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9. NIR ANALYSES
Near infra-red reflectance (NIR) spectroscopy has been used for more than
35 years to rapidly analyze grains, animal feeds and forages. The first application of
NIR spectroscopy was developed by Norris and associates in the early sixties to
measure water content in grains and seeds (Givens et al., 1997). Since these early
developments NIR spectroscopy has matured to a well established and broadly
accepted method to measure a wide array of chemical compounds in feed and food
ingredients or diets.

Concerning soybean products, the largest and most evident application is in the
rapid determination of proximate components previously carried out by the time-
consuming and laborious conventional wet chemistry. The potential of NIR to carry
out more evolved analysis such as protein quality and ANF is a real possibility since
the technique has been used to measure characteristics of similar complexity such
as digestibility of individual amino acids (van Kempen and Bodin, 1998). However,
despite the rapid answers and the major time savings made possible by NIR, the
development of the calibrations required for protein solubility and ANF have as yet
received little attention.

NIR spectroscopy is based on the principle that infra-red radiation of a sample

results in the reflection or transmittance of the radiation that is not absorbed by the
sample. The characteristics of the reflected or transmitted radiation can be used to
describe certain chemical characteristics of the sample. Since this relationship is not
mathematical, the relationship between the reflected radiation and the chemical
compound of interest must be based on a calibration. In this calibration the amount
of light reflected (or absorbed) at one or more wavelengths are related to a specific
chemical compound or compounds. More precisely, it is the chemical bonds and
functional groups of the compound that are related to the reflectance at a specific
wavelength. Consequently, molecules characterized by a repetitive bond and
structure are often more suited for detection by NIR. The choice of a wavelength or
a combination of wavelengths to detect a chemical compound is not necessarily
constant. The optimum choice of wavelengths to correlate with a specific compound
differs not only among ingredients but also among laboratories, equipment and even
years. Also, the scattered reflectance from other compounds leads to interference.

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9. NIR Analyses.

Consequently, the wavelength best related to the compound of interest is the one at
which absorption by the compound is maximized and interference by reflectance of
other sample constituents is minimized.

A number of items interfere with the near infra-red reflectance spectra. The
reflectance obtained from a sample is characterized by scatter due to instrument
type and function, sample preparation (grinding and thus particle size), temperature,
water content and interference of reflectance from other compounds. Variations in
water content of the sample are important because water absorbs radiation strongly.
In order to increase the precision of NIR analyses the factors interfering with the NIR
spectra need to be standardized when analyzing an ingredient or they need to be
eliminated through the application of mathematical corrections on the spectrum or
calibrations. Since standardization of sample preparations is not always practical and
since it reduces the major benefit of NIR analyses (time savings) preference is given
to mathematical corrections. A series of mathematical tools have been developed to
correct the spectral data and improve the predictive capacity of the calibrations.
The choice and application of these corrections differ considerably among the
constituents to be analyzed. The range of mathematical tools that is available to treat
spectral data is increasing rapidly thus improving the quality of the analysis and the
requirements for sample preparation.

Before routine analyses can be carried out equations need to be developed

for each individual constituent and often the individual ingredients. Sometimes, a
common equation can be developed for ingredients and/or their by-products.
In the case of soy products a single equation can by used for a number of products
if they are sufficiently alike in composition and preparation. This is for instance the
case for all soybean meals. However as a general rule of thumb it may be said that
the larger the physical and chemical differences among ingredients, the greater the
need to develop separate equations.

NIR calibrations are equations developed from a dataset composed of the

component of interest analyzed by a standard reference method (i.e. crude protein)
and the infra-red reflectance spectra. Least square multiple linear regression analysis
are used to develop the prediction equation (calibration) i.e. chose the equation
that provides the best fit between the analytical component and reflectance or
absorption at one or more wavelengths. The calibration data set should include
samples that represent the total chemical, physical and spectral variation normally

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9. NIR Analyses.

found in the population of samples that will be analyzed with the calibration. For
instance in the case of a calibration to measure crude protein in all soybean meals
the calibration dataset should include samples of SBM ranging from 42 to 50 % crude
protein. Calibration sets should have the widest possible range in composition but
above all they should be representative of all samples to be routinely analyzed with
the equation. It is generally not recommended to include samples with extreme
values (Shenk and Westerhaus, 1991). Extrapolation beyond the range of values
covered in the calibrations is not acceptable. Thus for most soybean products
separate equations will need to be developed for groups of products with similar
characteristics and values (i.e. Full fat soybeans, SBMs, SPCs, oils etc.).

