Conclusion for Spectrophotometer (I need Conclusion For Spectrophotometer) INTRODUCTION The spectrophotometer is an instrument used to measure the spectra of microscopic samples and areas. It is used to measure UV visible NIR spectra of microscopic samples. It measure transmission, absorption, reflectance, polarization and fluorescence of a given sample.It is designed as an optical microscope combined with UV visible NIR spectrophotometer. It measures up to 1 x 1 micrometre range of samples. With the help of software 3D mapping measurements and calorimetry can be done. Non contactmicrospot
Conclusion for Spectrophotometer (I need Conclusion For Spectrophotometer) INTRODUCTION The spectrophotometer is an instrument used to measure the spectra of microscopic samples and areas. It is used to measure UV visible NIR spectra of microscopic samples. It measure transmission, absorption, reflectance, polarization and fluorescence of a given sample.It is designed as an optical microscope combined with UV visible NIR spectrophotometer. It measures up to 1 x 1 micrometre range of samples. With the help of software 3D mapping measurements and calorimetry can be done. Non contactmicrospot thin film thickness measurement is possible with this. It has a great influence in the field of research and industry. Since it have great spectral range it is used by scientist and engineers to get spectra of extremely small sample areas. Even it works in the field where the visible light and infrared does not work. For example a forensic scientist okr chemist uses it matching fibre or paints of microscopic samples. A Geologist can use it for qualification of gems LEARNING OUTCOME 1. The basic mechanics operation of the spectrophotometer secomam PRIM. 2. The basic principles of spectrophotometry including the relationship/calculation of transmittance (T) and absorbance (A) value. 3. To identify the maximum absorbance (A max) of a compound and how it is determined. 4.To understand the use of protein standard. METHODLOGY A.Determine the absorbance spectrum and maximum absorbance wavelength The instrument that would be used in this practical is the Secomam PRIM. 1.Spectrophotometer is turned on. Warmed up the machine for 15 minute. 2. The Val button was pressed to activated. 3.Transmittance button has been selected, by the used of button A 4. The wavelength has been set at 375nm. 5. The cuvette was cleaned and dried 6. The blank solution was and cuvette is placed on sample holder of spectrometer. 7. Zero button was pressed. 8.The cuvette with the blank solution has been removed. 9. Another cuvette was cleaned and dried. 10. Standard solution A was poured in cuvette and cuvette is placed in sample holder of spectrometer. 11. Transmittance is recorded on result paper. 12. The cuvette with standard solution A was removed. 13. Wavelength is changed to 425 nm 14. The cuvette with the blank solution was placed in sample holder of spectrophotometer. 15. Zero button was pressed. 16. The cuvette with the blank solution was removed. 17. The cuvette with standard solution A was placed in sample holder of spectrometer. 18. Transmittance is recorded on result paper. 19. The cuvette with standard solution A was removed. 20. Step 13 to step 19 for different wavelengths: 490, 530, 540, 550, 580, 625 and 700nm. 21. Absorbance was calculated on result paper. 22. The graph of absorbance and wavelength was recorded on the result paper. B. Plot a protein standard curve 1. Absorbance mode was selected by pressing button A. 2.Optimum wavelength has been selected based on absorbance of spectrum (?=max). 3. The selected wavelength was set by entering the value directly using numerical keys and confirmed by VAL. 4. Cuvette is cleaned and dried. 5. protein (albumin) was prepared in serial concentrations (0, 20, 40, 60, 80, and 100 mg/ml). 6. The standard protein solution with 0 mg/ml concentration 7. Zero button was pressed. 8.Absorbance is recorded on result paper. 9. The standard protein solution (0 mg/ml) was removed. The cuvette was cleaned with distilled water and wiped dry. 10.Protein solution(20mg/ml) is poured in cuvette, and cuvette is placed on sample holder of spectrometer. 11. Read the absorbance (A) value. The data was recorded in Table 2. 12.Standard protein solution(20mg/ml) is removed, cuvette is cleaned and dried. 13. Repeated step 10 to step 12 to measure A value of the standard protein solutions at 40mg/ml, 60mg/ml, 80mg/ml, 100mg/ml and 580mg/ml concentrations, respectively. 