Aquaphotomics & NIR hyperspectral imaging tools for understanding the role of water in foods aoife.gowen@ucd.ie School of Biosystems Engineering, University College Dublin, Ireland.
Key Questions What does water look like in the NIR? What can hyperspectral imaging show us? How can we use this to understand the role of water in foods?
H 2 O molecule
H 2 O molecule Nonlinear molecule: 3N-6 vibrational modes 3 vibrational degrees of freedom ν as 3657 cm -1 2735 nm ν s 3756 cm -1 2662 nm δ s 1595 cm -1 627 nm Band locations from Chs 1 & 9 in Siesler, Ozaki, Kawata and Heise, 22, Near-Infrared Spectroscopy: Principles, Instruments and Applications, Wiley GIFs from http://chemwiki.ucdavis.edu/physical_chemistry/spectroscopy/vibrational_spectroscopy 4
NIR spectrum of Water 4 1.5 3.5 Absorbance (log (1/T)) 3 2.5 2 1.5 1 Absorbance (log (1/T)) 1.5.5 1 15 2 25 12 13 14 15 16 17
2 nd derivative NIR spectrum of Water Absorbance (log (1/T)) 3 x 1-3 2 1-1 λ(nm) Tentative assignment [expected λ] 1343 2v s [1331-1371] 1412 2v as [1367-148] 1431 2v δs + v s [144-152] 1463 v as + 2v δs [146-1525] 1647 v as + 2v δs [146-1525] -2-3 12 13 14 15 16 17 Siesler, Ozaki, Kawata and Heise, 22, Near-Infrared Spectroscopy: Principles, Instruments and Applications, Wiley
NIR spectra of Water: temperature 1.5 7 65 Absorbance (log (1/T)) 1.5 Temperature 6 55 5 45 4 35 13 135 14 145 15 155 16 165 3 1425 143 1435 144 1445 145 Peak Blue Shift : as T λ
Hydrogen Bonding Normal modes are perturbed when the vibrating O-H bond senses another water molecule oriented so that its lone e s face H of the vibrating bond. Proton Donor Proton Acceptor Frequency of v s decreases from 377 cm -1 in the isolated molecule to 3628 cm -1 in liquid water and to 3277 cm -1 in ice. *Adapted from Ch 2 (p. 14) in Franks, 2, Water: A matrix of life 8
Suggested Assignments of Water Bands Assignment 2v s 2v as Free OH in free water molecule 1342-1383 1373-1415 Free ( Dangling ) OH 1351-1392 1381-1423 H bond in dimer 141-1453 1455-1499 H bond in trimer 1424-1467 - H bond in tetramer 1475-1519 1533-158 Indicates blue shift of bands when less H bonding between H 2 O molecules *Observed in solid N2 matrix. Adapted from Luck, W.A.P. Infrared studies of Hydrogen Bonding in Pure Liquids and Solutions, in Franks, 1973, Water: A comprehensive treatise (Ch 4 p. 276-9) 9
Water bands in NIR Assignment Min Max 3v s 887 121 v as +2v s 895 913 2v as +v s 93 921 3v as 911 148 Assignment Min Max 2 vs 1331 1371 2V as Free OH 1343 1383 v as +v s 1349 2v as Dangling OH 1352 1392 2v as 1367 148 2v s Free OH 1374 1415 2v s Dangling OH 1381 1423 2v as H bond in dimer 1411 1453 2v as H bond in trimer 1425 1467 Assignment Min Max 2v as H bond in tetramer 1475 1519 2v as H bond in polymer 15 1544 2 v s H bond in tetramer 1534 158 2 v s H bond in polymer 1553 1599 887-148 nm 198-115 nm 1331-1467 nm 144-1525 nm 1475-1599 nm λ Assignment Min Max v ds +2v s 198 1125 v as +v ds +v s 111 2v as +v ds 1122 115 Assignment Min Max 2v ds +v s 144 152 2 v s H bond in dimer 1456 1499 v as +2v ds 146 1525
Aquaphotomics Aims to extract water absorbance patterns to describe biological systems e.g. animals, plants Prof. Roumiana Tsenkova Kobe University, Japan www.aquaphotomics.com How to extract water absorbance pattern (WAP) from NIR Spectrum? 11
Spectra of water over 1 hour at constant temperature of 28 o C 1.4 1.2 1 Log (1/T).8.6.4.2 13 135 14 145 15 155 16 12
PC1 of water over 1 hour at constant temperature of 28 o C.2.15 92.7 % Explained Variance.1 PC 1.5 -.5 -.1 -.15 13 135 14 145 15 155 16 13
Expt. 1: Salts in Water 4 salts: NaCl, KCl, MgCl 2 or AlCl 3 Concentration range:.2.1 Mol.L -1 Control: Millipore water x 3 Gowen et al, Talanta, 214
Experimental Design A. Gowen (Dublin) NIR Systems 65 S. De Luca (Rome) Nicolet 67 FT-NIR Y. Tsuchisaka (Kobe) NIR Systems 65
Water spectra
PC 1 Water only
PC 1 Salts
PC 2 Salts
Question How to remove effect of temperature in spectra? Extended Multiplicative Signal Correction!
