FLOW CHART
Prepare serial dilutions (5 ppm, 15 ppm, 25 ppm, 35 ppm and 45 ppm) in 50 ml volumetric flask.
Fill a cuvette with 45 ppm dilution and another cuvette with blank solution, insert them in sample compartment. Wipe clean sides of cuvettes but avoid touching clear surface. Do wavelengths scan and obtain max. Record data.
For photometric scan, fill cuvette as before but use the serial dilution prepared and scan one by one. Record absorbance reading and look at standard calibration graph produced.
Calculate volume needed, V1 from the 100 ppm food dye stock using dilution formula.
To prepare a dilution, measure exact volume of V1 and pour into 50 ml volumetric flask. Then add distilled water until the meniscus Then, shake volumetric flask properly.
Instructor will brief on operating procedure of PerkinElmer UV/Vis Spectrophotometer Lambda EZ210.
Repeat the process for all dilutions.
Determine the concentrations of Unknown 1 and Unknown 2.
Record all data completely and clean workstation properly.
Analysis of Data:
Use the spectrum obtained from wavelength scan to identify max for Carmoisine and then record.
Record different concentration of standards absorbance and construct standard calibration curve. (Concentration vs Absorbance)
Measure Unknown 1 and Unknown 2 concentration peak in the soda samplechromatograph, and use standard calibration curve to determine concentration of Unknown 1 and 2.
RESULTS
Part A: Wavelength Scan
(i)
Instrument Parameters Starting Wavelength = _______700.0__________ nm Ending Wavelength = ________400.0_________ nm Path length = ________10.0_________ mm
(ii)
Result for Wavelength Scan Sample used = _____________Carmoisine stock____ Concentration of the sample = _______100__________ ppm max obtained = _____540____________ nm
Part B: Photometric Scan
(i)
Instrument Parameters Wavelength, max = _____537.0____________ nm Path length = _______10.0___________ mm
(i)
Results for Photometric Scan (Creating a Standard Calibration Curve)
No.
Sample
Table 1: Data Carmoisine Concentration (ppm), x-axis
Absorbance (nm), y-axis
1
Std 1
5.000
0.091
2
Std 2
15.000
0.242
3
Std 3
25.000
0.416
4
Std 4
35.000
0.592
5
Std 5
45.000
0.720
6
Unknown 1
20.614
0.338
7
Unknown 2
42.180
0.692
DISCUSSION
In the analysis of food colour experiment, there are 2 objective that need to be focus on. The first objective was to determine λ max of Colourant (wavelength scan) and the second objective was to prepare a serial dilution and generate a standard calibration graph for sample quantification. Ultra-Visible Spectrophotometer is used in this experiment to determine the maximum wavelength of Carmoisine solution. Carmoisine is one of permitted colors that can be used in food. It is red in color, which is natural that usually used as colorant in jellies. The 100ppm stock Carmoisine solution was been diluted to 5 different concentration which are 5pm, 15ppm, 25ppm, 35ppm and 45ppm. When analyzing by using UV-VIS Spectrophotometer, the blank solution used was distilled water. For sample solution, the technician prepared the student with two samples for analyzing it using UV-VIS Spectrophotometer.
The experiment was continuing by putting the sample in a cuvette. A cuvette is a small tube of circular or square cross section, sealed at one end, made of plastic, glass, or fused quartz (for UV light) and designed to hold samples for spectroscopic experiments. Disposable plastic cuvettes are often used in fast spectroscopic assays, where speed is more important than high accuracy. Some cuvettes will be clear only on opposite sides, so that they pass a single beam of light through that pair of sides; often the unclear sides have ridges or are rough to allow easy handling. Cuvettes to be used in fluorescence spectroscopy must be clear on all four sides b ecause fluorescence is measured at a right-angle to the be am path to limit contributions from beam itself. The rough ones can be touched by bare fingers and the other ones, which are the smooth ones shouldn’t be touched by fingers. This is because th e smooth sides of the cuvette are where the light will go through the sample from the source. If the smooth sides of cuvette were stick with fingerprints, the light might be diffused to another way.
