Title of Experiment Determination of Erythrosine Concentration Using Uv-Visible Spectrophotometer

INTRODUCTION A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. When a photon encounters an analyte molecule (the analyte is the molecule being studied), there is a chance the analyte will absorb the photon. This absorption reduces the number of photons in the beam of light, thereby reducing the intensity of the light beam. Click on the Start button to start the simulation and the Stop button to stop the simulation. The light source is set to emit 10 photons per second. The intensity of the light reaching the detector is less than the intensity emitted by the light source. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200nm - 2500nm using different controls and calibrations. Within these ranges of light, calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination. An example of an experiment in which spectrophotometry is used is the determination of the equilibrium constant of a solution. A certain chemical reaction within a solution may occur in a forward and reverse direction where reactants form products and products break down into reactants. At some point, this chemical reaction will reach a point of balance called an equilibrium point. In order to determine the respective concentrations of reactants and products at this point, the light transmittance of the solution can be tested using spectrophotometry. The amount of light that passes through the solution is indicative of the concentration of certain chemicals that do not allow light to pass through.

OBJECTIVE 1. 2. 3. 4. 5. To obtain the absorption spectrum wavelength for the dye solution To determine the wavelength of maximum absorbance (max) from the spectrum To produce a standard calibration curve from the series of standard solution To use the standard curve to determine the concentration of an unknown solution To compare between single beam and double beam spectrophotometer

PROCEDURE PREPARATION OF STANDARD SOLUTION 1. (200 X 10-4 g/100ml ) of dye stock solution ( erythrosine ) was diluted into a series of different concentration as below : BLANK: 5 X 10-4 g/100ml 10 X 10-4 g/100ml 15 X 10-4 g/100ml 20 X 10-4 g/100ml 25 X 10-4 g/100ml

2. An unknown concentration of the dye solution is provided

OBTAINING THE ABSORPTION SPECTRUM 1. The spectrophotometer was turn on (Perkin-Elmer lambda 35 ) 30 minutes earlier to allow it to warm up . 2. The cuvette is filling about 2/3 full with the blank solution and another with 10 X 10-4 g/100ml concentration of the dye solution. 3. The uv visible absorbance spectrum for erythrosine was determined .the range of wavelength to use is 400 700 nm. 4. The absorbance maximum wavelength was identifying.

DETERMINATION OF STANDARD CURVE (BEER LAMBERTS LAW) Spectrophotometer type: Single beam Spectronic Palmer 1100 Double beam Perkin Elmer lambda 35

1. The spectrophotometer wavelength was set using the max obtained. 2. The absorbance for each standard solution and the unknown solution was measured.

CALCULATION M1V1=M2V2 a. 5 X 10^-4 g/100 ml ( 200 )( V1 ) = ( 5 )( 100 ) V1 = 2.5 ml

b. 10 X 10^-4 g/100 ml ( 200 )( V1 ) = ( 10 )( 100 ) V1 = 5.0 ml

c. 15 X 10^-4 g/100 ml ( 200 )( V1 ) = ( 15 )( 100 ) V1 = 7.5 ml

d. 20 X 10^-4 g/100 ml ( 200 )( V1 ) = ( 20 )( 100 ) V1 = 10.0 ml

e. 25 X 10^-4 g/100 ml ( 200 )( V1 ) = ( 25 )( 100 ) V1 = 12.5 ml

GRAPH SINGLE-BEAM SPECTROPHOMETER ABSORBANCE

Concentration Vs Absorbance
0.16 0.14 0.12
R = 0.9721

Absorbance

0.1

0.08
0.06 0.04 0.02 0 0 2 4 6 8 10 12 14

Concentration (mL)

GRAPH DOUBLE-BEAM SPECTROPHOMETER ABSORBANCE

Concentration VS Absorbance
0.14 0.12
R = 0.1297

Absorbance

0.1 0.08 0.06 0.04 0.02 0 0 2 4 6 8 10 12 14

Concentration (mL)

