Abstract The main objective of this practical was to demonstrate the use and the calibration of a spectrophotometer encompassed

on the need to proof conformity to Beer-Lambert law by use of a Spectronic 20. Initially, the absorption maximum of methyl orange was determined. Absorbance readings of methyl orange were taken at 10nm intervals from 600nm to 400nm. A cell containing distilled water was used as the blank. There was then a need to demonstrate BeerLambert law in which tubes were set up containing different volumes of methyl orange. All tubes were then read off at 460nm using a tube with distilled water as the blank. The absorbance of the unknown dye was also obtained in which its concentration was then interpolated. In calibrating the spectrophotometer, serial dilutions were done of potassium chromate in sulphuric acid were made. The absorbances of the solutions were then determined at 350nm. The molar absorbance index was then calculated and from this, the correction factor for the particular instrument. The absorption maximum of methyl orange was seen at 460nm. The concentration of the unknown dye was calculated to be 2.7g/ml and due to the linearity of the obtained curve, it was deduced to follow Beer-Lamberts law. An average value of 1493.3 was obtained in the calibration of the spectrophotometer leading a value of 2.12 for the correction factor which was out of the acceptable range. The unknown chromate was calculated to be 0.094mM when CF was used and 0.09375mM when CF is not used. Introduction/literature review 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. Spectrophotometry is widely used for quantitative analysis in various areas (e.g., chemistry, physics, biology, biochemistry, material and chemical engineering, clinical applications, industrial applications, etc.). Any application that deals with chemical substances or materials can use this technique. The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. This measurement can also be used to measure the amount of a known chemical substance. Spectrophotometry is one of the most useful methods of quantitative analysis (www.antoine.frostburg.edu). Spectrophotometers, electronically quantify the amount and kinds of light that are absorbed by molecules in solution. In its simplest form, a spectrophotometer has a source of white light (for visible spectrophotometrysome use Ultra Violet) that is focused on a prism or diffraction grating to separate the white light into its individual bands of radiant energy(www.chemwiki.ucdavis.edu). Each wavelength (colour) is then selectively focused through a narrow slit. The width of this slit is important to the precision of the measurement; the narrower the slit, the more closely absorption is related to a specific wavelength of light. Conversely, the broader the slit, the more light of different wavelengths passes through, which results in a reduction in the precision of the measurement. This monochromatic (single wavelength) beam of light, called the incident beam, then passes through the sample being measured. The sample, usually dissolved in a suitable solvent, is contained in an optically selected cuvette, which should be standardized to have a light path 1 cm across (Hames and Hooper, 2005).

After passing through the sample, the selected wavelength of light (now referred to as the transmitted beam, strikes a photoelectric tube. If the substance in the cuvette has absorbed any of the incident light, the transmitted light will then be reduced in total energy content. If the substance in the sample container does not absorb any of the incident beam, the radiant energy of the transmitted beam will then be about the same amount as that of the incident beam. When the transmitted beam strikes the photoelectric tube, it generates an electric current proportional to the intensity of the light energy striking it. By connecting the photoelectric tube to a device that measures electric current (a galvanometer), a means of directly measuring the intensity of the transmitted beam is achieved. In the spectrophotometers that we used (called the Spec-20), the galvanometer has two scales: one indicates the % transmittance (%T), and the other, a logarithmic scale with unequal divisions graduated from 0.0 to 2.0, indicates the absorbance (A) (Koolman and Roehm, 2005). Because most biological molecules are dissolved in a solvent before measurement, a source of error can be due to the possibility that the solvent itself absorbs light. To assure that the spectrophotometric measurement will reflect only the light absorption of the molecules being studied, a mechanism for subtracting the absorbance of the solvent is necessary. To achieve this, a blank (the solvent) is first inserted into the instrument, and the scale is set to read 100% transmittance (or 0.0 absorbance) for the solvent. The sample, containing the solute plus the solvent, is then inserted into the instrument. Any reading on the scale that is less than 100% T (or greater than 0.0 A) is considered to be due to absorbance by the solute only (Koolman and Roehm, 2005). There are two major classes of devices when it comes to spectrophotometry: single beam and double beam. A double beam spectrophotometer compares the light intensity between two light paths, one path containing a reference sample and the other the test sample (www.ruf.rice.edu). A single-beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted. Single-Beam spectrophotometers used in this practical, are often sufficient for making quantitative absorption measurements in the UV-Vis spectral region. The concentration of an analyte in solution can be determined by measuring the absorbance at a single wavelength and applying the Beer-Lambert Law (www.princeton.edu). Of use in this practical was the Spectronic 20. The Spectronic 20 is a spectrophotometer developed by Bausch & Lomb and launched in 1953. The design utilizes an analog dial for readout of transmission from 100%T to 1%T, 0A - 2A. Using the original instrument requires manual setting of the wavelength and making readings from a moving-needle analog display (Hames and Hooper, 2005). Beer-Lambert Law (also known as Beer's Law) states that there is a linear relationship between the absorbance and the concentration of a sample (www.teaching.shu.ac.uk). For this reason, Beer's Law can only be applied when there is a linear relationship. Beer's Law is written as: A =cl

