FINAL PROJECT (TF 1381) CHARACTERIZATION OF SPECTRAL ABSORBANCE FOR DETERMINATION OF PROTEIN CONTENT’S REDUCTION IN COW’S MILK DUE TO HEATING EFFECT NOVIA DWI IRIANTI NRP 2406 100 062 Supervisor: Dr.rer.nat. Ir. Aulia MT Nasution, M.Sc DEPARTMENT OF ENGINEERING PHYSICS Faculty of Industrial Technology Institut Teknologi Sepuluh Nopember Surabaya 2010
BACKGROUND Protein is an important substances in milk. Heating treatment causes the denaturation of protein in milk. Denaturation will reduce the protein content. Analysis of the light interaction with matter will be used to determine the protein content of milk. In this final project, Spectrophotometric method used to identify the effect of heating treatment on proteins denaturation by the information of spectral absorbance.
PROBLEM How to apply Ultraviolet (UV) Spectrophotometric method to determine the spectral absorbance of protein in cows milk? Whether the protein denaturation will affected the spectral absorbance? How to determine significant spectral range to measure the content of milk protein? How to apply customized measurement system based on spectral finger print to detect the protein denaturation with high sensitivity and linearity characteristics?
GOAL To detect the proteins denaturation due to heating by using absorption spectrofotometric method. And to characterize the spectral absorbance information to determine protein content due to heating treatment. This characterization result will be used to build customized measurement system based on spectral absorbance information to detect the protein denaturation with high sensitivity and linearity characteristics.
BASIC THEORY
PROTEIN Protein is an organic macromolecules that contain carbon, hydrogen, oxygen, nitrogen, and usually sulfur and are composed of one or more chains of amino acids. Primary structure Secondary structure Denaturation involves Tertiary the disruption on this structure structure Quartenary structure
PROTEIN DENATURATION Denaturation is a process in which protein or nucleic acid lose their tertiary , secondary , and quartenary structure. When natural proteins are subjected with physical or chemical treatment, their structure will change, and they become 'unnative' or 'unnatural'. We call that as denaturation process. Acids Heating Solvent/enzime Changes in pH Machanical treatment Figure 2.5 Protein interaction due to heating treatment because of denaturation
SPECTROPHOTOMETRIC METHOD Spectrophotometry is an analytical method based on measurement of monochromatic light absorption by a sample on specific wavelength using prism or diffraction grating monochromator with a fototube detector. Spectrophotometry Visible (VIS) Spectrophotometry (Wavelength region 380-800nm) Ultraviolet (UV) Spectrophotometry (Wavelength region 190-380nm) Infra-Red (IR) Spectrophotometry (Wavelength region 2.5-1000μm) UV-VIS Spectrophotometry (Wavelength Region (190-800nm)
Working principle of spectrophotometer Absorbance as a function of wavelength
OPTICAL CONFIGURATION UV-VIS SPECTROPHOTOMETRE Grating schematic in spectrophotometre
MATERIAL & METHOD
EQUIPMENT Figure 3.1 UV-VIS Beckman DU-7500 Figure 3.2 Magnetic Stirrer Spektrophotometre. Yellow MAG HS7 Figure 2.8 The cuvete Figure 3.7 The chamber to places the cuvete
EXPERIMENTAL FLOWCHART
TREATMENT VARIATION Heating treatment flowchart Start Dilute samples Equipment and Magnetic Stirrer materials preparation Homogen Magnetic Stirrer Setting (temperature and RPM) Heating and homogenizing the sample in various temperature and heating time Samples precipitation Finish
DATA PROCESSING Data tabulation Microsoft Excel MATLAB , A (λ) Data plot into graph Gaussian Fitting MATLAB M-file Derivative Gaussian Function MATHCAD Calculation of % of protein reduction Calculation of protein absolute concentration
DATA ANALYSIS
