SPECTROPHOTOMETRY

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ABSTRACT Spectrophotometric analysis for determining the amount of an inorganic compound in solution involves a reaction between an organic reagent and an analyte to form a colored complex. The reaction can be used to determine analyte concentrations assuming the color intensity and absorbance is proportional to the analyte concentration, the complex is stable, and the reagent does not signicantly react with other constituents thereby causing interferences. A spectrophotometer is the specic device which measures the absorption of a monochromatic light beam by a sample and added reagent. The objective of this laboratory exercise is to become familiar with a typical spectrophotometric analysis and to examine the effect of an interfering substance. The inorganic analyte being considered in this particular analysis is phosphate and the interfering substance is arsenic. INTRODUCTION A study of the interaction of light (or other electromagnetic radiation) with matter is an important and versatile tool for the chemist. Indeed, much of our knowledge of chemical substances comes from their specific absorption or emission of light. In this experiment, we are interested in analytical procedures based on the amount of light absorbed (or transmitted) as it passes through a sample. Suppose you look at two solutions of the same substance, one a deeper color than the other. Your common sense tells you that the darker colored one is the more concentrated. In other words, as the color of the solution deepens, you infer that its concentration also increases. This is an underlying principle of spectrophotometry: the intensity of color is a measure of the amount of a material in solution. A second principle of spectrophotometry is that every substance absorbs or transmits certain wavelengths of radiant energy but not other wavelengths. For example, chlorophyll always absorbs red and violet light, while it transmits yellow, green, and blue wavelengths. The transmitted and reflected wavelengths appear greenthe color your eye sees. The light energy absorbed or transmitted must match exactly the energy required to cause an electronic transition (a movement of an electron from one quantum level to another) in the substance under consideration. Only certain wavelength photons satisfy this energy condition. Thus, the absorption or transmission of specific wavelengths is characteristic for a substance, and a spectral analysis serves as a fingerprint of the compound. In recent years spectrophotometric methods have become the most frequently used and important methods of quantitative analysis. They are applicable to many industrial and clinical problems involving the quantitative determination of compounds that are colored or that react to form a colored product.
LIGHT AND THE PERCEPTION OF COLOR

Light is a form of electromagnetic radiation. When it falls on a substance, three things can happen: the light can be reflected by the substance it can be absorbed by the substance certain wavelengths can be absorbed and the remainder transmitted or reflected Since reflection of light is of minimal interest in spectrophotometry, we will ignore it and turn to the absorbance and transmittance of light. The color we see in a sample of solution is due to the selective absorption of certain wavelengths of visible light and transmittance

of the remaining wavelengths. If a sample absorbs all wavelengths in the visible region of the spectrum, it will appear black; if it absorbs none of them, it will appear white or colorless. We see the various colors when particular wavelengths of radiant energy strike our eyes. For example, the wavelength we perceive as green is 0.0000195 inches or, expressed more scientifically, 495 nanometers. Suppose we shine a beam of white light at a substance that absorbs blue light. Since the blue component of the white light gets absorbed by the substance, the light that is transmitted is mostly yellow, the complementary color of blue. This yellow light reaches our eyes, and we see the substance as a yellow colored substance. The table below gives pairs of complementary colors and the corresponding wavelength ranges.
Wavelength Color Absorbed Color Observed

1. 2. 3. 4. 5.

0.576 meter 0.600 meter 0.598 meter 0.620 meter 0.567 meter

These value are absorbance of the crystal violents and we calculated the unknown concentration by graph. RESULT

400 435 495 560 650 800

violet blue green yellow orange red

yellow-green yellow purple blue greenish blue bluish green

You should remember, of course, that the visible range is only a very small part of the electromagnetic spectrum. Ultraviolet and infrared spectrophotometric methods are suitable for many colorless substances that absorb strongly in the UV or IR spectral regions. MATERIAL-METHOD We took; 1. 0.1 M crystal violet 2. 0.2 M crystal violet 3. X M crystal violet 4. 0.3 M crystal violet 5. Y M crystal violet Then we placed these liquid in cuvette. Then we switch on the spectrophotometer. And we placed one by one in spectrophotometry. We can reported absorbance of different concentration in below:

DISCISSION In conclusion, chlorophyll inside of an intact chloroplast that has absorbed light gives

different results when the chloroplast is not intact. (Campbell, Neil) When inside of a thylakoid membrane, chlorophyll acts in a photosystem. In a photosystem, the energy gained from the absorbed light is transferred to an electron transport chain. (Campbell, Neil) However, when the spinach was ground up, the chloroplasts were broken up. Therefore, the photosystems were not functioning, and the chlorophyll fluoresces since there were no electron acceptors to prevent the excited electrons from dropping back down to ground state. Initially, it was hypothesized that the wavelength with the greatest transmission % would be 550 nm. However, by analyzing the graph, it becomes apparent that although there was a peak in the graph in the region of 550 nm, the region of 700 nm contained the highest transmittance percentage. This unexpected peak can be accounted for by the fact that when the thylakoids were mashed up, the photosystems, which are best at absorbing wavelengths of light at 680 nm and 700 nm, were disabled. Therefore, the energy from the absorbed light that would normally be transferred by the photosystems to the electron transport chain was fluoresced, resulting in a higher transmittance percentage. In addition it was expected that the highest absorption % would be between 420-450 nm and 650-700 nm. 650-700 nm is the range in which photosystem 1 and 2 absorb light to being the light dependant reactions. It was true that for the purple and blue light, absorbance was very high. Also, as expected, absorbance in the green region was very low, since green light is the least effective wavelength that can be used in photosynthesis. (Campbell, Neil) However, the unexpected low occurred in the 650-700 nm regions, where absorbance was expected to be high. The rise in the absorbance graph in the range of 550 nm to 600 nm can be explained by the presence of xanthophylls (which absorbs yellow wavelengths) and carotene (which absorbs

yellow wavelengths orange-yellow), which are also present in spinach. This can be explained in the same way the unexpected high in the 650-700 nm range for transmittance percentage: the photosystems were disabled, and therefore that range of light was fluoresced. A comparison of the two graphs shows a relationship between transmittance percentage and absorbance. When the transmittance percentage increases, the absorbance tends to decrease. This relationship is represented by the equation Absorbance = log 100/ Transmittance Percent. REFERENCES http://www.123helpme.com/view.asp? id=121700 (SPECTROPHOTOMETRY) http://employees.oneonta.edu/kotzjc/LAB/Spec _intro.pdf (SPECTROPHOTOMETRY)