Photometry refers to all measurement methods that run with a photometer and use visible light. Photometry is used to perform quantitative measurements on solutions or other dyes. The most common method is this measurement via the transmission, i.e. the passage of light through the optical medium. However, measurements are also possible via reflection, i.e. the reflection of light, which is used more on solid surfaces.
Photometry – construction of a photometer
The following figure shows the structure of a photometer:
Figure 1: Schematic representation of a photometer
Let’s go through the figure together from left to right:
light source
On the left is a light source, which can be a light bulb, for example. (Warm) white light comes from an incandescent bulb and most other light sources. is white light polychromatic («multicolored») because it is made up of many different wavelengths with different colors that all appear white to us. The easiest way to see that light is polychromatic is to use a prism to diffract the light. If you see a spectrum, that is, light splitting into many different colors, you are looking at polychromatic light.
monochromator
The problem with the polychromatic light is that it is still useless for our measurement. Each sample absorbs light at different wavelengths. Neither the absorbed wavelength nor the amount of absorbed light can be measured. For this reason, a monochromator is placed in front of the sample, which allows a specific wavelength of light to pass through. The wavelength that is allowed to pass can be set beforehand.
There are different monochromators. The simplest consists of the ones already mentioned above prism. Imagine holding your prism in front of the light source and seeing the light split up. Now all you have to do is block out all other light rays with an aperture so that only the wavelength you want comes through. You already have monochromatic («single color») light!
sample
The light now passes through the sample with a certain initial intensity I0. This is located in a so-called cuvette. However, there are a few requirements for the test:
- The solution must be homogeneous with the sample: it should be clear and not cloudy. Otherwise, the light would scatter on the fine particles of the sample, just like the polychromatic light on a prism, and the measurement would be falsified.
- The sample should absorb light at the measured wavelength.
- The concentration should be low because at high concentrations the Beer-Lambert law no longer applies.
Now the light passes through the sample, loses intensity and therefore only has the intensity I.
detector
The light with the intensity I is now caught by the detector, which carries out an intensity measurement. From the initial intensity known to the photometer and the intensity after transmission through the solution containing the sample, the photometer calculates the value for the extinction.
Briefly summarized
- Photometry refers to all light-based measurement methods that run with a photometer.
- A photometer always has the same structure:
- light source
- monochromator
- sample in cuvette
- detector
Photometry – absorbance as the central value
So what is extinction?
The absorbance is the common logarithm of the ratio of the initial intensity and the intensity after sample passage.
As a formula, this looks like this:
You must not confuse extinction with absorption, because extinction includes all light-fading events.
The following light-fading events can occur in your sample:
- The absorption of the wavelength by the molecules of the sample in the solution,
- the refraction of light in an inhomogeneous, milky solution on the particles of the sample,
- the reflection at the liquid surface or the cuvette.
In order to be able to measure the absorption specifically, you have to consider the following things:
- Your sample should be well dissolved: There should no longer be any particles floating around that make your sample milky or inhomogeneous.
- You should perform a calibration measurement with the cuvette and your solvent. This means that you put your solvent (usually water) and cuvette in the photometer and press calibrate.
- The photometer now measures the extinction again. However, since there is no dye, only the reflection from the water and the cuvette is measured, which is subtracted from the absorbance in subsequent measurements.
If you have followed these steps, you have successfully measured the absorbance with the absorbance.
Photometry – Beer-Lambert law
But what can you do with the extinction now? Here comes that too Beer-Lambert law help. This now represents the extinction in connection with our substance, its concentration and the layer thickness of the optical medium.
That Beer-Lambert law states that the extinction at a certain wavelength is equal to the molar, decadic extinction coefficient ε, (specific constant for a wavelength λ and a substance) times the concentration c of the sample in the solution times the layer thickness of the optical medium.
The layer thickness is, so to speak, the width of the cuvette, which is usually standardized at 1 cm.
The formula for that Beer-Lambert law :
On the left is the definition of extinction using the decade logarithm of the intensity ratio. You can see that on the right Beer-Lambert law.
So if the layer thickness remains constant, as well as the molar, decadic extinction coefficient, there is a linear function here. This straight line increases as the concentration of the sample in the solution increases.
Important: Beer-Lambert law only applies to small concentrations of the dye. Seen in this way, there is an upper limit for the extinction. Think of it this way: Your solution is black, so no light can get through. If you add more dye, the concentration increases, but the solution cannot become any blacker.
Recording a spectrum with a photometer
In order to determine the concentration of an analyte in the sample, it is usually sufficient to measure the extinction or the absorption of the solution. However, if you want to characterize your analyte more precisely, you may have to record a spectrum.
As a rule, the extinction is measured individually for each wavelength at a specified concentration and layer thickness. But since you don’t want to stand at your photometer, calibrate each wavelength individually and then measure them, modern spectrometers are used today to do this job for you.
Incidentally, as soon as we are talking about spectra, we are no longer in simple photometry, but in spectroscopy or more specifically, UV/VIS spectroscopy. The name comes from the fact that these spectra are recorded from the UV to the visible (visible) range of light.
The UV range is at wavelengths from 100 to 380 nm and the visible range is at wavelengths from 380 to 780 nm.
Application example of photometry for the Beer-Lambert law
Imagine you have a solution with an unknown concentration of an analyte that you know, which we now call learning material. You can see that the solution with study substance looks red and you may also already know from the chapter on dyes that substances always appear in the complementary color of the absorbed colour.
You take a color wheel labeled with wavelengths and see that the complementary color to the red you see is blue-green and you therefore decide to carry out several measurements with samples you have prepared with learning material at different concentrations at a wavelength of 640 nm.
The measurement was a complete success, you carried out five measurements and received the following table of values:
Concentration of learning material in mol / l
extinction
0.1
0.09
0.3
0.27
0.6
0.54
0.8
0.72
1.0
0.9
Now try to draw a diagram from these values. Write the concentration on the x-axis. It makes sense to go here in steps of 0.1. You should plot the extinction on the y-axis, which by the way has no unit.
Figure 2: Sample absorbance measurement diagram
Your chart should look something like this. Now you finally measure your sample with an unknown concentration of learning material. For this you get an extinction of 0.7. You can easily read off the concentration of the sample from the diagram if you go along the y-axis at the level of absorbance 0.7 to the straight line. From there you go to the x-axis and read the value there. Finished? Here you should get about 0.78 mol / l for the concentration of the sample and it can be that easy!
By the way, you get the molar, decadic extinction coefficient here from the slope, but remember to divide this by your path length of 1 cm!
Photometry – The most important thing
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Photometry refers to all light-based measurement methods that run with a photometer.
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A photometer always has the same structure:
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light source
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monochromator
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sample in cuvette
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Detector.
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The Lambert-Beer law, together with the definition for extinction, reads:
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Extinction includes all light-fading events:
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Visible light ranges from 380 to 780 nm.
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The UV light is in the range from 100 to 380 nm.
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If one records spectra, one no longer speaks of photometry, but of UV/VIS spectroscopy.