Postharvest technologies and

product development
"Novel objective and non-invasive methods for food analysis"
– Basics of NIR spectroscopy and its practical use –
theory and practice

Dr. Zoltan Kovacs

Kovacs.Zoltan3@etk.szie.hu
Department of Physics and Control
The (very) short history and
basics of spectroscopy
 Spectroscopy: Lambert–Beer law

Not applicable in diffuse

spectroscopy (like NIRS)
because l is unknown
(because of intensive
light scattering)
A = lg(I0/I) = ε × l × c
Based on Dr. Bázár
György’s presentation
 Basics of NIR spectroscopy

Sir Isaac Newton Sir William Herschel

(1643-1727) (1738-1822)

Based on Dr. Bázár

György’s presentation
 Spectrophotometry
Compounds’ chemical bonds can be induced in the spectral range of
UV and VIS.
The absorption of light in the function of wavelengths → absorption
spectrum

Based on Dr. Bázár

NIR 800-250nm György’s presentation
Karl Norris, USDA, Beltsville, MD, USA (1950’s)

Based on Dr. Bázár

György’s presentation
 Based on Dr. Bázár
György’s presentation
 Based on Dr. Bázár
György’s presentation
 Based on Dr. Bázár
György’s presentation
Diffuse reflectance (B) and diffuse
transmittance (D) is used in NIRS

Based on Dr. Bázár

György’s presentation
 It Ir
T= R=
I0 I st

1 1
A = − lg T = lg A = − lg R = lg
T R

Based on Dr. Bázár

György’s presentation
 NIRS = Vibrational spectroscopy
Vibrations of the water molecule

Wavelength [nm] Wavenumber [cm-1] Cause of absorption

1900 nm 5260 Combination (av1 + v2 + bv3; a+b=1)
1470 nm 6800 1st overtone (av1 + bv3; a+b=2)
1200 nm 8330 Combination (av1 + v2 + bv3; a+b=2)
970 nm 10310 2nd overtone (av1 + bv3; a+b=3)
836 nm 11960 Combination (av1 + v2 + bv3; a+b=3)
739 nm 13530 3rd overtone (av1 + bv3; a+b=4)
660 nm 15150 Combination (av1 + v2 + bv3; a+b=4)
606 nm 16500 4th overtone (av1 + bv3; a+b=5) Based on Dr. Bázár
514 nm 19460 5th overtone (av1 + bv3; a+b=6)
György’s presentation
Fundamental and overtones

Based on Dr. Bázár

György’s presentation
The NIR spectrum is composed as a result of light absorption of the
C–H, O–H, N–H and S–H bonds of organic compounds at different
wavelengths.
Water at different temperature

Based on Dr. Bázár

György’s presentation
NIR spectrum of water at different temperatures

Raw spectrum

2nd derivative

Based on Dr. Bázár

György’s presentation
 bonded bonded
free free

30°C 50°C

Based on Dr. Bázár

György’s presentation
 Ground Glass
In Air
A. Fine
B. Medium
C. Coarse

Fine Ground Glass

A. In Air
B. In Water
C. In Oil

Based on Dr. Bázár

György’s presentation
 Based on Dr. Bázár
György’s presentation
 Physical spectral effects
Spectral differences caused by scattering of fat globules

High fat%

Low fat%

Based on Dr. Bázár

György’s presentation
 Importance of spectral pretreatments
Smoothing: moving average
Savitzky-Golay function

Scatter correction: MSC (multiplicative scatter/signal correction)

EMSC (extended MSC)
SNV (standard normal variate)
Detrend

Derviatives Norris (gap)

Gap-segment
Savitzy-Golay

Various tools to remove/decrease additive and multiplicative effects.

With caution!
Based on Dr. Bázár
György’s presentation
 Chemometrics
Near infrared (NIR) spectroscopy spectra can be
converted mathematically to precise quantitative
information of chemical and physical nature by
multivariate calibration.

This makes NIR analysis useful for a variety of „difficult”

sample types (powders, slurries), more or less without
any sample preparation.

Martens & Stark, Journal of Pharmaceutical & Biomedical Analysis, 1991, 9:8, 625-635.
Based on Dr. Bázár
György’s presentation
Multivariate data
Multidimensional space
Hyperspace
Etc.

Why is it good to have many dimensions

(variables)?