The sample was then been tested using the instruments. Two types of analysis were done, which are, wavelength scanning and photometric scanning. λ max was obtained by scanned the highest concentration of the dilution which are 45ppm. For the photometric scan, the different dilution of sample was been scan to produce standard calibration graph. The data of results consist of the concentration values of the five standards with their respective absorbance with a standard calibration graph and the standard deviation. The concentration of the unknown samples also were automatically computed and printed on the data of results. The data for absorbance and concentration can be found in Table 1. From the result obtained and the graph that has been plotted, the Standard Calibration Graph line is linear related to the Beer’s Law. So, that means, from this experiment we know that Beer’s Law theory is true that the relationship between the absorbance of the solution and the concentration at the absorbing species have been proved.
CONCLUSION
UV spectrophotometer is a device used to study the interaction between radiation and matter in regards to the wavelength of photons. Electromagnetic radiation in the UV-VIS portion of the spectrum ranges in wavelength from approximately 200 to 700 nm. The UV range is colorless to the human eye, while different wavelengths in the visible range each have the characteristic color, ranging from violet at the short wavelength end of the spectrum to red at the long wavelength end o the spectrum. Ultra-Visible Spectrophotometer is used in this experiment to determine the maximum wavelength of Carmoisine solution. The 100ppm stock Carmoisine solution was been diluted to 5 different concentration which are 5pm, 15ppm, 25ppm, 35ppm and 45ppm. The sample was then been tested using the instruments. Two types of analysis were done, which are, wavelength scanning and photometric scanning. From the result obtained, a standard calibration curve was plotted. The graph that was plotted is linear so that proves the Beer’s Law Theory is true that the relationship between the absorbance of the solution and the concentration at the absorbing species have been proved.
RECOMMENDATION
Some recommendations can be done in this experiment to improve its accuracy in the future. For example, when doing the dilution process, we must make sure to measure the exact volume needed from the 100 ppm food dye stock and make sure to fill up the volumetric flask with distilled water until the meniscus then shake it properly. Next, when wiping the sides of the cuvette, make sure not to touch the clear surface because it will affect the final results. Finally, make sure to operate the Perkin-Elmer UV/Vis Spectrophotometer Lambda EZ210 with the right procedure to avoid errors on the results and graph.
TUTORIALS
1. State the Beer’s Lambert Law. A= bc Where A is absorbance and it does not have units, is the molar absorptivity with units of L mol-1 cm-1. b is the path length of the cuvette in which contain the sample. The unit is in centimeters. c is the concentration of the compound in solution, expressed in M or mol L-1 .
2. What is the volume needed to prepare a 50 ppm of carmoisine from a 100 ppm of carmoisine in 100 ml volumetric flask? M1 V1 = M2 V2 (100ppm) (V1) = (50ppm) (100mL) V1 = 50 mL 3. Why we need to wipe the sides of the cuvette clear surface? Cuvettes to be used in fluorescence spectroscopy must be clear on all four sides because fluorescence is measured at a right-angle to the b eam path to limit contributions from beam itself. The rough ones can be touched by bare fingers and the other ones, which are the smooth ones shouldn’t be touched b y fingers. This is because the smooth sides of the cuvette are where the light will go through th e sample from the source. If the smooth sides of cuvette were stick with fingerprints, the light might be diffused to another wa y. 4. Describe the function of wavelength scanning and photometric scanning. The function of wavelength scanning is to detect things and to understand them in a better way. It is like an x-ray. Most of the time scanning refer to health and technologist. They can also have something to do with science or technology. It is also done to determine at what wavelength the carmoisine able to absorb in the range of 200 nm to 700 nm which we cannot seen by our vision. The use of photometric scan is to determine the concentration of an unknown sample, after getting a standard curve from a series of kn own concentration. In this experiment, there is two unknown sample that is use.
REFERENCES
1. Food Analysis, Third Edition, Kluwer Acedemic/Plenum Publishers, S. Suzanne Nielsen, 2003, New York, 2003 2. Darrel D. Ebbing, Steven D. Gammon, General Chemistry Ninth Edition, Houghton Mifflin Company (2009). 3. http://elchem.kaist.ac.kr/vt/chem-ed/spec/uv-vis/uv-vis.htm , Uv-Vis Spectroscopy 4. https://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visible_spectroscopy , Ultraviolet-visible Spectroscopy 5. Drisko,R.L Chemistry 108 LabTextbook.N.P. Pauline M. Hamilton,2002,Print Food Dye Chromotoagraphy “Flinn Scientific INC,8 August,2002,Web. 15 Sept. 2014