Spec 20 how does it work

A spectrometer is a device that produces, typically disperses and measures light. A photometer indicates the photoelectric detector that measures the intensity of light. You need a spectrometer to produce a variety of wavelengths because different compounds absorb best at different wavelengths.

For example, p-nitrophenol acid form has the maximum absorbance at approximately nm and p-nitrophenolate basic form absorb best at nm, as shown in Figure 3. Looking at the graph that measures absorbance and wavelength, an isosbestic point can also be observed. An isosbestic point is the wavelength in which the absorbance of two or more species are the same.

The appearance of an isosbestic point in a reaction demonstrates that an intermediate is NOT required to form a product from a reactant. Figure 4 shows an example of an 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.

Once you know the intensity of light after it passes through the cuvette, you can relate it to transmittance T. Transmittance is the fraction of light that passes through the sample. This can be calculated using the equation:.

Where I t is the light intensity after the beam of light passes through the cuvette and I o is the light intensity before the beam of light passes through the cuvette. Transmittance is related to absorption by the expression:. Where absorbance stands for the amount of photons that is absorbed. With the amount of absorbance known from the above equation, you can determine the unknown concentration of the sample by using Beer-Lambert Law. Figure 5 illustrates transmittance of light through a sample.

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. For this reason, Beer's Law can only be applied when there is a linear relationship. Beer's Law is written as:. The molar extinction coefficient is given as a constant and varies for each molecule. The path length is measured in centimeters. Guanosine has a maximum absorbance of nm. What is the concentration of guanosine?

What is the absorption coefficient? The absorption coefficient of a glycogen-iodine complex is 0. 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. How are bird eyes different from human eyes? The primary difference between bird eyes and human eyes is that bird eyes have at least four, and sometimes five types of color cones, whereas humans have only three.

In addition, birds can see UV wavelengths. One of the reasons for this is that birds have an additional type of photo receptor to the two that humans have, called double cones. Bird eyes also have no blood vessels, which contribute to light scattering. 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. Close lid. Read the absorbance lower scale OR transmittance upper scale as appropriate for your sample Fig.

Figure 7. Meter for reading percent transmittance upper scale or absorbance lower scale. NOTE: When taking several measurements at the same wavelength over a short time period, you do not need to reblank for each. IF, however, you change the wavelength, you must re-zero the instrument.

If you are taking readings over an extended period or sharing the instrument, re-zero for each measurement. Beer-Lambert Law. The Beer-Lambert law describes an important relationship that exists between absorbance A and two sample parameters - solute concentration c and length of the light path l. Simply put, the law states that absorbance, A , is directly proportional to c and l , and is represented by the following equation:.

For practical purposes, the light path is the interior diameter of the cuvette and is the same for all samples.