Chemistry lab report (SPECTROPHOTOMETRIC DETERMINATION OF A FOOD DYE)

Experiment 4: Spectrophotometric determination

of a food dye

Introduction

Spectrophotometry is a method used to measure the extent to which a chemical substance absorbs light of different radiations by measuring the intensity of light as a beam of light passes through sample solution containing a food dye. The basic principle that applies here is that each compound absorbs or transmits light radiations over a certain range of wavelength. Spectrophotometry is one of the most useful methods of quantitative analysis in various fields such as chemistry, physics, biochemistry, material and chemical engineering and clinical applications among others.

Every chemical compound absorbs, transmits, or reflects light (electromagnetic radiation) over a certain range of wavelength. Spectrophotometry is a measurement of how much a chemical substance absorbs or transmits or even reflects electromagnetic radiation. Any application that deals with chemical substances or materials can use this technique. In biochemistry, for example, it is used to determine enzyme-catalyzed reactions. In clinical applications, it is used to examine blood or tissues for clinical diagnosis. There are also several variations of the spectrophotometry such as atomic absorption spectrophotometry and atomic emission spectrophotometry.

A spectrophotometer is an instrument that measures the amount of photons (the intensity of light) absorbed after it passes through sample solution. With the spectrophotometer, the amount of a known chemical substance (concentrations) can also be determined by measuring the intensity of light detected. Depending on the range of wavelength of light source, it can be classified into two different types:

UV-visible spectrophotometer: It uses light over the ultraviolet range (185 - 400 nm) and visible range (400 - 700 nm) of electromagnetic radiation spectrum.

IR spectrophotometer: This type uses light over the infrared range (700 - 15000 nm) of electromagnetic radiation spectrum.

White light (the light we observe) contains all wavelengths of light in the visible region of the electromagnetic radiation. A substance appears coloured because it absorbs light at one or more wavelengths in the visible region and transmits the remaining wavelengths. The human eye, a type of electromagnetic radiation detector, sees the light transmitted, not absorbed. The colour of the light transmitted is complementary to the colour of light absorbed.

A linear relationship exists between absorbance and concentration for dilute solutions at a specific wavelength. This relationship is known as Beer's Law and is given by the equation:

A = εbc ……………………………………….. 1

Where:

A = the absorbance (measured).

ε = the absorptivity constant (unique for each substance to be measured).

b = the thickness of the sample (determined by the width of the sample curvet and usually 1 cm)

c = the concentration of the absorbing substance.

In analytical analyses, a wavelength of light that is strongly absorbed is selected. It is determined by finding the maximum absorbance that occurs in the electromagnetic spectrum being analysed. Since absorbance is directly proportional to the concentration for dilute solutions, a plot of absorbance versus concentration can be used to construct a linear plot. After the plot is constructed, the concentration of an unknown substance can be determined using the absorbance of the unknown substance and the graph.

Procedure

The procedure is applicable for the spectrum SP 1105 Spectrophotometer

Set the wavelength indicator to the desired wavelength using the knob on the top of the instrument. In this case, set the wavelength to 400nm,

Set the operating mode to Transmittance by pressing the mode selector until the T is illuminated on the display.

You will need a pair of matched cuvettes for this experiment. Fill one of the cuvettes just over halfway with distilled water. This is your reference cuvette

Open the sample compartment on the instrument (the hinged cover on the left). Wipe the reference cuvette clean with a Kimwipe, and place it in the sample holder closet to you. Make certain that frosted sides of the cell point towards you. Close the sample compartment. Make sure that the knob on the lower left of the instrument is pushed all the way in at this point.

Adjust the instrument to 0% T (infinite absorbance) as follows: Pull the knob on the lower left of the instrument one notch. Press the 0% T button. Make certain that the instrument reads 00.0You will now set 100%T with your reference cuvette.

Push the knob pushed in all the way, press the 0A button. The instrument will read BLA for a few seconds and then display 100.0Press the mode button until the A is illuminated. You are now in Absorbance mode.

Open the sample compartment and remove the reference cuvette. Fill another cuvette just over halfway with the solution whose absorbance you want to measure. (in this case, fill the cuvette just halfway with solution 10). Wipe the cuvette clean, place it in the instrument, close the sample compartment, and record the absorbance.

Steps 1-8 are general list of steps for acquiring absorbance data from the Spectrum- 1105. The two steps listed below are specific to this experiment.

Using solution 10, record the absorbance every 25 nm from 400 nm up to and including 5600nm. Remember that you must readjust 0%T AND 100%T (i.e repeat steps 1-7) each time you change the wavelength.

Once you have found the maximum absorbance in this way, go back and determine λmax to the nearest 5nm by investigating wavelengths that gave absorbance.

Calibration curve and unknown measurement

Adjust the wavelength knob to the value λmax determined above. Do not forget to check and readjust 0% T and 100%T.

At this wavelength, measure absorbance for each standard solution (solution 1, 5, 10) as follows. With the knob on the lower left pushed all the way in.

Open the sample compartment and remove the reference solution from the sample cell holder.

Fill a cuvette just over halfway with solution 1and place the cuvette in the sample cell closest to you. Place the cuvette containing solution 10 in the sample cell.Fill one more cuvette just over halfway with solution 15 and place the cuvette in the sample cell holder.

Results and analysis

Table1

Beaker	Stocky dye (ml)	Distilled water ( ml )	Concentration (drops/L)
1	15	0	Cf = stocky
2	15	5	Cf = (15/20) x
3	10	10	Cf = (10/20)
4	10	20	Cf = (10/30)
5	5	20	Cf = (5/25)
6	5	35	Cf = (5/40)

Sample calculation of concentration from the table;

Use the equation:

CiVi = CfVf,

Where

Ci = initial concentration = 10drops/L

Vi = initial volume = 15mL

Cf = final concentration

Vf = final volume = 15mL + 5mL = 2OmL

Solve for Vf:

Vf = (Ci/Cf) Vi

= (15mL/2OmL) 10 drops/L

= 7.5 drops/L

Table 2: Radiation colours with their corresponding wavelengths.

Colour	Wavelength
violet	380–450 nm
blue	450–495 nm
green	495–570 nm
yellow	570–590 nm
orange	590–620 nm
red	620–750 nm

Discussion

You need a spectrometer to produce a variety of wavelengths because different compounds absorb best at different wavelengths. For example, p-nitro phenol (acid form) has the maximum absorbance at approximately 320 nm and p-nitrophenolate (basic form) absorb best at 400nm, as shown below in the sketch.

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 is 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. This is shown in the sketch below.

 

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:

Transmittance (T)=It/Io ……………………………. 2

Where It is the light intensity after the beam of light passes through the cuvette and Io is the light intensity before the beam of light passes through the cuvette. Transmittance is related to absorption by the expression:

Absorbance (A) = −log (T) = −log(It/Io) ……………………. 3

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. For instance the sketch below shows this a calibration curve for a blue dye.

Conclusion

In analytical analyses, a wavelength of light that is strongly absorbed is selected. It is determined by finding the maximum absorbance that occurs in the electromagnetic spectrum being analyzed. Since absorbance is directly proportional to the concentration for dilute solutions, a plot of absorbance versus concentration can be used to construct a linear plot.

References

Atkins, Peter and Julio de Paula. Physical Chemistry for the Life Sciences. New York: Oxford University Press, 2006.

Chang, Raymond. Physical Chemistry for the Biosciences. USA: University Science Books, 2005.

Gore, Michael. Spectrophotometry & Spectrofluorimetry. New York: Oxford University Press, 2000.