Why is it good to check them simultaneously

(why is it good to combine the variables)?
Based on Dr. Bázár
György’s presentation
 Based on Dr. Bázár
György’s presentation
 Based on Dr. Bázár
György’s presentation
1. Quantitative analysis
Spectra of samples with known compositions → Calibration (PLS regression) →
Spectrum of unknown sample → prediction of composition
MANY SPECTRA + REFERENCE DATA → CALIBRATION → PREDICTION

2. Qualitative analyis
Spectrum library → classification algorithms (discriminant analysis) →
Spectrum of unknown sample → identification, classification
MANY SPECTRA (+ CLASSIFYING METHOD) → CLASSIFICATION → → PREDICTION

Rapid, cost effective prediction

Authentication
On-line measurements, process analytical techniques (PAT)
Based on Dr. Bázár
György’s presentation
NIR is an indirect (correlative) measurement,
which needs to be calibrated against a reference
method.

The usual approach is

- Take both spectral data and reference
measurements for a number of samples
(training set)
- Use this training set to establish a formula that
predicts reference from spectral data
- Validate the formula on further samples

Based on Dr. Bázár

© Tom Fearn, 2009, ICNIRSGyörgy’s presentation
There are too many predictors
The problem with calibration using spectra is that there are too
many predictors (multicollinearity also causes problems). If we just
use multiple linear regression (MLR), we will get an equation that
fits the training data perfectly but does not predict unknowns.
One solution is to select a few wavelengths and do MLR.
Another is to reduce the dimension of the spectral data whilst
trying to retain as much as possible of the information.

© Tom Fearn, 2009, ICNIRS

Principal component analysis (PCA)
A common way to reduce dimension is to construct
new variables as linear combinations of the original
spectral variables.
This is what PCA does.
Terminology:
- Loadings (one for each spectral variable): the
weight each original variable gets in the
construction of the new factor
- Scores (one for each sample): the values of the
new variables
The new variables are independent (orthogonal).
© Tom Fearn, 2009, ICNIRS
Principal component analysis
 Use of PCA
Multivariate data structure

Spectral outlier detection

Identification – PCA is not a real classification tool!

 Calibration and prediction
Quantitative modelling

Principal component regression (PCR)

Partial least squares regression (PLSR)
Quantitative calibration using PLSR and PCR
It is possible to see MLR, PCR and PLSR as variants of the same sheme
- Contruct factors as linear combinations of the spectral data
- Use these factors to predict y

The difference is how the factors are chosen

- MLR, to maximise correlation (with risk of over fitting)
- PCR, to maximise explained spectral variability
- PLSR, compromise between the two

MLR only constructs one factor, nothing linear will improve the
correlation.
PLSR and PCR contruct several factors, stopping when preditions (cross-
validation or test set) start to deteriorate.

© Tom Fearn, 2009, ICNIRS

Parciális legkisebb négyzetek regresszió (PLSR)

Camo Webinar, 2009-03-10

 The importance of validation
It is important to test the final calibration on a separate set of samples, a
validation set, NOT used to produce the calibration formula
- There is a risk of over fitting on the training set, especially with the more
sophisticated calibrtion algorithms

One problem is that most methods require some tuning, e.g. choice of number of
terms in the equation, and it is tempting to use the validation set for this. If you
do, it is not a validation set (even if it is called cross-validation).

Split the training samples into three sets: training (calibration), tuning (validation
with feedback to training), validation (independent test)

Often training and tuning is combined → cross-validation (see some slides later:
Modes of testing a model)

© Tom Fearn, 2009, ICNIRS

 Testing a method

Næs, T., Isaksson, T., Fearn, T., Davies, T. (2002): Multivariate Calibration and
Classification. NIR Publictions, Chichester, UK
Basics and applications of Near-Infrared
Spectroscopy and Aquaphotomics
 What is aquaphotomics?

Water
水

and
と
光

Light
by Roumiana Tsenkova 38
Conventional NIRS vs. aquaphotomics
approach
Aqueous solution of Lactose
100 mM

