solution changes, there are ionization in some of the molecules of the solution, then the structure of molecular changes, which will affect the determination of absorbance.
    As an example, for anthocyanin measurement of lycium ruthenicum, it is less accurate if measure the anthocyanin concentration in a single PH environment, because their color and absorbance changes with pH.

The anthocyanin is red form of 2-phenyl benzopyran when pH is 1.0, and colorless form of methanol off base in pH 4.5, and the absorbance of the former form is much higher than latter one. Therefore, for the same solutions but different pH, the concentration will be different due to the absorbance reading is different, the and the conclusion will be inaccurate even though actual concentration is the same.

As a result, pH-differential method is a better way to determine the concentration of anthocyanin of lycium ruthenicum.

  • Does temperature affect UV Vis absorbance analysis?
    Temperature affects absorbance values.
    Different solvents’ interact performance are not exactly the same at different temperatures. It’s vital for an effective measurement to control the heating temperature especially some reaction undergoing at specific temperature. Solution parameters will change with the temperature changes:

    Rate of reaction-The reaction rate changes when temperature changed. This can cause a change in the activity of the sample. Enzymatic/biomolecular reactions are very sensitive to temperature.
    Solubility of a solute-Solubility is affected with variations in temperature. Poor solubility may result in imprecise absorption.
    Expansion or contraction of the solvent-This may lead to a change in the concentration of the solution and affect the absorbance, as absorbance is linearly related to concentration.
    Schlieren effect-This effect may occur with temperature changes, leading to a series of convective currents which may change the true absorbance.

For measurement of total nitrogen in water with spectrophotometer, in alkaline aqueous solution (>60℃), Potassium persulfate can be decomposed to produce potassium bisulfate and atomic oxygen, then the NO2-, NH4-, organic nitrogen in sample can be oxidized to nitrate nitrogen by atomic oxygen at 120-124℃. And we can determine the absorbance and concentration with this solution.

But there will be residual potassium persulfate in the solution to affect the absorbance if the temperature-controlled fail to meet the requirement. Potassium persulfate has a strong absorption peak at 220nm, which coincides with the absorption wavelength of total nitrogen. This absorption characteristic gradually weakens with the continuous decrease of potassium persulfate.

Temperature control can be achieved using high-performance nomothermal System for UV Vis spectrophotometry.

  • How does stray light affect the optical density (OD) reading?
    Stray light is defined as light in the system at wavelengths (colors) other than the one intended, that is to say, stray light is not from the instrument’s light source and does not follow the optical path but reached the detector, which causing a deviation at the corresponding wavelength.

    Therefore, the optical density measured by the detector is higher than the true OD. In other words, the measured absorbance of samples is lower than the true absorbance due to the stray light contribution.
    There is one method to reduce stray light in these systems is the use of double monochromators.

6. How to reduce the effect on absorbance as mentioned above?

As we said before, different absorbance measurement requires different pH and temperature environment control, and stray light is a complex subject to figure out the exact problem to solve.

In this part, we will solve the influencing factors of cuvette for absorbance, right cuvette choosing, cuvette using and maintenance:

  • How to choose the right absorption cuvette for absorbance studies?
    In our previous article, 7 Things to Think About When Ordering a Cuvette, we have attempted to provide answers to choose the right cuvette, it is specified in the aspects of cuvette material, pathlength, Z-dimension, volume, cover type, cell shape and etc.
    There will be a further explanation on path length choosing of cuvette in the following part:
  • How to choose the right path length of cuvette?
    The proper pathlength of cuvette is to ensure absorbance values are within the dynamic range of the detector.
    Pathlength choosing of cuvette depends on the concentration of a sample and the chamber of instrument, which decide the upper light path length of detection.
  • What time will use “long path” cuvettes?
    When the concentration of the sample is too low to be measured using a standard 10mm optical path length cell, and concentration is difficult in cases where the sample vaporizes or undergoes a chemical or molecular structural change during the concentration process, then a cell with a longer optical path length is used to enable the optimum absorption sensitivity of absorption measurement.

For instance, a well-known study using a long pathlength cell is the turbidity analysis of water. 50mm and 100mm cuvettes are often used for analyzing samples with a low turbidity.

There are cuvettes with optical path lengths of 20mm, 30mm, 40mm, 50mm, 100mm, and other long path length cells, made of JGS1 Quartz, 200-2500nm.

Path LengthType1Inner Width1Code1Link1Type2Inner Width2Code2Link2
20mm20mm long path length cuvette10mmQS43Details20mm long pathlength quartz cuvette50mmQC3909Details
20mm20mm long pathlength black wall quartz cuvetteQF80Detailshttps://ecuvettes.com/product/qr11-quartz-reaction-cuvettes-30-40-50mm-pathlength-powder-fused-2-polished-windows/20mmQC3001Details
30mm30mm long pathlength quartz cuvette10mmQS30Details30mm long pathlength cuvette30mmQR1103Details
30mm30mm long pathlength black wall cuvette2mmQB21Details30mm long pathlength black wall flow cuvetteQB26Details
40mmhttps://ecuvettes.com/product/qc2801-50mm-path-customized-flow-cell-45x12-5x52-5mm-with-side-and-bottom-connectors/10mmQS32Details40mm long pathlength screw cap cuvette10mmQF13Details
100mm100mm long path length quartz cuvette10mmQS27Detailsqc2501-cylindrical-flow-cuvette (4)φ19mmQC2501Details
50mm50mm long path length quartz cuvette20mmQC51Detailsqc2801-spectrometer-flow-cell (4)10mmQC2801Details
100mm100mm long pathlength airtight cuvette10mmQC2701Details100mm long pathlength airtight black wall cuvette2mmQC2401Details

We also have long pathlength cuvettes made of optical glass, it range from 10-100mm. Click here to see more, and here to see the Glued* one .