EMSC Model.2.15.1 X b b X b I b I 1 2 1 3 PC 1.5 2... -.5 Interference Spectra -.1 -.15 13 135 14 145 15 155 16 Corrected spectrum Xˆ X b 1 b b 2 b I 1 1 b 3 b I 1 2 21
PC 1 EMSC Salts LOD ~.1 % (mass/mass)
How watery is food? 5% 6% 7% 8% 9% % water
Hyperspectral Imaging Single wavelength image λ Y 1.5 1.5 X 2 4 6 8 1 12 14 16 18 2 Pixel spectrum 24/35
Hyperspectral Imaging Equipment Objective Slit Spectrograph Camera Mirror Wavelength range (nm) 95 165 Spectral resolution (nm) 7 Detector InGaAs
Expt. 2: Mushrooms 5 vibration times => induce surface damage x 24 mushrooms x 3 reps
Mushroom Curvature
EMSC on Mushrooms Raw Spectra: 1 mushroom EMSC(Reflectance) Reflectance 1.2 1.8.6.4.2 EMSC Spectra: 1 mushroom 1 11 12 13 14 15 16 1.2 1.8.6.4.2 1 11 12 13 14 15 16
EMSC on Mushrooms Mean Spectra: 12 mushrooms Mean EMSC Spectra: 12 mushrooms 3.9 2nd derivative (EMSC) 2.5 2 1.5 1.5 2nd derivative (EMSC).8.7.6.5.4.3.2 1 11 12 13 14 15 16.1 1 11 12 13 14 15 16
EMSC Mushrooms: difference spectra.8.2.6 6s 12s 3s 6s.15.1 Difference EMSC (log(1/r)).4.2 -.2 PC1 EMSC (log(1/r)).5 -.5 -.1 -.15 -.4 -.2 -.6 1 11 12 13 14 15 16 -.25 1 11 12 13 14 15 16 Blue shift indicating more free water as damage proceeds
PC1 EMSC Mushrooms PC1 EMSC (log(1/r)).2.15.1.5 -.5 -.1 -.15 λ (nm) 2D Tentative assignment 155 -I 3v as 1384 -I 2v as dangling OH or 2v s Free OH in free water molecule 1454 -I 2v ds +v s or v as in dimer -.2 -.25 1 11 12 13 14 15 16
Abs Peaks Mushroom Abs 155 nm.25.2.195.19.185.18 Abs 1384 nm.57.56.55.54.53.52.51.5 Abs 1454 nm.85.84.83.82.81.8.79.78.77.175 1 2 3 4 5 6 Vibration time.49 1 2 3 4 5 6 Vibration time.76 1 2 3 4 5 6 Vibration time 3v as 2v as dangling OH 2v ds +v s or v as in dimer EMSC corrected images at 1384 nm Undamaged Damaged
Free v s Bound water Free water: can be extracted easily from foods by squeezing or cutting or pressing Bound water: cannot be extracted easily Bound water molecules can t escape as vapor => Even upon dehydration food contains bound water.
