Samples are contained in round cuvettes that resemble common test tubes. It is best to work with two cuvettes with the spec 20, one containing your solvent and the other your solvent your reference and sample. Step 2. Step 3. Step 4. The Cary 50 is a lower cost instrument that competes well against more expensive double beam instrument for many applications. This instrument has been succeeded by the Agilent Cary The Cary 50 gets all its power from a computer and it an especially versatile instrument with many accessories that are especially useful in biological chemistry laboratories.
How is the human eye like a spectrometer? While eyes are commonly compared with cameras, an examination of the eye structure shows that it can also be seen as a small, simplified spectrometer. The images below show the structure of the human eye, and a detail of the retina, where the rods and cones are found.
Rods and cones are photoreceptive cells that translate the incoming light into signals that get transmitted to the brain, via the optic nerve, to be translated into colors, and subsequently coelesced into images.
Rods translate intensity and cones translate color. Humans have three types of color cones, blue, red and green. Remember the basic function of a spectrometer see above. In the case of the eye, the light source is the emitted light from an object. All substances absorb electromagnetic radiation light and transmit EMR at a complementary wavelength. This emitted light passes through the cornea, which acts as a refracting lens.
The light then travels through the retina, where the rods and cones act as the prism, diffracting the light and sending the data to the optic nerves to be collected by the brain. Set wavelength using the dial on top of the Spec 20 Fig. Figure 2. Wavelength control. Figure 1. Refer to your experimental protocol. If not etched in to the cuvette, use a wax pencil or Sharpie to make a small vertical mark at the top of each cuvette for alignment in the sample holder Fig. Figure 3. Figure 4. Raise the sample holder trapdoor and insert the cuvette such that the line on the cuvette lines up with the line on the sample holder Fig.
Close the lid. Using the right front knob Fig. This step is called setting the "full scale". Figure 6. Use right knob to set full scale against the blank. If your experiment involves multiple reaction tube formulations, each one may need its own blank and the Spec 20 must be rezeroed for each. Then a wavelength selector slit transmits only the desired wavelengths, as shown in Figure 1. Photometer : After the desired range of wavelength of light passes through the solution of a sample in cuvette, the photometer detects the amount of photons that is absorbed and then sends a signal to a galvanometer or a digital display, as illustrated in Figure 1.
Figure 2: A single wavelenth spectrophotometer You need a spectrometer to produce a variety of wavelengths because different compounds absorb best at different wavelengths.
Figure 3: Absorbance of two different compounds Looking at the graph that measures absorbance and wavelength, an isosbestic point can also be observed. Figure 4: An example of isosbestic point Referring back to Figure 1 and Figure 5 , the amount of photons that goes through the cuvette and into the detector is dependent on the length of the cuvette and the concentration of the sample.
Figure 5: Transmittance illustrated by Heesung Shim. Beer-Lambert Law Beer-Lambert Law also known as Beer's Law states that there is a linear relationship between the absorbance and the concentration of a sample.
Example 1 Guanosine has a maximum absorbance of nm. Solution To solve this problem, you must use Beer's Law. Solution Using Beer-Lambert Law, we can compute the absorption coefficient.
Example 4 In example 2 above, what is the molar absorption coefficient if the molecular weight is ? Solution It can simply obtained by multiplying the absorption coefficient by the molecular weight. Example 5 The absorption coefficient of a glycogen-iodine complex is 0. Solution It can also be solved using Beer-Lambert Law. References Atkins, Peter and Julio de Paula. Physical Chemistry for the Life Sciences. New York: Oxford University Press, Chang, Raymond. Physical Chemistry for the Biosciences.
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