Absorbance is a measure of the material-dependent reduction in the intensity of a light beam due to absorption or scattering after the beam has passed through matter (Fig. 1).1
In laboratories, absorbance measurements are carried out with the aid of a spectrometer and are used to determine various substances or their concentrations.
In biochemistry, absorbance measurement is used, for example, to determine the concentration of coenzymes, DNA or protein solutions, and to determine the cell density of bacterial cultures, for example.1
Fig. 1 Schematic representation of the passage of a light beam through turbid matter in a cuvette. A substance-dependent attenuation of the intensity of the beam occurs, which is measured at the sensor. Figure adapted from: Chemieunterricht.de, das Lambert-Beersche-Gesetz, https://www.chemieunterricht.de/dc2/rk/rk-lbg.htm [30.05.2022]
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The measurement of light intensity and reduction in photometry
In photometry, a light beam is sent through a solution, suspension or emulsion using a photometer and the absorbance is measured. The absorbance indicates how much light has been scattered or absorbed by the sample. The measured absorbance values can then be used to determine the concentration of dissolved substances in a liquid.
The physical processes of absorption and scattering are generally involved in the attenuation of light radiation as it passes through a sample.
If the particles in a solution or suspension are large in relation to the wavelength of the light, the light waves can no longer bypass them unhindered. This results in a strong deflection or scattering of the light. Such scattering is observed, for example, in the photometric measurement of blood. The erythrocytes in the blood are too large for the light to be guided around them unhindered.2
Furthermore, absorption of light by the particles in the liquid can occur. This occurs due to the interaction between the electromagnetic waves of the light and the electron shell of the dissolved molecules within the solution. Light is usually converted into heat in the process. Depending on the chemical structure of the molecules in the sample, different wavelength ranges of light can be absorbed to different degrees. For example, if a liquid that appears red is irradiated with white light, the yellow, green and blue light components are absorbed, while the red light shines through. This can be explained by the fact that while, for example, green light consists of green color, blue light of blue, etc., the white light is composed of all colors. Thus, the red liquid is irradiated with all colors (optically recognizable as white), causing some of them to be absorbed, and the red light to pass through.
Photometric measurements are usually performed in the wavelength range of ultraviolet (UV), visible (VIS) or infrared (IR) light. An absorbance measurement over different wavelengths is called spectroscopy or photometry, e.g., UV/VIS spectroscopy or infrared spectroscopy.2
Determination of the concentration by absorbance measurements
Thus, extinction measures the reduction in the intensity of light at a given wavelength. It is defined by the equation3:
E = lg(I0/I)
- E - measured extinction
- I0 - intensity of the light entering the solution
- I – intensity of the outgoing light
In an absorbance measurement to determine the concentration of a dissolved substance, the prerequisite is that the light-absorbing substance is in an optically transparent liquid, such as water. In this case, the concentration can be determined using what is known as Lambert-Beer's Law3:
E = ε*c*d
- E - measured extinction
- ε - molar extinction coefficient characteristic for each substance
- c - concentration of the solution
- d - layer thickness
The determination of the concentration by measuring the absorbance is therefore based on the so-called Lambert-Beer law, which states that there is a linear relationship between absorbance and concentration. However, there are also deviations from Lambert-Beer's law, especially at high substance concentrations. Therefore, a calibration curve is usually created before the actual measurement to check the linearity. Here, the absorbance of various standard solutions is plotted against their concentration3. Read our text on calibration curves to learn more.
Measurement method for determining the absorbance value
Photometric measurements measure the transmission of monochromatic light. The transmission describes the permeability of a medium for electromagnetic radiation, e.g. visible light.4
The measuring principle of photometric analyses
Photometric measurements are performed on photometers. The main elements of the instruments are a light source, a filter or monochromator for light separation, a detector and a display system. The cuvette containing the solution to be determined is placed in the beam path of the photometer.
A beam of a specific wavelength is emitted from the light source of the photometer and selected by the filter. The light beam reaches the cuvette, where it is reflected, absorbed or scattered. The remaining light arrives at the photocell, to which an intensity indicator is connected, where it is measured. Finally, the absorbance is displayed.5
What is the difference between single beam photometers and dual beam instruments?
A distinction is made between single-beam and double-beam photometers. In the case of single-beam photometers, there is a beam path within which both the blank cuvette and the measuring cuvette are brought individually one after the other into the beam path for the blank adjustment (Fig. 2).