where A is the measure of absorbance (no units), is the molar extinction coefficient or molar absorptivity (or absorption coefficient), l is the path length, and c is the concentration. The molar extinction coefficient is given as a constant and varies for each molecule. Since absorbance does not carry any units, the units for must cancel out the units of length and concentration. As a result, has the units: Lmol-1cm-1. The path length is measured in centimeters. Because a standard spectrometer uses a cuvette that is 1 cm in width, l is always assumed to equal 1 cm. Since absorption, , and path length are known, we can thus calculate the concentration of the sample (www.files.chem.vt.edu). Spectrophotometer calibration is a process in which a scientific instrument known as a spectrophotometer is adjusted to confirm that it is working properly. This is important, as it ensures that the measurements obtained with the instrument are accurate (www.scribd.com). The procedure varies slightly for different instruments, with most manufacturers providing a detailed calibration guide in the owner's manual so that people know how to calibrate the equipment properly. In spectrophotometer calibration, a reference solution is used to zero out the equipment. This solution provides a base or zero reading (www.wisegeek.com). The device is calibrated by placing the reference solution inside the spectrophotometer, zeroing out the settings, and running the instrument. Then, samples of an actual test material can be subjected to spectrophotometry in confidence that the machine has been calibrated and is working properly. In a single beam spectrophotometer, a single beam of light is generated, and the device must be recalibrated for each use (Voet and Voet, 2011). Methyl orange is a pH indicator frequently used in titrations. It is often used in titrations because of its clear and distinct colour change. In the basic form of methyl orange, a hydrogen ion is lost from the -NN- bridge between the rings, and the electrons formerly used to bind the hydrogen neutralize the positive charge on the terminal nitrogen, so that it is no longer able to pi-bond. Solutions of the methyl orange appear yellow in alkaline solution. The molecule absorbs bluegreen light, which makes its solution appear red in acidic conditions. The yellow form has an absorption peak at about 440 nm, whilst the red form has an absorption peak at about 520 nm (www.chemspider.com). Method Determination of absorption maximum of methyl orange Distilled water was placed in one cell labelled B and 10g/ml methyl orange placed into another tube labelled S. the spectronic 20 was then set to 600nm and the instrument was zeroed using cell B. absorbances were then measured at 10nm intervals until 400nm using cell B. The instrument was zeroed using cell B at each wavelength. Demonstration of the Beer-Lambert law Test tubes were set up containing 0.0, 2.0, 4.0, 6.0, 8.0, and 10.0, of 10g/ml of methyl orange respectively. Tubes were made up to 10.0ml by use of water. Tube 1, containing 10ml of water

