1. COMPARATIVE STUDY BETWEEN NEAR- INFRARED(NIR) SPECTROMETERS IN THE MEASUREMENT OF SUCROSE CONCENTRATION

2. INTRODUCTION

3. NIR Spectroscopy I A spectroscopic method that uses the near-infrared region of the electromagnetic spectrum (from about 800 nm to 2500 nm). NIR spectra have only a few significant peaks, but they are exceptionally information-rich due to the number of overlapping absorption bands.

4. NIR Spectroscopy II NIR can typically penetrate much further into a sample than mid infrared radiation due to the low coefficient of absorbance. NIR radiation has less energy/photon but does excite molecular vibrations. NIR measurements are non-destructive and samples are not altered and can be reused.

5. The Importance of Measuring Sugar Concentration in Fruit I Commonly used in the wide range of crops. The important aspect to test the maturity of fruit and obtain the right time to harvest it. The percentage of sugar ( o Brix), indicates the sweetness of the fruit.

6. The Importance of Measuring Sugar Concentration in Fruit II Contribute to the calculation of sugar-acid ratio which is one of the step needed to test the maturity of fruit. One critical element of the ripening involves the conversion of starches to sugars. The example of starch test is shown in Figure 1.Figure 1

7. Objectives 1.To compare response analysis between lower and same range of NIR wavelength (Jaz and QE65000) spectrometer. 2.To identify response analysis of higher range of wavelength (NIRQuest) spectrometer. 3.To compare response analysis between lower and higher range of NIR wavelength spectrometer.

8. Problem Statement The NIR spectrometers are widely used in the spectroscopy field. The calibration transfer between NIR spectrometers should be practiced in spectroscopy field to improve the efficiency in energy, time and work. The comparative study between NIR spectrometers will give huge contribution to the calibration transfer.

9. MATERIALS AND METHOD

10. Apparatus and Material Background I The apparatus used in the experiment are: QE65000 Spectrometer (650 – 1100 nm), Figure 2.Figure 2 NIRQuest512-2.2 Spectrometer (900 – 2200 nm), Figure 3.Figure 3 Handheld Refractometer, Figure 4.Figure 4 The apparatus used in the reference taken: JAZ-COMBO Spectrometer (650 – 1100 nm), Figure 5.Figure 5 *** The characteristics and properties of NIR spectrometers are shown in the Table 1.Table 1

11. Apparatus and Material Background II The material used in the experiment is: Sucrose: The molecule is a disaccharide sugar composed of the monosaccharides glucose and fructose with the molecular formula C 12 H 22 O 11. *** The skeletal formula for sucrose is shown in the Figure 6.Figure 6

12. Experiment Setup The overall experimental setup was conducted using spectroscopic instrumentations from Ocean Optics. The setup is illustrated in Figure 7.Figure 7 The response was due to mixture between water with sucrose for different type of spectrometer. The characteristics of sucrose sample used in the experiment are tabulated in Table 2.Table 2

13. Methodology The flow chart of the experiment is shown in the Figure 8.Figure 8 The experiment was conducted only for the calibration data.

14. RESULTS AND DISCUSSIONS

15. Response Analysis between Jaz and QE65000 Spectrometer I Significant results were managed to be located at wavelength between approximately 940 and 985 nm. Figure 9(a) and Figure 9(b) shows the resultant linear regression generated between absorbance and sucrose concentration by using Jaz and QE65000 spectrometer respectively.Figure 9(a) Figure 9(b)

16. Response Analysis between Jaz and QE65000 Spectrometer II λ = 959 nm managed to generate the highest coefficient of determination for sucrose by using: Jaz spectrometer : R 2 = 0.9794; RMSE = 1.43 QE65000 spectrometer : R 2 = 0.956 ; RMSE = 2.15 The pattern behaviour of linear relationship between absorbance and the sucrose concentration against wavelengths for Jaz and QE65000 spectrometer are shown in Figure 10(a) and Figure 10(b) respectively.Figure 10(a) Figure 10(b)

17. Response Analysis between Jaz and QE65000 Spectrometer III It can be seen that the correlation between absorbance and sucrose concentration starts to loose its linearity once it has moved further than 960 nm. Further analysis through the application of Multiple Linear Regression (MLR) has successfully improved the correlation for sucrose measurement.

