Near Infrared Spectroscopy An Overview

Background
Theory
Uses
-Industries
* Agriculture
* Chemical Industry
- Production vs. Research
* Centralized machine
* Online
Learning Objectives
- List the needs to successfully predict sample composition using NIRS
- Compare and contrast the advantages and disadvantage of using NIRS vs. wet chemistry
- Apply NIRs to your project
- Anticipate potential benefits, obstacles, and biases
Caution : I am not a physicist but a user who has gathered this information through the
course of my use!

Near Infrared Spectroscopy Theory

Near Infrared Spectroscopy NIRS
Uses the near-infrared region of the electromagnetic spectrum

Near Infrared Spectroscopy An Overview

BASICS:
-

Specific chemical bonds absorb energy in the NIR spectrum.

The amount of energy absorbed by the compound is related to the amount in the
sample (i.e. it is quantitative)
Samples are scanned using a near infrared spectrophotometer that both emits NIR
light and detects it.
Samples are measured for composition of interest.
Statistically predictive equation(s) may be developed by combining the NIRS
absorbance data with the chemical measurement.
Related to hyperspectral imaging but many more wavelengths
Any trait of interest (protein, micronutrients, etc.) can be attempted, but a successful
calibration depends on many things.
Tradeoff in accuracy for speed and cost compared to wet chemistry

Near Infrared Spectroscopy An Overview

Advantages:
- No consumables other than lamps ($50 - $1000)
- Non-destructive , non-invasive measurement
- Sample can therefore be small
-

Nearly instantaneous measurement

- Minimal sample preparation

- Continuous real time data can be obtained

- Can penetrate far into samples
- Can positively ID a pure compound if a library of compounds is developed
Disadvantages:
- Machines are expensive ($5,000 - $100,000)
- Must still measure reduced set of samples with wet chemistry
- Calibration is less accurate than wet chemistry
- Measurement outside of range of calibration samples is invalid
- Small calibration sample sizes can lead to overconfidence

Generalized Different Approaches Taken Toward NIRS

Chemistry,
Plant Biology

Agriculture
(soil science, agronomy,
plant breeding)

Type of samples analyzed

Pure compounds

Complex, heterogeneous
samples

Calibrations made using

Peak height or peak area

like HPLC / GC

Complex statistical packages

and algorithms

Type of instrument used

Fourier Transformed Near

Infrared Spectroscopy
(FT-NIRS)

Scanning monochrometer
(FOSS generally)

Reported as

Wavenumbers
(1 / wavelength)

Wavelength
(Nanometers, nm)

GENERALLY

Because of this, different people who work on NIRS can

speak different languages

www.winisi.com/NIRS_theory.htm

NIRS Theory

Thermo Scientific

higher energy near-IR,

approximately 14000
4000 cm1 (0.82.5 m
wavelength)
mid-infrared, approximately
4000400 cm1 (2.525 m)
far-infrared, approximately
40010 cm1 (251000 m)

NIR light, approximately 140004000 cm1 (0.82.5 m wavelength) can excite

overtone or harmonic vibrations in compounds.

NIRS Reflectance vs. Transmission

Two primary modes
- Reflectance
* Bounces off samples
* Best for solid objects (grain, soil, stover)
- Transmission
* Pass through samples
* Must be liquid or at least not completely opaque
* Usually much better calibrations may be developed
than reflectance
Penetration of samples is much deeper than visible light.

Near Infrared Spectroscopy Integrating Sphere

Hyperspectral imaging uses filters (like on a camera) to take pictures that include only
certain wavelengths
NIRS cover all wavelengths but only at the one point on a detector.
If you are scanning a heterogeneous sample (such as corn) if the detector is on the
embryo you will get a different result than if it is on the endosperm.
To average the light over a larger region an integrating sphere is used.
Integrating spheres are optical cavities that provide diffuse reflectivity, they diffuse light.
The more perfect your integrating sphere the higher your signal-to noise ratio will be

http://www.thermo.com/
eThermo/CMA/PDFs/Prod
uct/productPDF_7545.pdf

Near Infrared Spectroscopy Uses and Applications

Crops
- Forage quality and composition
- Pasture management http://cnrit.tamu.edu/ganlab/GANlab_webpage_files/analysis.htm
- Fecal nitrogen (FN) and phosphorus (FP) http://cnrit.tamu.edu/ganlab/GANlab_webpage_files/analysis.htm
- Approved method of the American Association of Cereal Chemists to measure to
moisture and protein content in wheat (AACC. 2002. Approved methods of the AACC, Methods 39-10, 39-11, 46-30 and 08-01. St. Paul,
Minn.: American Association of Cereal Chemists.)

- Ability to measure carotenoids in maize Brenna OV, Berardo N (2004) Application of near-infrared reflectance spectroscopy (NIRS)
to the evaluation of carotenoids content in maize. J Agric Food Chem 52:55775582.