14. The absorbance (at ?max) versus concentration (mg/ml) was plotted on a piece of graph paper. The concentration was placed on the horizontal axis. C. Determine the concentration of an unknown solutions using protein standard curve 1. Absorbance was arranged with selected wavelength. 2. The cuvette was cleaned and dried. 3. The standard protein solution(0mg/ml) was. poured, and cuvette was placed on sample holder of spectrometer. 4. Zero button was pressed. 5. Unknown solution was poured into the cuvette and cuvette was placed into the sample holder of the spectrophotometer. 6. Absorbance A has been recorded. 7. Concentration of unknown solution was measured based on protein standard on curve B. RESULT Table 1: Percent Transmittance/absorbance of the Standard Solution A at various wavelengths 375 78.5 0.105 425 85.0 0.021 490 73,6 0.133 530 64.6 0.190 540 63.7 0.196 550 65.0 0.187 580 77.6 0.110 625 112.5 -0.052 700 162.1 -0.21 The amount of light that is absorbed by a solution is commonly expressed either in terms of percent transmittance (%T), as in this experiment, or terms of absorbance (A). Absorbance is defined as follows: Absorbance = 2-log of percent transmittance A= 2-log%T Table 2: Protein (albumin) Standard Curve *Standard protein (ml) Biuret Reagent (ml) Total Volume (ml) Absorbance (at ?max) *0 mg/ml (Blank solution) 2.5 2.5 5.0 0.000 *20 mg/ml 2.5 2.5 5.0 0.069 *40 mg/ml 2.5 2.5 5.0 0.103 *60 mg/ml 2.5 2.5 5.0 0.203 *80 mg/ml 2.5 2.5 5.0 0.396 *100 mg/ml 2.5 2.5 5.0 0.485 Discussion 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: uses light over the ultraviolet range (185 – 400 nm) and visible range (400 – 700 nm) of electromagnetic radiation spectrum. IR spectrophotometer: uses light over the infrared range (700 – 15000 nm) of electromagnetic radiation spectrum. In visible spectrophotometry, the absorption or the transmission of a certain substance can be determined by the observed color. For instance, a solution sample that absorbs light over all visible ranges (i.e., transmits none of visible wavelengths) appears black in theory. On the other hand, if all visible wavelengths are transmitted (i.e., absorbs nothing), the solution sample appears white. If a solution sample absorbs red light (~700 nm), it appears green because green is the complementary color of red. Visible spectrophotometers, in practice, use a prism to narrow down a certain range of wavelength (to filter out other wavelengths) so that the particular beam of light is passed through a solution sample. The amount of light transmitted through a solution is referred to as transmittance (T). The transmittance is defined as the ratio of the light energy transmitted through the sample (I) to the energy transmitted through the reference blank (I0). Since the compound being tested is not present in the reference blank, the transmittance of the reference blank is defined as 100%. For most biological applications however, we measure absorbance (Al, also referred to as Optical Density or ODl, where l is the wavelength used for the measurements), the amount of light absorbed by a solution. Absorbance is related logarithmically to transmission thusly. A = -log T, a reference blank is used. In this case, to zero out any light absorbed by anything in the solution other than the compound of interest. By definition, the absorbance of the reference blank is set at zero. As we can see from the result of the experiment, the peak of the absorbance is at 540nm, but before the peak finally reached 540 nm, we had some problems. We did the procedure accordingly, but the peak of the wavelength is at 580nm, and then, other group also had the same problem. Since all of us had the exact same problem, we repeated the experiment, again the peak of the absorbance doesnt change. The sample has changed into the unknown sample, and then the procedure repeated accordingly, and finally the result appeared as expected. The peak of the absorbance is at 540nm. Supposedly, sample A has the same result as unknown sample, but there is some factor that affected the result, such as contamination, most probably sample A is contaminated by other organism or other things, so its affected the absorbance of the wavelength. Its something that happened commonly when someone doing a research, some of the result its not turns out as you expected. Protein albumin standard curves Standard curves represent the relationship between two quantities. To calculate concentration based on the standard curve, first you find the concentration for each sample absorbance on the standard curve then you multiply the concentration by the dilution factor for each sample. Conclusion ?? .
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