18. Response Analysis between Jaz and QE65000 Spectrometer IV The highest efficiency algorithm has been identified by using different wavelengths: Jaz spectrometer: 730, 830, 915, and 960 nm QE65000 spectrometer: 909 and 960 nm

19. Response Analysis between Jaz and QE65000 Spectrometer V The calibration algorithm, R 2, and RMSE for calibration (RMSEC) for both spectrometers are as follows: Jaz spectrometer: Sucrose concentration (°Brix) = 122 + 1375 ƛ 730 - 942 ƛ 830 + 855 ƛ 915 - 736 ƛ 960 (R 2 = 0.992; RMSEC = 0.907 °Brix);

20. Response Analysis between Jaz and QE65000 Spectrometer VI QE65000 spectrometer: Sucrose concentration (°Brix) = 135 + 777 ƛ 909 - 824 ƛ 960 (R 2 = 0.995; RMSEC = 0.760 °Brix) The linearity of the calculated model are illustrated in Figure 11(a) and Figure 11(b) for Jaz spectrometer and QE65000 spectrometer respectively. Figure 11(a) Figure 11(b)

21. Response Analysis of NIRQuest Spectrometer I Significant result is managed to be located at wavelength between approximately 980 and 1700 nm. Figure 12 shows the resultant linear regression generated between absorbance and sucrose concentration by using NIRQuest spectrometer.Figure 12

22. Response Analysis of NIRQuest Spectrometer II λ = 1363 nm managed to generate the highest coefficient of determination and lowest RMSE for sucrose by using: NIRQuest spectrometer : R 2 = 0.813; RMSE = 4.64 The pattern behaviour of linear relationship between absorbance and the sucrose concentration against wavelengths for NIRQuest spectrometer is shown in Figure 13.Figure 13

23. Response Analysis of NIRQuest Spectrometer III The correlation between absorbance and sucrose concentration starts to loose its linearity once it has moved further than 1400 nm. Further analysis through the application of Multiple Linear Regression (MLR) has successfully improved the correlation for sucrose measurement.

24. Response Analysis of NIRQuest Spectrometer IV The highest efficiency algorithm has been identified by using different wavelengths: NIRQuest spectrometer : 980, 1156, 1163, 1195, 1337, 1350, 1395, 1606, 1670, 1676, and 1682 nm.

25. Response Analysis of NIRQuest Spectrometer V The following are the calibration algorithm, R 2, and RMSEfor calibration (RMSEC): NIRQuest spectrometer Sucrose concentration (°Brix) = 171 + 79 ƛ 980 + 2909 ƛ 1156 - 1550 ƛ 1163 - 422 ƛ 1 195 + 445 ƛ 1 337 - 1783 ƛ 1350 + 310 ƛ 1395 – 41.8 ƛ 1606 + 298 ƛ 1670 - 298 ƛ 1676 + 195 ƛ 1682 (R 2 = 0.982; RMSEC = 1.613 °Brix) The linearity of the calculated model is illustrated by Figure 14.Figure 14

26. Response Analysis between NIR Spectrometers I By comparing the three spectrometers, all of them show the same properties : The absorption of a specific range of NIR wavelength decreases linearly with the increases of sucrose concentration. Further analysis through MLR has successfully improved the correlation for sucrose measurement.

27. Response Analysis between NIR Spectrometers II The ascending order of highest coefficient determination produced are by using NIRQuest, Jaz, and QE65000 spectrometers.

