Today i want to share my experiment report which is about spectrophotometer . You can understand how this machine works and also you can calculate how much wavelengths are absorved . Enjoy you reading. And do not use this report without show references please .
Name : Nazlican
- Cansu Abay
- Yasın Emre Dogan,
- Aleyna Yılmaz
Professor:Yrd.Doç.Dr. Ozlem Yalcın Capan
Subbitted to : Yrd.Doç.Dr. Ozlem Yalcın Capan
To understand how spectrophotometer works and make measurement with it.
Spectrophotometer is a device that measures the intensity of passing light that can pass through a specimen due to absorption or excitation. The spectrophotometer is based on the fact that compounds absorb light of a certain wavelength. The amount of light radiation absorbed by the sample is measured by the spectrophotometer. Light absorption is directly related to the concentration of the color compound in the sample.
- Light source: Light produced on the solution will be dropped on the lamp. . It produces light between 20 nm and 700 nm wavelength.
- Input slit: A light line of light from the source of light to the system is a small transition area that allows you to enter the state.
- Monochromator: Only on the light tub of certain wavelength.
- Output slit: Anyone who prevents the arrival of thetransition interval.
- Cuvette: Special tube for colored solution.
- Detector: Unit measuring light emitted by the bathtub.
- Meter: It is the part that enables to quantify the amount of light measured.
Figure 1 illustrates the basic structure of spectrophotometers. It consists of a light source, a collimator, a monochromator, a wavelength selector, a cuvette for sample solution, a photoelectric detector, and a digital display or a meter.
The Mechanism of Spectropotometer
Between the sample spectrometer to be examined and the photometer, placed in a special tube made of hard plastic or quartz. Different samples different wave they need to find this space first because they absorb their size. For example, the known spectrum range of DNA is 260 nm. This is the amount of DNA range is used. This range can be found by spectrometry at all intervals the beam is sent. The graph obtained from the results obtained is analyzed it is understandable in which range the material absorbs light.
Calculations (Beer-Lambert Law )
intensity of light through sample (I)
Transmittance (T) = ——————————————————-
intensity of light through blank* (I0)
Absorbance (A) = log10 ——
- 0,0025 , 0,0050 , 0,0100 , 0,0150 , 0,0200 concentrations of bromophenol (BMP) dye solution.
- Plastic cuvettes.
- D h2o
1) Open the spectrophotometer.
2) Take 0,0025 , 0,0050 , 0,0100 , 0,0150 , 0,0200 and 2 unknown concentrations of bromophenol (BMP) dye solution
3) Take two plastic cuvettes. Fill one of the cuvette with distilled water to use as an empty water and fill the other cuvette with the BMP solution.
4) To automate the spectrophotometer, insert the empty tube, set the wavelength to 625 nm and press the autozero key. The counter will show zero absorption.Do it for every concentrations of bromophenol blue dye .
5) Remove blank and place cuvette containing 0,0025 M , 0,0050 M , 0,0100 M , 0,0150 M , 0,0200 M concentrations of bromophenol blue dye
6)Read and record the absorbance. Calculate unknowns by results which oncentrations of solutions are known .
|Concentration(M) of bromophenol(x axis)||ABS(625)
Figure 2 .. Table of concentrations .
Figure 3.. graph of concentrations.
By this graph , a formula has been founded.
0,466 – 0,077402430 =136,1902439024x
(0,466 – 0,077402430) /136,1902439024 = x x= 2,85334667 . 10^-3 M = Unknown 1’s concentration and,
2,348 – 0,077402430=136,1902439024x
(2,348 – 0,077402430) / 136,1902439024 =x x=1,6672248 . 10^-2 M Unknown 2’s concentration
1) Why did we use plastic cuvettes and not quartz cuvette?
Quartz cuvettes are suitable for both UV and visible wave lengths. Plastic cuvettes can be used easily between 200-700 nm. This experiment needs to cuvettes which can measure 625 nm .
2) Why does DNA absorb highest energy at 260nm?
Absorption in DNA and proteins occurs by the rings in chemical structures. The photon from the light source stimulates the pi orbitals in the annular structures and is converted to energy by photon absorption. For this reason, spectrophotometric DNA measurements take place in cyclic molecules with pi-conjugated systems. In the case of nucleotides, this absorption takes place by the annular structures on the nitrogenous base and mainly absorbs the light at a wavelength of 260 nm. Since the nucleotides that make up DNA and RNA show absorbance, especially at 260 nm, the amount of DNA is also analyzed using this wavelength in DNA measurements.