The blank is a cuvette that contains only the medium in which the substance to be measured is dissolved. The blank measurement is performed to measure the absorbance of the medium and to calculate it with the measured absorbance of the substance.5
With the double-beam photometer (Fig.3), on the other hand, the blank cuvette and the measuring cuvette can be irradiated simultaneously, as here the light beam splits into two beams. This allows automatic compensation for blank absorption. This makes manual adjustment of the blank value superfluous when recording spectra at different wavelengths.6
Fig.2 Setup of a single-beam photometer. The empty and measuring cells are measured individually one after the other. Figure adapted from: Lexikon der Biologie – Spektrum akademischer Verlag, Photometer, https://www.spektrum.de/lexikon/biologie/photometer/51334 [30.05.2022]
Fig. 3 Set-up of a double-beam photometer. The blank and measuring cuvette can be measured simultaneously. Figure adapted from: Lexikon der Biologie – Spektrum akademischer Verlag, Photometer, https://www.spektrum.de/lexikon/biologie/photometer/51334 [30.05.2022]
What insights do the absorbance measurements provide?
Absorbance measurement is relevant for several areas. For example, the determination of absorbance can be applied for the qualitative identification of the measured sample. Furthermore, the concentration of an absorbing sample can be determined via calibration curves.7
In addition, absorbance measurements play a key role in kinetic reactions, where the concentration of the absorbing sample changes over time. The decrease in concentration of the reactant occurs as the sample is catalyzed by the addition of enzymes and a new product is formed. As the reaction proceeds, the reaction rate can be measured by the change in absorbance over time.8
These capabilities make absorbance measurement applicable to diverse industries.
Impulses for biochemistry
In biochemistry, absorbance measurements are also an important tool for the quantitative determination of numerous biomolecules.
Purity and concentration (DNA and RNA), for example, can be determined photometrically. The measurement is carried out in the UV range at 260 or 280 nm.7
In an absorption spectrum of 230 to 320 nm, extracted DNA has an absorption maximum at 260 nm (A260). Other organic substances such as proteins or carbohydrates also have their absorption maximum in the UV range, namely at 280 nm (A280). In order to evaluate the quality or purity of nucleic acids, an additional absorbance measurement at 280 nm is therefore often performed. The quotient of A260/A280 is a measure of the purity of the sample. If the value is in a range of 1.8-2.0, the DNA or RNA is considered pure and cleanly extracted. If the determined value is below this, a clear contamination of the sample by proteins is thus often indicated.7
The calculation of the concentration c [μg/mL] of the respective nucleic acid is carried out by the formula resulting from a conversion of Lambert-Beer's law:
c = A260*VF*UF
- c - concentration of nucleic acid
- A260 - absorbance at 260 nm
- VF - dilution factor
- UF - conversion factor
The conversion factor results from the inverse of the extinction coefficient and is specific for each sample: for double-stranded DNA it is 50 and for RNA 40.
In biochemistry, the quantitative yield of protein can also be determined photometrically. There are a number of possibilities for this. First, the absorption spectrum of a protein solution can be measured directly at 280 nm. In this case, the concentration is directly linear to the absorbance. On the other hand, however, the determination can be made indirectly, via colorimetric assays.
A dye is added to the protein solution to be analyzed, which forms a complex with the proteins contained and thus leads to a discoloration of the solution. An example of this is the Bradford assay, in which Coomassie Brilliant Blue G-250 is used as the dye. This has an absorption maximum at 470 nm. However, when complexed with the protein, the maximum shifts to 595 nm.7
Possibilities in environmental analysis
Photometry is also used in environmental analysis, as it is a particularly sensitive and selective analysis method that has proven to be very accurate and reliable.9
Thus, photometry is often used to study the quality of water. It is very important that there are no pollutants in groundwater or that the content of some components, such as nitrate, does not exceed a certain value. The examination of water not only serves its use in nature and the environment, but is also of great importance in industry, municipal water supply or fish farming. 10
The analysis involves the use of colorimetric methods, in which the components of water are colored with special chemicals. Coloration occurs due to the formation of chemical compounds between the ions to be measured, which are contained by the pollutants, and the chemicals. The intensity of the color depends on the concentration of the ions and makes the photometric measurement possible.10
Prerequisites for absorbance measurements
The absorbance should be measured in compliance with the following criteria.
- high accuracy
- sensitivity
- Rreproducibility
- linearity
- easy handling
Furthermore, photometers or spectrometers should offer the possibility to automatically calculate the sample concentration based on stored calibration curves from the measured absorbance.
How does the absorbance measurement with the fluidlab R-300 work?
Standard cuvettes (10 mm) with a filling volume of 4.5 ml or 3 ml, which are filled with a sample volume of 2.5 ml or 1.5 ml for the measurement, are suitable for absorbance measurement with the R-300. The liquid to be measured should be well mixed for this purpose.
The cuvette is now placed in the special adapter of the instrument. The adapter allows the light to pass the front side in a small beam angle. Inside the cuvette, reflection occurs due to particles in the liquid.
The beam finally passes the back side of the adapter and is detected, whereupon the absorbance can be read on the display of the R-300.