The quality of a calibration depends greatly on the number of samples and

the choice of the samples. The number of samples required to develop a reliable
equation remains a subject of discussion. No definite numbers can be provided as
the size of the calibration dataset is related to the variability within a set and the
range of values that needs to be covered. Under most conditions applicable to
soybean products, the number of samples will be no less than 40. The larger the set
of well prepared and selected samples the stronger the calibration will be. Once the
calibration established, validation of the calibration will be necessary. Samples for
validation are subject to the same criteria for representation and number as those
used for samples to establish the equation. Generally a smaller number are allowed
when samples are representative of the population. Routine procedures to verify
the validity and quality of the equation need to be established. The calibration can
and should be strengthened through a continuous updating and expansion of the
calibration set by adding critically selected samples.

A number of statistical measures are used to describe the quality of a calibration

or evaluate its predictive capacity. Most of these refer directly to the least square
multiple linear regression techniques used to develop the equations. Most common
measures are the regression coefficient (R2), the standard error of prediction or
estimate (SEP) and bias (D). The R2 is a measure of the variability in the reference
data accounted for by the regression equation; the SEP is the variability between
predicted values and reference values when the equation is applied to the data
other than the calibration set, and D is the average difference between the predicted
and reference values. Ideally R2 should be as close as possible to 1.0 while SEP and
D should be as small as possible.

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9. NIR Analyses.

Analyses obtained by NIR are potentially subject to a large number of errors

related to the equipment, the calibration and validation process or sample
preparation (Williams, 1987). Not all errors are of equal importance and their
occurrence and impact is being reduced by the development and installation of
more sophisticated NIR techniques and equipment. Users have learned to manage
the equipment better and increased their understanding of the special requirements
needed for NIR analysis. While the routine use of the equipment is quite simple, the
maintenance and development of calibrations require a high level of expertise.
For proper operation and in order to reduce errors clear protocols should be drawn
up and implemented at all levels of NIR operations. It is important that these
protocols assure continuity between the use of NIR for routine analytical functions
and the development of new calibrations. When used for routine quality assurance
analyses, it is important to provide a separate dust-free environment. This is often
difficult to realize in operations dealing with commodities and feed production.

An important number of the errors that can occur in NIR are related to the
equipment. There is a relatively large variation between NIR equipments.
Consequently, in the case of monochromatic equipment for instance calibrations
cannot be transferred directly from one NIR to another without adjustments or
corrections followed by a series of validations. Universal calibrations have been
developed to solve the problem of transferability of calibrations. These equations
are based on a larger dataset than normal covering often different regions and years.
Results of these calibrations are often less accurate that those of equipment-specific
calibrations. More recently the concept of cloning or networking NIRs has been
developed. In these networks and through a series of mathematical corrections the
NIRs are calibrated to provide identical spectral results. This of course facilitates
enormously the transfer of calibrations and the verification of the different NIRs in
the network.

While in principle all organic compounds of a feed or feed ingredient can be

analyzed by NIR, for most ingredients and especially for soybean products, best
results in terms of accuracy and precision are obtained for humidity, crude protein
and lipids. NIR results for fiber components and non-fiber carbohydrates (starch,
sugars) normally give larger SEPs and biases and lower R2 values. NIR cannot be used
for the analyses of minerals although a rough estimate for ash and minerals may be
obtained by relating the reflectance at specific wavelengths to the organic matter or
components of the organic matter (Givens et al., 1997). NIR can be used to analyze

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9. NIR Analyses.

other organic compounds such as amino acids (van Kempen and Bodin, 1998) ANF
or fatty acids in soy products, however, the number of publications on this subject is
limited and more work is needed.

Equipment required for NIR analysis of soy products:

• Drying equipment (force draught oven).
• Wet chemistry laboratory (to conduct analyses for reference values used in
calibration development – see previous sections).
• Grinder (preferably Retch grinder but this is optional; calibrations can be
developed for un-ground, homogeneous material).
• NIR equipment.
Procedure (calibration development).
• Dry sample to constant weight (see Section 8.1).
• Grind (optional).
• Split sample in 2 sub-samples, one for reading on NIR equipment and one for
analysis by the reference method(s) (wet chemistry).
• Fill sample holder (as described in manual).
• Insert sample holder in NIR and read reflectance or analyte concentration.
• Obtain analytical results for analyte of interest by reference method
(see Chapter 8).
• Using a statistical software perform multiple linear regression analysis between
wavelength spectra (independent variable) and results of chemical analysis
(dependent variable).
• Establish regression equation (high R2, low SE); beware of over-parameterization
(use of too many wave lengths).
• Validate equation with samples not used to establish equation.
• Re-evaluate calibration regularly.

Procedure (application):
• Dry sample to constant weight (see Section 8.1).
• Grind (optional).
• Fill sample holder (as described in manual).
• Insert sample holder in NIR and read reflectance or analyte concentration.
(Modern apparatus have integrated computers that will give a direct reading of
the component concentration).