was then used as the blank to standardize the instrument with the subsequent recording of the absorbance of each solution at 460nm. The absorbance of the unknown dye was also computed. Calibration of the spectrophotometer Two serial dilutions were made of potassium dichromate dissolved in 1L 0.018N sulphuric acid so that each test tube contained 8ml of solution. The absorbances of the 3 solutions at 350nm using 0.018M sulphuric acid as the blank. The molar absorbance index for each of the solutions was then calculated as was the correction factor for the particular spectrophotometer. Results The absorption maximum of methyl orange is 460nm. The concentration of the unknown dye was calculated to be 2.7g/ml and due to the linearity of the obtained curve, it was deduced to follow Beer-Lamberts law. An average value of 1493.3 was obtained in the calibration of the spectrophotometer leading a value of 2.12 for the correction factor. The unknown chromate was calculated to be 0.094mM when CF was used and 0.09375mM when CF is not used. Discussion An absorption spectrum is plot of the functional amount of radiation absorbed at each wavelength or frequency as a function of wavelength or frequency. A materials absorption spectrum shows the fraction of incident electromagnetic radiation absorbed by the material over a range of frequencies or wavelengths (or electromagnetic spectrum, broken by a specific or characteristic pattern of dark lines or bands, observed when radiation traverses a particular absorbing medium). The absorption pattern is unique and can be used to identify and/or quantify the material (www.chemspider.com). The absorption spectrum of methyl orange is as shown in figure 1.2. it was noted that the absorption maxima was 460nm whereas literature reviews that it should be 440nm. Various possible explanations for this deviation exist, includingthe series of steps (human error), transfers and also due to error of illusion and parallax errors. In a single beam absorption spectrophotometers fluctuations of light fluxes of both line and continuum sources also introduce error into the measurement of optical density. The obtained absorption maxima was however close to the theoretical value and thus this can be used to assay the dye. The obtained spectra of methyl orange has a single absorption band with maximum at 440 nm. When plotted with the respective concentrations, a clear conformation of the methyl orange absorbances to the Beer-Lambert law is shown. The absorbance of the unknown sample was 0.0126 and by extrapolating this absorbance value with dye concentration in Fig 3 an approximate concentration of 27g/ml was obtained. The method was noted to be laborious and cumbersome; the actual wavelength of maximum absorption might be in-between the intervals so it is open to error as the value becomes assumptive. This process may thus result in overlapping peaks. A better way would be to use automated spectrophotometers which scan through the relevant wavelengths with no room for

human error and another way is to use derivative spectrophotometry which can give improved resolution of overlapping peaks. The figure given below is a representation of the structures of methyl orange at pH 3.1 and 4.4 respectively. At pH 3.1 the dye is yellow and red at pH 4.4 (www.scribd.com).

Figure 1.1 structures of methyl orange at ph 3.1 Choosing a wavelength at an absorption maximum minimizes deviations from Beers law, which assumes is constant. A peak in the absorption spectrum where the analyte is the only compound absorbing light is selected, or a wavelength is chosen where the analyte has the largest difference in its absorbance relative to other sample components (Voet and Voet, 2011). The maximum absorption of 350nm wavelength was obtained and this wavelength was selected for the measurement of potassium dichromate dilutions. Results from the determination of the absorbance values of the potassium dichromate dilutions were performed using this maximum wavelength and the obtained absorbances are shown in table 1.1 with the subsequent concentration values. The CF value (2.12) was outside theacceptable range of 0.95 to 1.05 but due to time constraints other dilutions and instrumentation checks were not done as per procedure. The unknown dichromate gave an absorbance reading of 0.14, which was extrapolated to a concentration of 0.109375mM. Using the CF value the concentration was calculated to be 0.094mM. This constituted a 14.1% variation. Without using the CF value the concentration is 0.09375mM constituting a variation of 14.3%. References Hames .B.D., Hooper N.M, 2005, Instant notes in Biochemistry, 2nd Edition, Taylor e-Library, Leeds. Koolman .J. Roehm .K.H., 2005, Color Atlas of Biochemistry, 2nd Edition, Thieme Stuttgart, New York. Voet .D. and Voet .J.G., 2011, Biochemistry, 4th edition, John Wiley & Sons Inc., New Jersey. http://antoine.frostburg.edu/chem/senese/101/acidbase/faq/methyl-orange.shtmlaccessed 17/03/13 at 13:00 http://www.chemspider.com/Chemical-Structure.16736152.htmlaccessed 17/03/13 at 13:00