Water activity W J Scott (1952) established that it was not water content that correlated with bacterial growth in foods, but Water activity: ratio of the water vapor pressure of the food to the water vapor pressure of pure water under the same conditions
Moisture Content Vs Water Activity http://vssweb1.landfood.ubc.ca/courses/fnh/31/water/wprin.htm
Expt 3. Aqueous solutions Transflectance Cell 3 μl Aqueous solutions: Salt Aw LiCl.25 LiCl.5 NaCl.76 NaCl.92 KCl.98 W 1
Mean Sample Spectrum No pretreatment EMSC 4.2.25.5.76.92.98 1.7.25.5.76.92.98 1 4.6 3.8.5 Log(1/Reflectance) 3.6 3.4 Log(1/Reflectance).4.3 3.2.2 3.1 2.8 1 11 12 13 14 15 16 1 11 12 13 14 15 16
Difference Spectrum (EMSC (Water -)).1.5.5.25.5.4 EMSC(W) - EMSC(AW) -.5 -.1 EMSC(W) - EMSC(AW).3.2.1 -.15.98.92.76 -.1 -.2 9 1 11 12 13 14 15 16 17 -.2 9 1 11 12 13 14 15 16 17
Difference Spectrum EMSC Aw >.5.1 2D λ (nm) Tentative assignment.5 I+ 114 v δs +2v s EMSC(W) - EMSC(AW) -.5 -.1-1167 2v as + v δs + 1384 2v as or 2v s dangling OH M- 1433 2v as in dimer or trimer I+ 1517 v as + 2v δs or 2v as in tetramer -.15.98.92.76 -.2 9 1 11 12 13 14 15 16 17
Difference Spectrum EMSC Aw<=.5 EMSC(W) - EMSC(AW).5.4.3.2.1.5.25 2D λ (nm) Tentative assignment -I 145 2v as or 2v s dangling OH -I 1489 2v ds +v s or 2 v s H bond in dimer or v as +2v ds -.1 -.2 9 1 11 12 13 14 15 16 17
Expt. 4: Water temperature 1ml water pipetted on ceramic tile Glass slide heating wire placed through centre of drop Glass slide placed on top =>thin layer of water Heating wire switched on Images obtained every minute for a total of 15 mins. White tile Heating wire
Expt. 2: Water temperature 4.2 EMSC (log(1/r)) 3.5 3 2.5 2 1.5 1.5 PC1 EMSC (log(1/r)).1 -.1 -.2 1 11 12 13 14 15 16 -.3 1 11 12 13 14 15 16 Classic effect of temperature
PC 1 EMSC PC1 EMSC (log(1/r)).2.1 -.1 -.2 λ (nm) 2D Tentative assignment 145 I- 2v as dangling OH 1475 I- v as +2v ds Or 2v as in tetramer -.3 1 11 12 13 14 15 16
Absorbance at PC1 peaks 82 2.82 3.15 3.15.8 2.8 78 2.78 76 74 72.7 68 Abs 145 nm 2.76 2.74 2.72 2.7 2.68 Abs 1475 nm 3.1 3.5 Abs 1475 nm 3.1 3.5 66 64 2.66 2.64 62 2.62 2 24 46 68 1 8 1 12 12 14 14 16 16 Time Time (min) (min) 2v as dangling OH 3 3 2 24 46 68 1 8 1 12 12 14 14 16 Time Time (min) (min) v as +2v ds or 2v as in tetramer
PC1 Score images
Expt. 5: Hydration of Dry foods Coffee Hydrate to different MC & AW Wafer Soybeans Sample # Sample size Coffee 33 2g Wafer 21.6g Soybeans 42 2 kernels Measure NIR HSI, MC, AW
AW/MC Wafer 14 12 1 MC 8 6 4 2.2.3.4.5.6.7 AW
AW/MC Coffee 8.5 8 7.5 7 MC 6.5 6 5.5 5 4.5.2.3.4.5.6.7 AW
Soybean MC 18 17 16 15 14 13 12 11.65.7.75.8.85.9.95 AW
Correlation between Aw, MC: wafer 1.8 MC Aw Correlation Coefficient.6.4.2 -.2 -.4 -.6 -.8-1 1 11 12 13 14 15 16
Correlation between Aw, MC: coffee 1.8 Correlation Coefficient.6.4.2 -.2 -.4 -.6 -.8-1 1 11 12 13 14 15 16
Correlation between Aw, MC: soybean 1.8 MC Aw Correlation Coefficient.6.4.2 -.2 -.4 -.6 -.8-1 1 11 12 13 14 15 16
PC 1 EMSC Spectra.5.4 Coffee Wafer Soybean.3 PC1 EMSC (log(1/r)).2.1 -.1 -.2 1 11 12 13 14 15 16
Difference EMSC (subtracting T) Wafer Difference Spectrum.6.5.4.3.2.1 -.1 -.2 7.9% 1.1% 11.2% 11.3% 11.8% 11.9% -.3 9 1 11 12 13 14 15 16 17
