IN COWS MILK DUE TO HEATING EFFECT NOVIA DWI IRIANTI NRP 2406 100 - - PowerPoint PPT Presentation

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.

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?

PROBLEM

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.

Secondary structure Tertiary structure Quartenary structure Primary structure Denaturation involves the disruption on this 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 Solvent/enzime Changes in pH Machanical treatment Heating Figure 2.5 Protein interaction due to heating treatment because of denaturation

Spectrophotometry is an analytical method based on measurement of monochromatic light absorption by a sample on specific wavelength using prism

• r diffraction grating monochromator with a fototube detector.

SPECTROPHOTOMETRIC METHOD

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 Spektrophotometre. Figure 3.2 Magnetic Stirrer Yellow MAG HS7 Figure 3.7 The chamber to places the cuvete Figure 2.8 The cuvete

EXPERIMENTAL FLOWCHART

Homogen Dilute samples

Equipment and materials preparation Magnetic Stirrer Setting (temperature and RPM) Heating and homogenizing the sample in various temperature and heating time Samples precipitation Start Finish

Magnetic Stirrer

TREATMENT VARIATION

Heating treatment flowchart

Data tabulation Data plot into graph Gaussian Fitting Derivative Gaussian Function Calculation of % of protein reduction Calculation of protein absolute concentration

Microsoft Excel MATLAB , A(λ) MATLAB M-file MATHCAD

DATA PROCESSING

DATA ANALYSIS

Protein Spectral Absorbance Using Spectrophotometer

• A. Protein Absorbance of samples with various dilution

Table 1. Protein absorbance of sample with various dilution Figure 1. Protein Absorbance of sample with various dilution

DATA

λ(nm) Protein Absorbance of Sample With Various Dilution 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

λ(nm) Spectral Absorbance of Protein with Various Temperature (heating time 1000s) 80oC 90oC 100oC 110oC 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

Table 2 Protein absorbance of sample with various temperature Figure 2 Protein Absorbance of sample with various temperature

• B. Protein Absorbance of samples with various temperature ( heating time 1000s )

DATA

Table 3. Protein absorbance of sample with various heating time Figure 3. Protein Absorbance of sample with various heating time

• C. Protein Absorbance of samples on 80oC temperature with various heating time

DATA

Data Processing ( Derivative Spectrophotometry Technique )

Gaussian Sampling (Gaussian fitting of the curve 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 (filtering process) To obtain the derivative spectra

DATA

Spectral Absorbance Gaussian Fitting Curve Fig 15. Gaussian fitting of the curve of Spectral absorbance

After Zooming Fourth derivative spectral absorbance of protein samples in various heating time (at T=90oC)

Specific peaks at 290nm

Fig 16. Fourth derivative spectral absorbance

• f sample in 90oC

Fig 17. Fourth derivative spectral absorbance of sample in 90oC (after zooming)

Fourth derivative spectral absorbance of protein samples in various temperature (at t=100s) After Zooming

Specific peaks at 290nm

Fig 17. Fourth derivative spectral absorbance of sample in 100s heating time Fig 18. Fourth derivative spectral absorbance of sample in 100s heating time (after zooming)

Variasi Berat jenis protein (gr/ml) Tanpa pemanasan 0,0261 Pemanasan 80oC, 10s 0,0221 Pemanasan 90oC, 10s 0,0121 Pemanasan 100oC, 10s 0,0085 Pemanasan 110oC, 10s 0,0038 Perlakuan Parameter uji Hasil (%) Berat Jenis (gr/ml) Murni Protein susu murni 78,82 7,882 Pengenceran 300x Protein susu 2,61 0,0261 Pemanasan 80oC, 10 sec Protein susu (hasil pengenceran) 2,15 0,0215 Pemanasan 90oC, 10 sec Protein susu (hasil pengenceran) 0,93 0,0093 Pemanasan 100oC, 10 sec Protein susu (hasil pengenceran) 0,96 0,0096 Pemanasan 110oC, 10 sec Protein susu (hasil pengenceran) 0,42 0,0042

KJELDAHL METHOD SPECTROPHOTOMETRY

COMPARED

CALCULATION OF ABSOLUTE CONCENTRATION OF PROTEIN

To determine the absolute concentration of protein, it is known that :

• Initial concentration of protein: 7.882 g/ml
• After the protein concentration was diluted

300x: 0.0261 gr/ml

• The sample used in testing is a sample that

was diluted 300x

Fig 14 Protein absolute concentration in sample with various treatment Table 8. Percentage of protein content reduction and protein absolute concentration in sample with various treatment Perlakuan Sampel Variasi Waktu Protein tersisa (%) Konsentrasi (gr/ml) Tanpa Pemanasan

• 0,0261

Pemanasan 80oC 10s 84,6 0,0221 100s 71,0 0,0185 300s 57,4 0,0150 1000s 58.,5 0,0153 Pemanasan 90oC 10s 46,5 0,0121 100s 40,3 0,0105 300s 37,2 0,0097 1000s 34,6 0,0090 Pemanasan 100oC 10s 32,7 0,0085 100s 25,6 0,0067 300s 22,5 0,0059 1000s 17,9 0,0047 Pemanasan 110oC 10s 14,4 0,0038 100s 9,90 0,0026 300s 5,70 0,0015 1000s 3,70 0,0010

CONCLUSION

• 1. Absorbance measurements using ultraviolet (UV) Spectrophotometry can be used to

determine the protein content of cow's milk.

• 2. Fourth derivative UV Spectrophotometry is used because there are no shift at gaussian peak

spectra of protein on different variation. The first, second and third order of derivative spectral have an inconsistent peak ,so it can't used for analysis of protein content determination.

• 3. The most specific wavelength for identification of spectral absorbance characteristic to

determine protein content is 290 nm.

• 4. Test results and data processing proved that the higher heating temperature and the longer

heating time of sample causes the decrease protein absorbance.

• 5. Based on data measurement, the protein denaturation will occur at temperature 80oC, and the

largest protein denaturation occurs at temperature 110oC with heating time 1000s, where it decrease untill 96.3%. And the remaining protein concentration was 0,29 gr/ml.

• 6. UV spectrophotometry method is very suitable and useful for determining the material

content of a substance.

The conclusions are as follows:

REFERENCES

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• f

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THANK YOU…