Article
NIR news
‘Specific Normalization’ method for
NIRS calibrations
Jose Antonio Cayuela-Sánchez
Abstract
In this study, we present a chemometric procedure called ‘Specific Normalization’. It upgrades the calibration for use in a
specific set of new samples by normalizing together the spectra of both the calibration set and a new measured sample set.
The proposed procedure is shown for near infra-red spectroscopy measurement of olive oil acidity, as an example. This
procedure would need its integration as software application for routine analysis.
Keywords
Calibration, normalization, routine, specific, validation
Introduction
Near infra-red spectroscopy (NIRS) methods usually
consider setting a calibration that will predict a certain
chemical or even physical characteristic of a material.
The accuracy and precision of such predictions depend
on the protocol used for calibration and for their use.
A NIRS calibration is a predictive tool, the results of
which need periodic proof through timely
confirmation.
Elaborating a NIRS calibration implies using che-
mometric methods, that is, pre-treating the spectral
data. On some occasions, the explained variables
should also be treated previously by specific methods
to fix correlations with the spectral variable, as in the
frequent case of analysing compositional features.1 In
this study, we will focus only on the spectral data.
The chemometrics for NIR spectra usually begins by
averaging the replicates from each sample. Once the
spectra and their possible singularities are analysed, it
is convenient to reduce the variables, since they usually
present repetitive information in most of their bands.
After that, it is usual to apply some normalization
method to minimize the influence of external influen-
ces, such as temperature or light, and also to minimize
the noise of the equipment. The most common method
is mean normalization, although there are other diverse
choices, such as maximum normalization, unit vector,
etc. Normalization has important impact on the cali-
bration fitness, which reflects on its performance.2 The
next step is often to transform the data from reflec-
tance or transmittance to absorbance. Then, first- or
second-order derivatives with different choices are
usual, in addition to smoothing or other possible
treatments.
Selecting the wavelength intervals that best fit the
calibration is another important issue in NIRS.3
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Setting those completely defines the calibration. The
calibration fit is assessed by its squared coefficients of
calibration R2c and cross-validation R2cv 4 The literature
shows multiple examples of studies that consider such
statistics enough for assessing a NIRS calibration.
However, R2cv is a good statistic to show as to how to
perform a NIRS model only when the features of the
predicted samples are closely similar to those used to
calibrate. Many of the practical applications using
NIRS are associated with agri-food products. These
biological products have great variability. It is one of
their characteristics, that may be due to many causes,
such as edaphological, climatic, genetic, and agronom-
ic. It so often happens that target samples present dif-
ferences from those used for calibration. For these
reasons, in agri-food products, it is advisable to carry
out predictive exercises to assess the NIRS calibrations
used according to external validations. This means pre-
diction on sets of samples that are not included in the
calibration.
Predictions made with a calibration, either in a pre-
dictive exercise for external validation or for routine
use, suppose using the spectra from a set of new sam-
ples. Unlike routine use, validation needs analytical
determination of reference values by the usual labora-
tory method. Using a calibration as a measurement
method implies analysing the reference values only in
the validations of a periodic proof protocol. Both in
Department Biochemistry and Molecular Biology of Plant Products,
Instituto de la Grasa (CSIC), Sevilla, Spain
Corresponding author:
Jose Antonio Cayuela-Sánchez, Spanish Scientific Research Council,
Campus University Pablo de Olavide, Ed. 46, Crtra. Utrera, km 1, Sevilla
41013, Spain.
Email: jacayuela@ig.csic.es
8 NIR news 34(3–4)
the routine use and in validation, calibration provides
predictions from the supplied spectra. The chemometric
treatment applied to these spectra has an important influ-
ence on the accuracy and precision of the result held.2
In the routine use of a laboratory, analysing sample
sets from different sources, a calibration projects the
target feature from a set of spectra. The same chemo-
metric treatments should be used sequentially with the
calibration spectra. Therefore, pre-treating the valida-
tion set is applied to the calibration set in the same
way, but separately.
In this study, we present a chemometric procedure
called ‘Specific Normalization’ for normalizing the cal-
ibration spectra. It upgrades the calibration for use in a
specific set of new samples. The proposed method may
seem impractical for the daily routine analysis of
diverse sample sets. However, it might be feasible to
integrate the procedure as a software routine, which
would be of great interest. The example of NIRS mea-
surement of olive oil acidity explains below the specific
normalization procedure proposed.
Material and methods
Olive oils
Two different sets of olive oil samples provided by
Sovena Spain S.A. base the example that shows the
procedure. The calibration setup was from 100 sam-
ples. The provider sent separately the 24 new samples
used as the validation set. Both sets of samples were
within the Recupera 2020 1.4.4 research project.
Free acidity analysis
The technological center AINIA (Paterna, Spain) mea-
sured the acidity values. AINIA developed a techno-
logical service for the referred project, including this
determination. Instituto de la Grasa (IG-CSIC) sent
the olive oil samples to AINIA for developing the
said service.