1/T

39
Conventional NIRS vs. aquaphotomics
approach
Aqueous solution of Lactose
100 mM

1/T

40
 Conventional NIRS vs. aquaphotomics
approach
Aqueous solution of Lactose
100 mM

1/T

remove water

Crystallized lactose

41
 Conventional NIRS vs. aquaphotomics
approach
Aqueous solution of Lactose
100 mM

1/T

remove water

Crystallized lactose Pure water

1/T

42
 Conventional NIRS vs. aquaphotomics
approach
Pure water

1/T

remove water
Aqueous solution
of Lactose 100 mM - Pure
water

Crystallized lactose

Subtracted

1/T
spectrum of
Aqueous solution
of Lactose 100 mM

43
Aquawater + Photonlight + Omicstech-platform
If we think differently about water then a different outcome can be expected.
Broad idea: Everything in nature interacts with its environment.
Interaction → Change → Systematic treatment → Analytical criteria
from D. Kojić
Shifting the paradigm:
a) from water as passive medium towards
b) water as an active solvent,
Water is now recognized as an active solvent,
adapting its structure to the solutes that it
accommodates, and in biological systems a kind
of biomolecule in its own right with an active
role in the dynamics of biomolecular and
cellular processes.
P. Ball 2008
44
Aquaphotomics:
Water as a molecular mirror

Perturbations
WAMACS*
Spectral Activated Water
Data Base Absorbance Bands

water

Bio・
Aqueous
System Water Absorbance
NIR spectrum
Spectral Pattern,
WASP

*WAMACS = Water Matrix Coordinates, i.e. water absorbance bands in VIS-NIR range
spectral ranges where specific water absorbance bands related to respective functions
are found with the highest probability
by Roumiana Tsenkova have been confirmed by overtone calculations of already reported water bands in IR range 45
Water Mirror Approach in aquaphotomics

Aquaphotomics spectral analysis is often called “water mirror approach”

because of the indirect manner of acquiring information about
the solute composition or the surroundings
of aqueous system – by measuring the
changes in absorbance at the water
absorbance bands in the spectrum
of the aqueous system.
R. Tsenkova 2008; 2009 46
 Overview of the basic methodology of
aquaphotomics

Water Absorbance Bands

Tsenkova, R., Munćan, J., Pollner, B., Kovacs, Z. (2018) Essentials of

Aquaphotomics and Its Chemometrics Approaches. Frontiers in Chemistry 6, 363 47
 Visualization of Water Absorbance Pattern, WASP
AQUAGRAM
Aquagram displays normalized absorbance values at
specific water bands on the axes originating from the
center of the graph.
Absorbance values at the WAMACs are placed on the
respective radial axes.
“blue shift” with increasing temperature
temperature

Pure water measured at

different temperature

48
 Visualization of Water Absorbance Pattern, WASP
AQUAGRAM less hydrogen
Aquagram displays normalized absorbance values at
bounded water
specific water bands on the axes originating from the
vapor like
center of the graph.
Absorbance values at the WAMACs are placed on the highly hydrogen
respective radial axes. bounded water
ice like
“blue shift” with increasing temperature
S4-
temperature Water molecules
with 4 hydrogen
bond

S3-
Water molecules
with 3 hydrogen
bond

S2- S0
Water molecules with
Pure water measured at 2 hydrogen bond

different temperature
S1-
Water molecules with
1 hydrogen bond
free water49 49
WAMACS RANGE, NM ASSIGNMENT REFERENCE
C1 1336-1348 ν3, H2O asymmetric stretching vibration Ozaki 2002
C2 1360-1366 Water solvation shell, OH-(H2O)1,2,4 Robertson et al. 2003, Xantheas 1995
C3 1370-1376 ν1 + ν3, symmetrical stretching fundamental vibration and H2O Ozaki 2002
asymmetric stretching vibration
C4 1380-1388 Water solvation shell, OH-(H2O)1,4 and superoxide, O2-(H2O)4 Weber et al. 2000, Xantheas 1995
C5 1398-1418 Free water and free OH- (S0) or trapped water Iwamoto, Uozumi, and Nishinari
1987, Kaffka et al. 1990, Kojić et al.
2014, Maeda et al. 1995
C6 1421-1430 Water hydration, H-OH bend and O…O Esquerre et al. 2009, Tsenkova 2009,
Tsenkova et al. 2004, Williams 2009
C7 1432-1444 Water molecules with 1 hydrogen bond (S1) Cattaneo et al. 2009, Ozaki 2002,
Segtnan et al. 2001b
C8 1448-1454 ν2 + ν3, Water solvation shell, OH-(H2O)4,5 Cattaneo et al. 2009, Ozaki 2002
C9 1458-1468 Water molecules with 2 hydrogen bonds (S2) Franks 1973, Tsenkova et al. 2004
C10 1472-1482 Water molecules with 3 hydrogen bonds (S3) Franks 1973, Ozaki 2002
C11 1482-1495 Water molecules with 4 hydrogen bonds (S4) Gowen et al. 2009, Ozaki 2002,
Segtnan et al. 2001b
C12 1506-1516 ν1, ν2, symmetrical stretching fundamental vibration, and doubly Gowen et al. 2009, Headrick et al.
degenerate bending fundamental or strongly bound water 2005, Segtnan et al. 2001a
AQUAPHOTOME – a comprehensive database of WASPs

51
Focus on subsystems vs. Collective approach

52
 What aquaphotomics can provide

Applying the benefits of NIRS

fundamental - rapid measurement

applied
- non invasive measurement
research - cost effective research
- etc.