How to perform a measurement with the R-300 and how to use the cuvette is shown in more detail in this introduction video to the anvajo fluidlab R-300.
What are the advantages of absorbance measurement with the fluidlab R-300 compared to conventional photometers?
Compared to other photometers, the fluidlab R-300 has the advantage of being a very small and handy instrument that can be operated intuitively. It covers a wavelength range of 375 nm-700 nm with a photometric measuring range of 0-2.5, resulting in a very high linearity.
High linearity
The fluidlab R-300 has a photometric accuracy of 0.01. The high linearity of the instrument results from the small spectral bandwidth of 2 nm.
In direct comparison with one of the most commonly used spectrometers, the R-300 exhibited linearity over a wider range of values (Fig.4). This property allows very accurate evaluation of assays in high concentration ranges (see performance report on the spektrometer).
Fig. 4 Comparison of the linearity of a trypan blue dilution series between the fluidlab R-300 and a widely used competitor. The linearity of the R-300 is higher than that of the competitor over the range of values considered.
Fulfillment of photometric accuracy standards according to the requirements of pharmacopoeia
It is important that photometers meet certain guidelines concerning the spectral and photometric accuracy of the measurement system. In this way, a uniform measurement is to be generated with photometers.
The calibration of the instruments is usually carried out using clearly defined reference materials. For this purpose, glass cuvettes filled with specific liquids or cuvettes with special glass filters are often used.
The fluidlab R-300 is calibrated during production using certified reference filters from Hellma Analytics. Tests regarding accuracy, as well as reproducibility and linearity could show that the anvajo fluidlab R-300 fulfills the requirements of the pharmacopoeia (see performance report on the spectrometer).
You can read more details about this in our performance report of the R-300.
Multiple options for assay testing and analysis
All in all, the fluidlab enables a wide range of colorimetric and turbidimetric analyses in the 375 nm-700 nm range. For colorimetric analyses, it is important that the substance to be measured has a distinct coloration or is converted into a colored substance and has a defined relationship to the original substance.11
Turbidimetric analyses, such as measurements of optical density at 600 nm (OD 600), in which the growth of bacteria is determined, can also be performed with the fluidlab R-300.
The high linearity over a wide range of values even allows accurate evaluation of assays in high concentration ranges, which is not possible with most photometers. The fluidlab also has the unique ability to perform a live observation of the entire spectrum, displaying the absorbance of the measured substance at other wavelengths.
In addition, the fluidlab R-300 offers a kinetics application to automatically perform absorbance measurements over different time intervals.
Scientific sources
1Mayerhöfer, T. G., Pahlow, S., & Popp, J. (2020). The Bouguer-Beer-Lambert Law: Shining Light on the Obscure. Chemphyschem: a European journal of chemical physics and physical chemistry, 21(18), 2029–2046.
2Schoenberg, E. (1929). Theoretische Photometrie. Grundlagen der Astrophysik. Handbuch der Astrophysik, Springer, Berlin, Heidelberg, 2, 1-280.
3Abitan, H., Bohr, H., & Buchhave, P. (2008). Correction to the Beer-Lambert-Bouguer law for optical absorption. Applied optics, 47(29), 5354–5357.
4Proske, O., Blumenthal, H., Ensslin, F. (1953). Photometrie. Analyse der Metalle. Springer, Berlin, Heidelberg, 2, 1228–1240.
5Da Costa, G. S. (1992). Basic photometry techniques. In Astronomical CCD Observing and Reduction Techniques, 23, 90-102.
6Rugg, F. M., Calvert, W. L., & Smith, J. J. (1951). The Design and Performance of a Double-Beam Infrared Spectrophotometer. JOSA, 41(1), 32-38.
7Arnemann, J. (2019). DNA-/RNA-Konzentrationsbestimmung: Lexikon der Medizinischen Laboratoriumsdiagnostik. Springer Reference Medizin. Springer, Berlin, Heidelberg, 1, 719 ff.
8Buchholz, K. (1989). Immobilisierte Enzyme–Kinetik, Wirkungsgrad und Anwendung. Chemie Ingenieur Technik, 61(8), 611-620.
9Frenzel, W. (2015). Photometrie: Einsatzmöglichkeiten in der Umweltanalytik, Wiley Analytical Science, https://analyticalscience.wiley.com/do/10.1002/gitfach.14006 [05.04.2022].
10Berndt, V., Ottleben, I. (2017). Die Wasserqualität anforderungsgerecht mittels Photometrie prüfen, Laborpraxis, https://www.laborpraxis.vogel.de/die-wasserqualitaet-anforderungsgerecht-mittels-photometrie-pruefen-a-590075/ [05.04.2022].
11Willard, H.H., Furman, N.H., Grubitsch, H. (1950). Kolorimetrische Analyse: Grundlagen der quantitativen Analyse.Springer, Wien, 1, 375–382.