NIR spectra
Recording the NIR spectra was done at the IG-CSIC
using a Luminar spectrometer (Brimrose, Maryland),
with its liquid probe. The spectrum-recording proce-
dure, the characteristics of the spectrometer, and opti-
cal arrangement were reported in Cayuela and Garcıa.5
Luminar 5030 (Brimrose Corp., Baltimore) is an
acousto-optic tunable filter (AOTF) NIR spectropho-
tometer, equipped with a transflectance post-dispersive
optical configuration and an InGaAs (1100–2300 nm)
detector. The instrument takes the reference automat-
ically. The beam divides before leaving the instrument,
and a small portion goes to a second detector that
makes the reference. The scanning speed of Luminar
5030 is 60 ms. The instrument’s liquid probe equipped
a threaded interchangeable optical path ‘0.5’. The
probe and path are made of stainless steel. The NIR
radiation coming from the optical fiber goes through
the liquid sample and gets reflected on the polished
stainless steel surface of the optical path’s cylinder.
Then, it goes back through the liquid sample, heading
through the spectrometer detector; this process is called
‘transflectance’. The spectrometer set is complete with its
computer unit. The instrument registered the whole
signal, with each spectral variable matching to a 2 nm
interval. The repeatability, expressed as standard devia-
tion on the average absorbance of 1100–2300 nm of five
measures of a white tile, is 6.76 10
4 cm
1 mol
1. The
software used to register the signals was Acquire
(Brimrose Corp., Baltimore, Maryland, USA). The
Unscrambler 9.7 averaged the repeats to set up a spec-
trum for each sample, resulting from a total of 100 spec-
tra matching to two measures of 50 spectra each.
Calibration procedure
Before setting up the multivariate correlation between
the spectral variables that boost the model and the free
acidity of the olive oils, we use The Unscrambler 9.7 to
apply chemometric pre-treatments to the spectra. First,
we average each of the replicated spectrum. Then, after
visual proof that said compression of the spectral data
did not imply relevant loss of information, the spectral
variables were reduced into 8 nm intervals. Then, the
mean of the transmittance intensities of the spectra are
normalized, using two alternative procedures. In the
first alternative procedure (Procedure I, MI), the mean
normalization was applied to the spectra of the calibra-
tion set independently from the validation set, which
was separately normalized as usual. The second alterna-
tive (Procedure II, MII) consisted of applying identical
treatments to the calibration and validation sets, but in
this case, the means of the spectra of both sets are nor-
malized. After normalization, the transmittance values
were transformed to absorbance, followed by the first-
order Savitzsky–Golay derivative. The result was also
alternatively proved with the second derivative of the
same type.
The predictive model of the free acidity of olive oils
was established by multivariate PLS correlation of
selected spectral variables with said acidity values.
Calibration fitness was evaluated according to the close-
ness between the squared coefficient of calibration (R2c)
and the squared coefficient of cross validation (R2cv).
Model validation
Assessing the PLS model performance was mainly
done according to the residual predictive deviation
(RPD). The RPD is defined, according to Williams
and Sobering,6 as the ratio between the standard devi-
ation of the reference data from the validation set (SD)
and the standard error of model performance in the
validation (SEP).
RPD ¼ SD=SEP (1)
Cayuela-Sánchez
The r from external validation exercises was consid-
ered also to aid interpreting the calibration perfor-
mance. This is the correlation coefficient of the
simple linear regression between the analyzed and pre-
dicted values.
Results
Olive oil NIR spectra
The description of the visible and NIR spectra of olive
oils has been reported previously,7 and the virgin olive oil
spectra from the samples analysed in this work agree with
the previously indicated reports. The C-H first overtone
at 1700 nm induces a related high intensity area that can
be seen clearly. This happens similarly with the combina-
tion of fundamental vibrations of the C-H groups,
producing a combination band at 1880–2100 nm, and a
high intensity absorbance peak at 2300 nm.
Olive oil acidity models
The predictive calibration of the olive oil acidity car-
ried out according to MI, where the spectra of the
Figure 1. Olive oil acidity model MII.
Figure 2. Spectral variables that set up model MI.
9
calibration set was normalized independently from
the validation set, provided R2c ¼ 0.98 and R2cv ¼ 0.97.
The olive oil acidity calibration corresponding to
MII, which supposes normalizing together the spectra
of both sets of calibration and validation, is depicted
in Figure 1. MII showed identical statistics as MI
(R2c ¼ 0.98 and R2cv ¼ 0.97). Nevertheless, it was possi-
ble to distinguish slight differences between the actual
and predicted values of the data, as well as significant
differences in the wavelengths optimizing both models
MI (Figure 2) and MII (Figure 3).
Predictive exercises
The models MI and MII were tested on the same val-
idation set. The external validation with both models
differed only in the referred Procedures I and II for the
spectra normalization. Even the statistics R2c and R2cv of
MI and MII being identical, the predictive exercises
provided validation statistic with MII (Figure 4)
as fairly best (RPD ¼ 8.81) compared to MI
(RPD ¼ 7.65). This result revealed that MII enhanced
the performance of the olive oil acidity NIRS predic-
tive model.
10
Figure 3. Spectral variables that set up model MII.
Figure 4. Predictive exercise for external validation of MII.
Conclusions
This study proved the propriety of the suggested
‘Specific Normalization’ procedure. Normalizing
together by the mean and the spectra of both the cal-
ibration set and a new measured set upgrades the cal-
ibration for use in a specific set of new samples.
Therefore, it can be used for both validation and rou-
tine use. To be of practical utility, this procedure would
need its integration into a software routine.
Acknowledgements
The author thanks Sovena Spain for providing the olive oils
used in this study.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The author thanks the Ministry of Economy and
Competitiveness of Spain and the Spanish Council for
Scientific research (CSIC) for financing the Recupera 2020
1.1.4 project.
NIR news 34(3–4)
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