http://www.scribd.com/doc/38584941/Calibration-of-UV-Spectrophotometeraccessed at 13:00

17/03/13

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htmaccessed 17/03/13 at 13:00 http://www.files.chem.vt.edu/chem-ed/spec/beerslaw.html accessed 17/03/13 at 13:00 Appendix

0.6 0.5 0.4 0.3 0.2 0.1 0 0 100 200 300 400 500 600 700

Absorbance

Wavelength(nm)

Figure 1.2 Figure 1.2 shows the absorption spectra for methyl orange.

0.6 0.5 0.4 0.3 0.2 0.1 0 0 2 4 6 8 10 12

Absorbance

conc(g/ml)

Figure 1.3 Figure 1.3 shows the plot of absorbance vs. dye concentration (g/ml).

0.6 0.5 0.4 0.3 0.2 0.1 0 0 20 40 60 80 100 120

Absorbance

conc(g/tube)

Figure 1.4 Figure 1.4 shows the plot of absorbance vs. dye concentration (g/tube). Table 1.1 Dilution 1 2 Concentration 0.0625 0.125 Absorbance 0.03 0.16 - value 480 1280

3 0.25 0.68 2720 Table 1.1 is the representation of the dichromate dilutions, concentrations, absorbances and the calculated value. Average is therefore = CF = =2.12 = 1493.3. Using this as :

The unknown chromate gave an absorbance reading of 0.14. This was extrapolated using: to give a concentration of 0.109mM. Using the correction factor, the unknown chromate was calculated as: = 3160/2.12 giving as 1490.566. Using = Concentration = by: When the CF is not used, Concentration = given by: = 0.09375mM. Therefore, the variation is the concentration was calculated to be 0.094mM. = 0.094mM. Therefore, the variation when CF is used is given

Answers to questions for experiment 2.2.1 1. The curve conforms to Beer-Lambert law since it is linear. 2. Beer-Lambert Law (also known as Beer's Law) states that there is a linear relationship between the absorbance and the concentration of a sample. Beer's Law is written as: A =cl where A is the measure of absorbance (no units), is the molar extinction coefficient or molar absorptivity (or absorption coefficient), l is the path length, and c is the concentration. The molar extinction coefficient is given as a constant and varies for each molecule. Since absorbance does not carry any units, the units for must cancel out the units of length and concentration. As a result, has the units: Lmol-1cm-1. The path length is measured in centimeters. Because a standard spectrometer uses a cuvette that is 1 cm in width, l is always assumed to equal 1 cm. 3. The concentration is 2.7g/ml. This was interpolated from Figure 1.3 4. /ml is the better concentration term because it is more standard and is noted to be a scientific term; it also give a better graphical representation.

5. A standard curve is an ideal and imaginary line that perfectly obeys Beer-Lambert law. It is also a quantitative research tool used in the plotting of assay data to be used in determining the concentration of an unknown substance. 6. The figure shows a curve obeying Beer-Lambert law.

NAME

TINASHE W. MANGWANDA

STUDENT NO

N0110270A

COURSE

ANALYTICAL BIOCHEMISTRY

PROGRAMME :APPLIED BIOLOGY AND BIOCHEMISTRY

LECTURER

PROF SIWELA

PRACTICAL WRITE UP: Use and calibration of spectrophotometers