53
 Fields of aquaphotomics applications
Application References
Fundamental Sugars (Bázár et al., 2015; Cui et al., 2017a), proteins (Tsenkova et al., 2004; Chatani et al., 2014), DNA (Goto et al., 2015),
biochemical studies salts (Gowen et al., 2013a; Gowen et al., 2015), alkali-metal halides (Kojić et al., 2014), acids (Omar et al., 2012), metal ions
(Sakudo et al., 2006b; Tsenkova et al., 2007a; Putra et al., 2010)
of water solutions
Water quality Water filtration process (Cattaneo et al., 2011), detection and quantification of pesticides (Gowen et al., 2011),
discrimination of mineral waters (Munćan et al., 2014), detection of contaminants (Gowen et al., 2015), holistic water
monitoring (Kovacs et al., 2016)
Food quality Various foodstuff (Gowen, 2012), cheese(Atanassova, 2015), honey (Bázár et al., 2016), mushrooms (Gowen et al., 2009a),
bacteria in food (Nakakimura et al., 2012), milk (Tsenkova, 1994; Tsenkova et al., 2001a; Tsenkova et al., 2001b), food
packaging influence (Cattaneo et al., 2016; Barzaghi et al., 2017)
Materials and Soft contact lenses (Munćan et al., 2016b; Šakota Rosić et al., 2016) fullerene based nanomaterials (Matija et al., 2012;
nanomaterials Matija et al., 2017), polystyrene particles (Tsenkova et al., 2007b)
Microbiology Bacteria (Nakakimura et al., 2012; Remagni et al., 2013; Slavchev et al., 2015; Slavchev et al., 2017), HIV virus (Sakudo et al.,
2005)
Plant biology Mosaic virus detection in soybean (Jinendra et al., 2010), abiotic and biotic stress (Jinendra, 2011)
Animal medicine Mastitis in cows (Tsenkova et al., 2001a; Tsenkova et al., 2001b; Tsenkova et al., 2001c; Tsenkova and Atanassova, 2002;
Tsenkova et al., 2005; Atanassova et al., 2009; Meilina et al., 2009), udder health (Tsenkova, 1994), ovulation period in
Bornean orangutan (Kinoshita et al., 2016), ovulation period in Giant Panda (Kinoshita et al., 2010; Kinoshita et al., 2012),
estrus detection in cows (Takemura et al., 2015),tissue discrimination (Sakudo et al., 2006a)
Human medicine DNA mutations (Goto et al., 2015), HIV virus detection (Sakudo et al., 2005), tissue discrimination (Sakudo et al., 2006a), the
state of metals in tissues (Sakudo et al., 2007), prion protein disease (Tsenkova et al., 2004), skin cream effects (Matija et al.,
2013) dialysis efficacy monitoring (Munćan et al., 2016a), colorectal cancer diagnostics (Munćan et al., 2016a)
Tsenkova, R., Munćan, J., Pollner, B., Kovacs, Z. (2018) Essentials of
Aquaphotomics and Its Chemometrics Approaches. Frontiers in Chemistry 6, 363
Aquaphotomics Spectrum
Human
medicine
Food quality
Materials and
nanomaterials

Protein AQUAPHOTOMICS Water and solute

analysis analysis

Plant biology Microbiology

Veterinary
medicine 55
Food quality assessment
Tokaj wine Melon juice

Adulterated with grape must Grown and stored under

concentrate different conditions

Paprika powder Galacto-

Aquaphotomics oligosaccharides
Adulterated with corn flour at Direct monitoring of the GOS
different concentration levels formation

Tomato concentrate Yogurt

Grown with different strains of
Adulterated with starch, paprika
lactobacillus using different
seed powder, NaCl and sucrose
water

Honey Mineral water

Adulterated with rice and With different mineral content

beetroot syrup mixed and analyzed
56
“Water is the driving force in nature”
Leonardo da Vinci

https://static.tumblr.com

We would like to thank

our collaborators.