  • When will use “short path” cuvettes?
    With regards to some high-concentration samples, it allows measurement with a 10-mm standard path length cell after diluting.
    But for some high concentration samples and can’t be diluted easily, for example, due to the analyte may interact with the solvent, diluting a sample may cause a change in the absorbance (i.e., shift in the peak wavelengths). In this case, a short path length cuvette is used to measure high-concentration samples without diluting effectively.

For instance, A well-known study using a short path length cell is solution analysis in the NIR region. If a 10mm standard pathlength cell is used for absorbance measurement in the NIR region, saturation often happens because light absorption by the solvent, making it impossible to determine the absorption of the analyte. Then a short path length cell is used to prevent absorption saturation because of the solvent.

There are short-path cells with optical path lengths of 0.1mm, 0.2mm, 0.5mm, 1mm, 2mm, 5mm, and etc.

Path LengthType1Code1Volume1Link1Type2Code2Volume2Link2
0.1mm0.1mm short pathlength cuvetteQS4135ulDetails0.1mm-short-panthlength-flow-cellQF776ulDetails
0.2mm0.2mm short pathlenth cuvetteQS2370ulDetailsQF92 0.2mm 16ul Flow Through Cell02QF9216ulDetails
0.5mmQS3103-0.5mm-175uL-Ultra-Short-Path-Length-Quartz-CuvettesQS31170ulDetailsqm47-micro-flow-cell (8)QM47150ulDetails
1mm1mm short pathlength cuvetteQS21350ulDetails1mm short pathlength flow cellQC50860ulDetails
2mm2mm short pathlength cuvetteQS26700ulDetails2mm Short pathlength flow cuvetteQF8660ulDetails
5mm5mm short pathlength cuvetteQS241.75mlDetails5mm short pathlength flow cellQC3701650ulDetails

We also have short pathlength cuvettes with macro window, air-tight, or narrow width as below.

Path LengthType1Code1Volume1Link1Type2Code2Volume2Link2
0.2mm0.5mm demoutanble short pathlength cuvetteQM2760ulDetails0.2mm Dual Channel FlowCellQF89Details
1mm1mm Air-Tight short pathlength CuvetteQC47350ulDetails1mm short pathlength macro window cuvetteQM261.5mlDetails
1mm1mm short pathlength screw cap cuvetteQF11350ulDetailshttps://ecuvettes.com/product/qf11-1mm-spectrophotometer-cuvette-with-screw-cap-0-35ml-absorption-cuvette-quartz/QM48350ulDetails
2mm1mm short pathlength screw cap cuvetteQF12700ulDetails2mm short pathlength stopper cuvetteQM36700ulDetails
5mm5mm short pathlength screw cap cuvetteQF141.75mlDetails5mm short pathlength long tube screw cap cuvetteQC14011.75mlDetails
5mm5mm short pathlength narrow width cuvetteQM67350ulDetailsQM86 5mm 7ml Macro Window Cuvette02QM86017mlDetails

We have more short path length cuvettes, such as 1.5mm, 2.5mm, 3mm, 6mm, and optical glass cuvette is also in stock. 

If you want to buy quality and affordable cuvette, please contact us or go to our web ecuvette.com.

After we studied the cuvette choosing of material, pathlength, volume, Z-Dimension and ordered the right cuvette, it’s time for us to learn how to clean the cuvette if we didn’t buy the disposable one.

  • How to clean to the outer window of cuvette?
    The cuvette should be replaced immediately if there are cracks, chips or scratches on the window, or result in poor performance, less effective result and waste your sample and time, it is much expensive than the cost of a replaced cuvette.

For fingerprints on cuvette windows, proper cuvette cleaning is very essential, otherwise you will get a higher absorbance reading than its actual performance.
Using high quality lens paper to wipe off the fingerprints or other stain on the window of cuvettes is safe to use to keep cuvette window clean.
Special Notice: Do not use paper towels, Kimwipes or other similar types of paper products. Because abrasives are used on most paper products, which will scratch the cuvette´s surfaces. Scratches will cause light scattering and will affect the measurements ultimately.

At the end of the day, clean your cuvettes thoroughly, air dry completely and store them in a cuvette rack or other suitable container. If the cuvettes didn’t dry completely and stored wet, they may dry with residue sample of previous application on the measuring surfaces, which will affect subsequent measurements.

cuvette rack
QA30 10mm standard cuvettes racks ,16 positions04

DO NOT use ultrasonic cleaning bath to clean your cuvettes. Or your cuvettes will be damaged or shattered because of the high frequencies used in these baths.

Therefore, a proper cleaning solutions for your cuvettes will increase their useful life and provide more consistent results.

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