Spectrophotometry refers to a method that is employed in the determination of the amount of light that is absorbed by a chemical substance. In chemistry, the term spectrophotometry refers to the determination of the reflection or transmission characteristics quantitatively material through a function of wavelength (NIST, 2012). The spectrophotometry analysis is carried out through the light intensity determination as a beam of light passes through a tube containing the sample solution. The process of analyzing solution concentration using spectrophotometer utilizes the fact that light possesses a dual nature. This is where the light may exist either as particle or as a wave (Keller, 2003).
Basically, the principle that is applied in the spectrophotometry process is that compound transmits or absorbs light at a given a wavelength range. The quantity of light absorbed by any substance is thus determined, and the value used in quantifying of the amount of any substance that has the ability to absorb light.
In the determination of the intensity of light that is absorbed by the substance after the light has gone through it, an instrument, which is referred to as spectrophotometer is used. Through the use of this instrument, the concentration of any chemical substance that is not known can easily be determined (Blauch, 2009). A spectrophotometer can be grouped into two distinct types. The grouping is dependent on the light wavelength range that is used. In one of the classifications called UV-visible spectrophotometer, the method makes use of light that has a wavelength that is in the range of ultraviolet light between 185 and 400 nm and the range of visible light which is from 400 and 700 nm of the electromagnetic radiation spectrum.
The second classification of spectrophotometer is the IR spectrophotometer, which uses light in the range of infrared light from 700 to 1500 nm of the spectrum of the electromagnetic radiation. In the case where visible spectrophotometry is employed, it is possible to detect the transmission or absorption that is done by a given substance using the observed color (Davis, 2010).
Those substances that have do not have ability to take in light are, however, very difficult to quantify. For one to quantify most of these substances, some modifications on the substance are necessary in order to make them detectable using spectrophotometer. The compounds that are employed in this process of modifying the compound should be able to result into a substance that has the properties that are needed for spectrophotometry. These properties include that the absorption of light must be intensively sufficient to enable determination of small quantities of the species to be analyzed. The species that is involved in light absorption should be created in a very rapid way and has to have a good stability. The reaction through which the absorbing species is formed should also be stoichiometric and specific or even reproducibly non-stoichiometric. The reaction that gives the absorbing species should be specific with the absorbing species having a stable form and should not be affected by either the pH of the solution or solution temperature.
An example of a substance that is used in the modification is the 1, 10-phenanthroline that is used in the photometric determination of iron (Bellér, Lente, & Fábián, 2010). 1, 10-phenanthroline has the capability of reacting with the iron ions to forming a complex [Fe(phen)3]2+ (deep red colored) through the following equation.
Fe2+ + 3(1, 10-phen) → [Fe (1, 10-phen)3]2+
The ions of iron that are in the ferric oxidation state are not able to work properly in the experiment and need to be reduced returning their oxidation state back to the ferrous state. The process of reducing the ions achieved through hydroxylamine hydrochloride addition into the reaction. The process of spectrophotometry has been one of the most helpful techniques used in the quantitative analysis in various fields of study including physics, biochemistry, chemistry, chemical and material engineering, as well as in clinical applications. In the determination of the concentration of an unknown sample, the experiment involved the use of standard solutions that had known concentrations. By determining the absorbance of the standard solutions using a spectrophotometer and plotting, a standard curve was drawn to determine the concentration of solutions with unknown concentration (Laminar, 1998).
In this experiment, iron concentration was determined using a chromogenic reaction with the reagent 1, 10-phenanthroline forming a colored complex with iron.
In a 50 cm3 volumetric flask, 5 cm3 of hydroxylamine hydrochloride (10% w/v) solution was added. In the same volumetric flask, 10 cm3 of 0.10 mol/dm3 acetate buffer and 5 cm3 of 1, 10-phenanthroline solution (0.25% w/v) were added. 20 cm3 of unknown iron solution was then added into the volumetric flask. The total content that was in the flask was diluted up to the line mark using deionized water, and the flask marked as solution A. The bottom of the meniscus was made sure to be level with the line and a dropping pipette was used in adding the last ml of the deionized in order to prevent overshooting. The procedure was repeated in a fresh 50 cm3 volumetric flask to make up solution B. The two solutions, solution A and B were taken to be the sample solutions. Four standard solutions and the blank were collected and the blank used in the calibration of the spectrophotometer. The absorbance of the standards was read at 510 nm, and their readings recorded. In the same way, the absorbance of the sample solutions was done at 510 nm using the blank as a reference.
