Spectrophotometer

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Introduction
What is Protein?
There are more than 300 amino acids in the nature. However, only 20 amino acids are joined by each other with peptide bonds in different sequences and numbers. These sequences are formed by helping of genetic information which is encoded in genes. So, each protein molecule has its own amino acid sequence. Proteins are most abundant long chain macromolecules in all cells and they serve as structure and function matter in cells of all living organisms such as hormones, enzymes, antibodies. [1]

Fig. 1 Fig. 2
General structure of amino acids Protein fragment showing peptide bond

Basis of Spectrophotometer
Measuring amount of substance in solution by helping of solution color is called colorimetry. Devices that used for this method is called colorimeter. In colorimetric measurements, color of solution is compared with color of standard solutions in different concentrations. Measuring amount of substance in solution by helping intensity of transmittance light from solution is called photometry. Devices that used for this method is called photometer. This kind of devices have filter to adjust wavelength of light. If the device has a prism to do this, it is called spectrophotometer. In photometric measurements, concentration of colorless solutions can be measured(by using UV lights).[2] Spectrophotometer is a kind of photometer which is used in molecular biology. It is used for determining the amount of substance in solution. Basic principle of the spectrophotometer is light transmittance with specific spectrums through the solution and determining the amount of absorbance. As amount of substance in solution increases, so more light is absorbed by solution. Spectrophotometer gives quantitative information about amount of substance in solution by determining intensity of not absorbing light. For example, to obtain growth of bacteria at different temperatures, when solutions which has bacteria are measured by spectrophotometer, more absorbance will be obtained in sample that has much bacteria. As a result, much bacteria means much substance in solution and this situation shows that absorbance is high. When calculate the concentration of substance with spectrophotometer, Lambert-Beer law is used.[3]

According to the this law; intensity of transmittance light is inversely proportional with solution concentration and travelling distance of light in solution logarithmically. Absorbed light is proportional with them. I0 : light intensity entering to sample

I : light intensity exiting from sample
I/I0 : transmittance
ε : molar extinction coefficient (l/mol.cm)
c : concentration of absorbing species (mol/l)
l : path length of light-absorbing sample (cm)
OD: absorbance ( -log T )
The Basic Components of a Spectrophotometer

Fig. 3
Real image of a spectrophotometer (Perkin Elmer LAMBDA 35 UV Vis System) A spectrophotometer device basically consists of light source, monochromator and detector. Optical signal which is converted to electrical signal in the detector is measured by a galvanometer. In addition to the main components, there are lenses and mirrors to gather, focus and reflect the incident light. A monochromator provides to make monochromatic light with one wavelength from the polychromatic light that comes from light source. To do this, prisms are used as monochromator in spectrophotometers.

Figure 3. The components of spectrophotometer
Bradford Assay
To measure concentration of proteins in solution, there are a lot of different methods. Every method has several advantages or disadvantages. The most commonly used three methods are basic absorbance method, Lowry assay and Bradford assay. Basic absorbance method is based on maximum absorbance of tyrosine and tryptophan amino acids at 280 nm. This method has an important disadvantage. If the proteins which are in same concentration...
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