Wafer: PC1 EMSC loading.5 PC1 EMSC (log(1/r)).4.3.2.1 -.1 -.2 1 11 12 13 14 15 16
PC1 EMSC Wafer 2D λ (nm) Tentative assignment.5 -M 197 v δs +2v s PC1 EMSC (log(1/r)).4.3.2.1 -.1 -.2 1 11 12 13 14 15 16 +I 1335 2v s -I 1412 2v s Free OH in free water molecule or 2v s Dangling OH -M 1454 2v s in dimer or 2v as in dimer -M 1573 2v δs in tetramer
Abs Peaks Wafer.695.718.93 1.125 1.28 Abs 197 nm.69.685.68.675.67.665 Abs 1335 nm.716.714.712.71.78.76.74.72.7 Abs 1412 nm.92.91.9.89.88.87 Abs 1454 nm 1.12 1.115 1.11 1.15 Abs 1573 nm 1.26 1.24 1.22 1.2 1.18 1.16 1.14.66 2 4 6 8 1 12 14 MC.698 2 4 6 8 1 12 14 MC.86 2 4 6 8 1 12 14 MC 1.1 2 4 6 8 1 12 14 MC 1.12 2 4 6 8 1 12 14 MC.695.718.93 1.125 1.28 Abs 197 nm.69.685.68.675.67.665 Abs 1335 nm.716.714.712.71.78.76.74.72.7 Abs 1412 nm.92.91.9.89.88.87 Abs 1454 nm 1.12 1.115 1.11 1.15 Abs 1573 nm 1.26 1.24 1.22 1.2 1.18 1.16 1.14.66.2.3.4.5.6.7 AW.698.2.3.4.5.6.7 AW.86.2.3.4.5.6.7 v δs +2v s 2v s 2v s Free OH in free water molecule or 2v s Dangling OH AW 1.1.2.3.4.5.6.7 AW 2v s in dimer or 2v as in dimer 1.12.2.3.4.5.6.7 AW 2v δs in tetramer
Difference EMSC (subtracting T) Coffee Difference Spectrum.6.5.4.3.2.1 -.1 -.2 5.1% 5.6% 5.8% 6.5% 6.6% 7.% -.3 9 1 11 12 13 14 15 16 17
Coffee PC1 EMSC loading.5 PC1 EMSC (log(1/r)).4.3.2.1 -.1 -.2 1 11 12 13 14 15 16
PC1: EMSC Coffee PC1 EMSC (log(1/r)).5.4.3.2.1 -.1 -.2 1 11 12 13 14 15 16 D2 λ (nm) Tentative assignment M- 197 v δs +2v s I+ 1342 2v s or 2v as Free OH in free water molecule I- 1419 2v s dangling OH or 2v as H bond in dimer
Abs Peaks Coffee Abs 197 nm.495.49.485.48.475.47 4.5 5 5.5 6 6.5 7 7.5 8 8.5 MC Abs 1349 nm.412.41.48.46.44.42.4.398.396.394.392 4.5 5 5.5 6 6.5 7 7.5 8 8.5 MC Abs 1419 nm.77.76.75.74.73.72.71.7.69.68 4.5 5 5.5 6 6.5 7 7.5 8 8.5 MC Abs 197 nm.495.49.485.48.475.47.25.3.35.4.45.5.55.6 AW v δs +2v s Abs 1349 nm.412.41.48.46.44.42.4.398.396.394.392.25.3.35.4.45.5.55.6 AW 2v s or 2v as Free OH in free water molecule Abs 1419 nm.77.76.75.74.73.72.71.7.69.68.25.3.35.4.45.5.55.6 AW 2v s dangling OH or 2v as H bond in dimer
Difference EMSC (subtracting T) Soybeans Difference Spectrum.4.3.2.1 -.1 12.6% 15.3% 14.9% 17.4% 14.6% -.2 9 1 11 12 13 14 15 16 17
Soybean: PC1 EMSC loading.5 PC1 EMSC (log(1/r)).4.3.2.1 -.1 -.2 1 11 12 13 14 15 16
PC1 EMSC Soybean 2D λ (nm) Tentative assignment PC1 EMSC (log(1/r)).5.4.3.2.1 -.1 I+ 1335 2v s -I 1412 2v s Free OH in free water molecule or 2v s Dangling OH -M 1587 2v δs in tetramer -.2 1 11 12 13 14 15 16
Abs Peaks Soybean.89 1.2 1.35 1.195 1.19 1.345 Abs 1349 nm.885.88 Abs 145 nm 1.185 1.18 1.175 1.17 Abs 1587 nm 1.34 1.335 1.165 1.33 1.16.875 11 12 13 14 15 16 17 18 MC 1.155 11 12 13 14 15 16 17 18 MC 1.325 11 12 13 14 15 16 17 18 MC.89 1.2 1.195 1.35 1.19 1.345 Abs 1349 nm.885.88 Abs 145 nm 1.185 1.18 1.175 1.17 Abs 1587 nm 1.34 1.335 1.165 1.33 1.16.875.65.7.75.8.85.9.95 1 AW 2v s 1.155.65.7.75.8.85.9.95 1 AW 2v s Free OH in free water molecule or 2v s Dangling OH 1.325.65.7.75.8.85.9.95 1 AW 2v δs in tetramer
Key Questions What does water look like in the NIR? What can hyperspectral imaging show us? How can we use this to understand the role of water in foods?
Acknowledgements 67