Protein Spectral Absorbance Using Spectrophotometer A. Protein Absorbance of samples with various dilution Figure 1. Protein Absorbance of sample with various dilution Table 1. Protein absorbance of sample with various dilution Protein Absorbance of Sample With Various Dilution λ(nm) 100x 200x 300x 400x 500x 270 0.7921 0.3842 0.2616 0.178 0.1345 271 0.7878 0.3864 0.2618 0.178 0.1332 272 0.7816 0.3845 0.2608 0.1763 0.1337 273 0.7863 0.3825 0.2602 0.1759 0.1336 274 0.7900 0.3816 0.2605 0.1749 0.1331 275 0.7882 0.3813 0.2604 0.1743 0.1327 276 0.783 0.3802 0.2584 0.1726 0.1321 277 0.7793 0.3782 0.2576 0.1729 0.1289 278 0.7787 0.3761 0.2525 0.172 0.1311 279 0.7711 0.3747 0.2522 0.1701 0.1295 280 0.7609 0.3717 0.2511 0.1678 0.1265 DATA
B. Protein Absorbance of samples with various temperature ( heating time 1000s ) Table 2 Protein absorbance of sample with various Figure 2 Protein Absorbance of sample with various temperature temperature Spectral Absorbance of Protein with λ(nm) Various Temperature (heating time 1000s) 80 o C 90 o C 100 o C 110 o C 270 0.1470 0.0934 0.0570 0.00971 271 0.1464 0.0928 0.0564 0.00967 272 0.1469 0.0933 0.0569 0.00962 273 0.1470 0.0928 0.0570 0.00964 274 0.1465 0.0920 0.0565 0.00982 275 0.1457 0.0912 0.0557 0.00987 276 0.1455 0.0907 0.0555 0.00964 277 0.1446 0.0910 0.0546 0.00972 278 0.1451 0.0905 0.0551 0.00976 279 0.1447 0.0897 0.0547 0.00974 280 0.1438 0.0896 0.0538 0.00963 DATA
C. Protein Absorbance of samples on 80 o C temperature with various heating time Figure 3. Protein Absorbance of sample with Table 3. Protein absorbance of sample with various heating time various heating time DATA
Data Processing ( Derivative Spectrophotometry Technique ) To obtain the derivative spectra Gaussian Fitting Curve Gaussian Sampling ( Gaussian fitting of the curve Spectral Absorbance The total fitting equation of gaussian would be derivated Gaussian Fitting Curve The program will generate a graph that has been approximated by Gaussian functions. Gaussian fitting was done to eliminate the noise Fig 15. Gaussian fitting of the curve of Spectral absorbance (filtering process) DATA
Fourth derivative spectral absorbance of protein samples in various heating time (at T=90 o C) Specific peaks at 290nm Fig 16. Fourth derivative spectral absorbance of sample in 90 o C After Zooming Fig 17. Fourth derivative spectral absorbance of sample in 90 o C (after zooming)
Fourth derivative spectral absorbance of protein samples in various temperature (at t=100s) Specific peaks at 290nm Fig 17. Fourth derivative spectral absorbance of sample in 100s heating time After Zooming Fig 18. Fourth derivative spectral absorbance of sample in 100s heating time (after zooming)
COMPARED Hasil Berat Jenis Berat jenis protein (gr/ml) Variasi Perlakuan Parameter uji (%) (gr/ml) Tanpa pemanasan 0,0261 Protein susu Pemanasan 80 o C, 10s 0,0221 Murni 78,82 7,882 murni 0,0121 Pemanasan 90 o C, 10s Pengenceran 0,0085 Pemanasan 100 o C, 10s Protein susu 2,61 0,0261 300x Pemanasan 110 o C, 10s 0,0038 Protein susu Pemanasan (hasil 2,15 0,0215 SPECTROPHOTOMETRY 80 o C, 10 sec pengenceran) Protein susu Pemanasan (hasil 0,93 0,0093 90 o C, 10 sec pengenceran) Protein susu Pemanasan (hasil 0,96 0,0096 100 o C, 10 sec pengenceran) Protein susu Pemanasan (hasil 0,42 0,0042 110 o C, 10 sec pengenceran) KJELDAHL METHOD
CALCULATION OF ABSOLUTE CONCENTRATION OF PROTEIN Table 8. Percentage of protein content reduction and protein absolute concentration in sample with various treatment To determine the absolute concentration of Protein protein, it is known that : Perlakuan Variasi Konsentrasi tersisa • Initial concentration of protein: 7.882 g/ml Sampel Waktu (gr/ml) (%) • After the protein concentration was diluted Tanpa 300x: 0.0261 gr/ml - - 0,0261 Pemanasan • The sample used in testing is a sample that was diluted 300x 10s 84,6 0,0221 Pemanasan 100s 71,0 0,0185 80 o C 300s 57,4 0,0150 1000s 58.,5 0,0153 10s 46,5 0,0121 Pemanasan 100s 40,3 0,0105 90 o C 300s 37,2 0,0097 1000s 34,6 0,0090 10s 32,7 0,0085 Pemanasan 100s 25,6 0,0067 100 o C 300s 22,5 0,0059 1000s 17,9 0,0047 10s 14,4 0,0038 Pemanasan 100s 9,90 0,0026 110 o C 300s 5,70 0,0015 Fig 14 Protein absolute concentration in sample 1000s 3,70 0,0010 with various treatment
CONCLUSION
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