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Once a set of reference samples is
obtained, it is advisable to divide these
into two sets; a set for calibration, and
a set to test the calibration often
referred to as the validation set. The
suitability of the samples used in the
calibration set is particularly important
since it must closely match the
product being manufactured at the
production site. A calibration set must
contain enough samples to enable the
estimation of the calibration constants,
and the range of the samples should
be representative of the range of
future samples.
A calibration, based on ‘ideal’
samples prepared in a completely
stable environment, may give very
small errors when used for similar
samples. However, when the
calibration is used to produce a value
for a real product, the accuracy is
often degraded. Furthermore, a tradeoff
exists between calibration sets
covering a very wide range of OH
number and the accuracy with which
OH number can be predicted.
Therefore, it has been found that the
accuracy increases when using a
method calibrated over a more
restricted (ca. 50) OH range.
The PLS (Partial Least Squares)
method available in the Perkin-Elmer
QUANT+ TM software uses the NIR
spectra recorded to derive a
calibration. This equation is then
used to predict the value of the
constituent of interest of future
samples using their NIR spectra. An
application that is analyzed by using
spectroscopy is more precise than
when measured by other methods.
However, the accuracy of the method
in predicting the true value of the
future samples is dependant upon
the accuracy of the reference method.
It is therefore important to stress
that the steps involved in setting up
the calibration are crucial to the
success of the NIR application. This
is particularly true for the collection
of suitable calibration standards,
and the analysis of the calibration
set by titration. This step should be
carried out with an objective of
producing the greatest possible
accuracy.
A suggestion for achieving this
accuracy is to perform the titration
in duplicate or triplicate. However,
it is more important to have as many
samples in the calibration set as
possible. In comparison, it is better
to collect twice as many calibration
samples than it is to perform the
reference analysis of the original
calibration standards in duplicate.
The most ideal, though costly and
time consuming, situation would
allow for the inclusion of a large set
of calibration standards, with each
sample analyzed in duplicate or
triplicate.
QUANT+ software was used to
derive a calibration matrix between
the calibration spectra and their
Figure 1: Extremes of calibration set.
reference OH values. The technique
of PLS extracts those spectral
contributions which are correlated
with OH value, and eliminates the
need to select specific wavelengths
for the calibration.
As previously stated, the near
infrared region consists of many
overlapping bands that contain
overtone and combination bands
involving the absorptions of mainly
OH, NH and CH bonds. The bands
of interest in the determination of
OH value are due to the OH first
overtone at 7150 – 6670 cm-1
(1399 – 1499 nm), the OH
combination bands of 5260 – 4760
cm-1 (1900 – 2100 nm), and
moisture content changes: ~5155
cm-1 (1940 nm).
Experimental
Fourteen standards of known OH
value were supplied to serve as a
calibration set. A further eight
samples were also supplied to act as
an independent validation set. These
were used to compare the reference
values with those values predicted by
the calibration equation. All samples
had been analyzed previously in
triplicate by the reference ‘wet’
chemistry method (titration).
The infrared spectra of all samples
were recorded on a PerkinElmer
FT-NIR Spectrometer. The samples
were scanned in an NIR quartz
transmission cell through a range
of 10000 – 4000 cm -1 at a resolution
of 8 cm -1 and strong apodization.
All spectra were recorded in ratio
mode using the shuttle accessory.
The shuttle automatically collects
alternate background and sample
scans to eliminate the effects of
water vapor in the air. A pathlength
of 5 mm was considered to provide
useful absorbance values. The
cuvettes were cleaned with acetone
and dried before refilling with the
next sample.
Two calibrated methods were
built using the reference data and
the spectra collected from the
FT-NIR Spectrometer. These were
setup using the QUANT+ software.
Both methods used the range 9000 –
4528 cm -1 with an upper threshold of
1.5 A. The offset option was
employed to minimize baseline drift