After the spectrophotometer was calibrated using the blank and the absorbance of the standard concentrations were measured, the results for the absorbance for the standard iron concentrations were obtained as in Table 1.
The absorbance of the two solutions A and B was measured and recorded in Table 2 below.
The results that were obtained for the absorbance of the standards solutions were used in plotting a graph of absorbance against concentration. The plotted graph is shown in Figure 1below.
The graph obtained was used to make a line of best fit and an equation that could provide a relation between the absorbance and the concentration of the iron solutions. From the attained equation, the concentration of the unknown solutions was determined. This was done by taking the Y in the equation to represent the absorbance and the x to represent the concentration. The calculations that were used in the concentration determination can be illustrated through the following steps.
Y= 7899.9x + 0.0046
The y and x can be replaced with the symbols for absorbance (A) and concentration (c) respectively and the equation written as,
The concentration for the sample solution A can be calculated as follows
The concentration for the sample solution B can be calculated as follows
Since the two solutions were the same, the average concentration for the two solutions can be determined to be 1.03723×10-4mol/dm3.
The solution that was used in the preparation of the working solution was made up of 20 ml of the solution of unknown iron concentration. This is an indication that the unknown solution had been diluted from was diluted in the making of the 50 ml of the solution. The initial iron concentration for the working solution can, therefore, be calculated using the formula C1×V1=C2×V2. In the equation, C2 is the concentration of the working solution while V2 is the volume of the sample solution. The C1, on the other hand, is the concentration of iron that was in the initial solution. The concentration of the unknown iron solution was thus calculated as follows
Suppose the unknown solution (total volume 100cm3) was made up by dissolving 2.3160 g of a solid in 100 cm3 of deionized water. Calculate the concentration of iron in the solid based on the results obtained above.
When the unknown solution having a total volume of 100cm3 is constituted by dissolving a solid that has a weight of 2.3160 g in 100 cm3 de-ionized water, iron amount that is in the solid can be determined using the following procedure:
The concentration of the unknown solution would still be the same in 100 ml as it was in the 20 ml of the solution that was used above. That is, the concentration would be 2.59 ×10-4mol/dm3.
For a volume of 100 ml, the moles of iron that were in the solution can be calculated by using the formula
moles=2.59 ×10-4mol/dm3×0.1L =2.59×10-5moles
One mole of iron is made up of 55.847 grams of iron content. The weight of iron that was in the solid can thus be calculated using the moles obtained and the relative atomic mass of iron.
Mass=2.59×10-5moles ×55.847 g =0.00145 grams
This experiment was aimed at determining the iron concentration through the use of a chromogenic reaction with the reagent 1, 10-phenanthroline forming a colored complex with iron. The experiment enabled a successful determination of the concentration of iron in the unknown solution samples. There was close similarity between the concentrations for the two samples since the values for their concentration were close to one another. This is a strong indication that the use of spectrophotometer in the determination of sample concentrations is a highly duplicable ion experiment. The results obtained from the experiment also enabled a successful determination of amount of iron that was in a solid sample.
The technique of using a spectrophotometer in analysis has various applications. The technique is used in monitoring sulfur compounds that absorb UV light strongly such as hydrogen sulfide, carbon disulfide and carbonyl sulfide for safety, comply with the environmental guidelines as well as for process efficiency (SensEvolution, 2013). The method is also employed in the determination of chemicals and compounds in substances such as food and drinks. Other fields where the technique is used include clinical, agricultural, pharmaceutical, and environmental (Mcgraw-Hill, 2011).
Bellér, G., Lente, G., & Fábián, I. (2010). Central Role of Phenanthroline Mono-N-oxide in the Decomposition Reactions of Tris (1,10-phenanthroline) iron(II) and -iron(III) Complexes. Inorganic Chemistry, 49(9), 3968–3970.
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Davis. (2010). Spectrophotometry. Retrieved June 4, 2013, from http://chemwiki.ucdavis.edu/Physical_Chemistry/Kinetics/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry
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Laminar. (1998). Example Lab Report: Spectrophotometric Analysis. Retrieved June 7, 2013, from http://www.public.asu.edu/~lwmays/classes/cee341/lab_example.pdf
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SensEvolution. (2013). UV-Visible Analysis Applications. Retrieved JUne 7, 2013, from http://sensevolution.baggi.com/spectroscopy-analysis-applications/9-uv-visible-analysis-applications.html