DISCUSSION In this experiment, we have learned how to determine the erythrosine concentration by using UV-visible spectrophotometer. Before the determination of absorbance, the standard solution must be freshly prepared and must be prepared with extreme care because the accuracy of analyte determination is depending on the accuracy of the standard. As the mineral elements concentration in foods are at trace level, thus it is essential to use highly pure chemical reagent and water for preparation of samples and standard solutions. Thats why the deionized water is used to dilute dye solution. In single-beam uv-visible absorption spectroscopy, obtaining a spectrum requires manually measuring the transmittance of the sample and solvent at each wavelength. Single-Beam spectrophotometers 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. Singlebeam spectrophotometers can utilize a fixed wavelength light source or a continuous source. The simplest instruments use a single-wavelength light source, such as a light-emitting diode (LED), a sample container, and a photodiode detector. Instruments with a continuous source have a dispersing element and aperture or slit to select a single wavelength before the light passes through the sample cell. The double-beam design greatly simplifies this process by measuring the transmittance of the sample and solvent simultaneously. The detection electronics can then manipulate the measurements to give the absorbance. The dual-beam design greatly simplifies this process by simultaneously measuring P and Po of the sample and reference cells, respectively. Most spectrometers use a mirrored rotating chopper wheel to alternately direct the light beam through the sample and reference cells.

The detection electronics or software program can then manipulate the P and Po values as the wavelength scans to produce the spectrum of absorbance or transmittance as a function of wavelength. The linearity of the Beer-Lambert law is limited by chemical and instrumental factors. Causes of nonlinearity include, deviations in absorptivity coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity. Secondly, scattering of light due to particulates in the sample and fluoresecence or phosphorescence of the sample. Next, changes in refractive index at high analyte concentration, shifts in chemical equilibria as a function of concentration. Lastly, non-monochromatic radiation, deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band and stray light. From our result it show that the higher the concentration, the higher the absorbance value. Linear regression (R^2) of single-beam spectrophotometer is 0.9721 higher than double-beam spectrophotometer that is 0.1297.In double-beam, error has occur because it suppose the higher the concentration, the higher the absorbance value. But, start at 10mL, the absobance value decrease. The error came from Spectrophometer (Perkin-Elmer Lambda 35). R^2 gives a formula for the line most closely matching those point. It also gives an R^2 value to say how well the resulting line matches the original data points. Single-beam spectrophotometer only have a single beam from the light source. The reference standard is measure to standardize the instrument. For single-beam configuration to perform well, the light source, detector and electronics must be reasonably stable over time.

The cuvette with erythrosine that is used must be placed in the right slot. The clear side must face the light source in order to allow the light fully penetrate the sample. While handling the cuvette, we must not let our fingers to touch the clear side or the light will just penetrate our fingerprint, not the sample. The advantage of using the single-beam spectrophotometer is that there are fewer moving part which make it easier to handle. The beam from the light source of double-beam spectrophotometer is split into two. One beam illuminates the reference standard while the other one illuminates the sample. The beam recombined before they reach the single monochromator. The cuvette containing the reference standard and sample can be placed together simultaneously but the result obtained will be a bit unorganized and might be confusing with the reading. Lastly the precaution step is the cuvette is put one by one. Although it takes longer time, but the result obtained is more organized and accurate. Double-beam spectrophotometer is more complicated compare to single-beam because there parts that are not static and moveable. So each time the parts is removed, it has to be tide back correctly to its place or the reading obtained will be inaccurate.

CONCLUSION At the end we know the graph absorbance versus concentration show a linear perpendicular graphs. As concentration increase absorbance also increase. Result show, the higher the concentration the higher the absorbance. Comparison between single and double beam have been determined. The objective achieved.

REFERENCE 1) www. hm.davidson.edu v e spe trophotometry pe trophotometry.html 2) http://www.files.chem.vt.edu/chem-ed/spec/uv-vis/singlebeam.html 3) http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UVVis/spectrum.htm 4) http://elchem.kaist.ac.kr/vt/chem-ed/spec/beerslaw.htm 5) http://www.files.chem.vt.edu/chem-ed/spec/uv-vis/uv-vis.html 6) http://www.chemguide.co.uk/analysis/uvvisible/spectrometer.html