28. CONCLUSIONS AND RECOMMENDATIONS

29. Conclusions I Wavelength that generate the highest coefficient of determination : Jaz spectrometer: 959 nm (R 2 = 0.9794; RMSE = 1.43) QE65000 spectrometer : 959 nm (R 2 = 0.956 ; RMSE = 2.15) NIRQuest spectrometer : 1363 nm (R 2 = 0.813; RMSE = 4.64)

30. Conclusions II Combination of NIR wavelength that produce the highest coefficient of determination: Jaz spectrometer : ƛ =730, 830, 915, and 960 nm (R 2 = 0.992; RMSEC = 0.907) QE65000 spectrometer : ƛ =909 and 960 nm (R 2 = 0.995; RMSEC = 0.760)

31. Conclusions III NIRQuest spectrometer : ƛ =980, 1156, 1163, 1195, 1337, 1350, 1395, 1606, 1670, 1676, and 1682 nm (R 2 = 0.982; RMSEC = 1.613) These wavelengths exert an important combination in development of calibration algorithm for individual spectrometer measurement.

32. Recommendations For future experiment : record the validation data as well in order to confirm the prediction. conduct the experiment by using the sugar content in fruit to obtain the real experience on how to improve the intrinsic quality of fruit.

34. Figure 1Figure 1 : The Example of Starch Test

35. Figure 2Figure 2 : QE65000 Spectrometer

36. Figure 3Figure 3 : NIRQuest512-2.2 Spectrometer

37. Figure 4 : Handheld Refractometer Figure 4

38. Figure 5Figure 5 : Jaz Spectrometer

39. Table 1Table 1 :The characteristics and properties of NIR spectrometers. JazQE65000NIRQuest Characteristics -Modular, stackable and autonomous components -Czemy-Turner optical bench -On-board microprocessor and OLED display -Replaceable slits and gratings -Ethemet and memory module -Battery and external memory module -200-1100 nm spectral range- grating dependent -Resolution 0.14-7.7 nm (FWHM) -Peak quantum efficiency 90% -Back thinned 2DCCD detector -Thermoelectric cooling -6 slit options -14 grating option -900-2050 nm spectral range -Less than 1 nm optical resolution FWHM -15000:1 signal to noise -On board thermoelectric cooling -16 bit USB A/D converter -Crossed czemy-Turner optical bench -Various trigger modest grating options Application-Fluorescence -Biotechnology -Raman spectroscopy -DNA sequencing -Remote sensing -Dosimetry -Spectroscopy -Medical -Biomedical imaging analysis -Fluorescence -Luminescence detection -Spectroscopy of emission and absorption lines spectroscopy

40. Figure 6 Figure 6 : Skeletal Formula for Sucrose

41. Figure 7Figure 7 : Experiment Setup for NIR Measurement (Top view)

42. Table 2Table 2 :The Sample Characteristics. Spectrometer Range of Sucrose (°Brix) Mean n (Calibration) Jaz0.9-35.017.150 QE650000.1-39.18.850 NIRQuest512-2.20.2-38.818.850

43. Figure 8 : Flow Chart of Experiment Figure 8 Apparatus setup Sample preparation Collect reference spectrum Reduce concentratio n of sucrose until 50 data Save data obtained Repeat experiment with other spectrometer Data analysis

44. Figure 9(a) Figure 9(a) : Linear relationship between absorbance and concentration of aqueous sucrose at λ = 959 nm by using Jaz spectrometer

45. Figure 9(b) : Linear relationship between absorbance and concentration of aqueous sucrose at λ = 959 nm by using QE65000 spectrometer Figure 9(b)

46. Figure 10(a) Figure 10(a) : Coefficient of determination generated at different wavelength for aqueous sucrose concentration by using Jaz spectrometer

47. Figure 10(b) Figure 10(b) : Coefficient of determination generated at different wavelength for aqueous sucrose concentration by using QE65000 spectrometer

48. Figure 11(a) Figure 11(a) : Calculated VS actual concentrations of sucrose by using Jaz spectrometer.

49. Figure 11(b) Figure 11(b) : Calculated VS actual concentrations of sucrose by using QE65000 spectrometer.

50. Figure 12 Figure 12 : Linear relationship between absorbance and concentration of aqueous sucrose at λ = 1363 nm by using NIRQuest spectrometer.

51. Figure 13 Figure 13 : Coefficient of determination generated at different wavelengths for aqueous sucrose concentration by using NIRQuest spectrometer.

52. Figure 14 Figure 14 : Calculated VS actual concentrations of sucrose by using NIRQuestspectrometer.