- Sugars in grapes and other fruits Jarn C, Ortuo JC, Arazuri S, Arana JI, Salvadores MC. Sugar determination in grapes using NIR
technology. Int J Infrared Milli 2001; 22: 15211530.

- Oleic acid in single peanut seeds Tillman, B. L.; Gorbet, D. W.; Person, G.Predicting oleic and linoleic acid content of single peanut seeds
using near-infrared reflectance spectroscopy Crop Sci. 2006, 46 ( 5) 2121 2126.

- Starch gelatinization temperature in rice. J. S. Bao, H. Corke: Pasting properties of -irradiated rice starches as affected by pH. J.
Agric. Food Chem. 2002, 50, 336341.

- Malting quality in barley Ratcliffe, M., and Panozzo, J. F. 1999. The application of near infrared spectroscopy to evaluate malting quality. J. Inst.
Brew. 105:85-88.

- ADF, NDF, lignin, protein in sorghum leaf and stem

Murray, S.C., Rooney, W.L., Mitchell, S.E., Sharma, A., Klein, P.E.,
Mullet, J.E., and Kresovich, S. 2008. Genetic Improvement of Sorghum as a Biofuel Feedstock: II. QTL for Stem and Leaf Structural Carbohydrates. Crop Science 48: 2180-2193.

- Starch, oil, protein, ADF, phosphorus in sorghum grain

Murray, S.C., Sharma, A., Rooney, W.L., Klein, P.E., Mullet, J.E.,
Mitchell, S.E., and Kresovich, S. 2008. Genetic Improvement of Sorghum as a Biofuel Feedstock: I. QTL for Stem Sugar and Grain Nonstructural Carbohydrates. Crop Science 48:
2165-2179.

- Greenseeker technology also uses NIR light

- Etc.

Near Infrared Spectroscopy FORAGE

VERSUS

http://www.dairyone.com/

Near Infrared Spectroscopy GRAIN

VERSUS

http://wardlab.com/

Near Infrared Spectroscopy Uses and Applications

Soils
- Soil calibrations have generally not performed well across soil types, but accounting for
soil organic matter has improved calibrations. Russell, C.A. (2003) Sample preparation and prediction of soil organic matter
properties by near infra-red reflectance spectroscopy. Communications in Soil Science and Plant Analysis, 34, 1557-1572.

- Reasonable results have been found with P, Ca, Mg, K, Fe, Mn, S and Na but not pH
within Canadian soils. Malley, D. F., Yesmin, L., Wray, D., and Edwards, S. (1999). Application of near-infrared spectroscopy in analysis of soil mineral
nutrients. Comm. Soil. Sci. Plant Anal. 30, 9991012.

Soil mineralization of nitrogen.

Russell, C. A. , Angus, J. F. , Batten, G. D. , Dunn, B. W. and Williams, R. L. (2002) The potential of NIR
spectroscopy to predict nitrogen mineralization in rice soils. Plant Soil 247 , pp. 243-252.

-Soil moisture and organic matter.

Hummel et al., 2001. J.W. Hummel, K.A. Sudduth and S.E. Hollinger , Soil moisture and organic matter
prediction of surface and subsurface soils using a NIR sensor. Computers and Electronics in Agriculture 32 2001 (2001), pp. 149165.

Water
-Comparatively few publications
- Phosphorus, total organic carbon, color, and pH performed marginally. E. Dbakk, M. Nilsson, P. Geladi,
S. Wold and I. Renberg. Water Res. 34 (2000), p. 1666.

- Monitoring bacteria species in water. http://www.icpmf.org/pp/S06_3_AM1010_Camara_Martosetal%28ICPMF09%29.pdf

- May benefit from spectral subtraction in aqueous solutions.

Near Infrared Spectroscopy Uses and Applications

Industrial / pharmaceutical
-Place probe in bioreactors to measure:
- Target blends in pharmaceutical industry
- Fermentation progess
- Oil and gas industry
Health
- Used to detect tissue oxygenation since 1977
- Human skull easily penetrated by NIR light and can
be used for neuroimaging of brain parameters
Strangman G, Boas D A and Sutton J P 2002a Non-invasive neuroimaging using near-infrared light Biol.
Psychiatry. 52 67993

- Pediatric Critical Care and Cardiac Intensive Care

https://www.acep.org/WorkArea/DownloadAsset.aspx?id=44550

http://www.thermoscientific.com/

How Do I Build a Calibration?

1. Identify your goals and the types of machines available.
2. Scan all samples into machine using appropriate settings (TBD) and under similar
conditions.
3. Use integrated software features to select most informative samples for wet chemistry.
4. Re-scan those same samples to make sure you are accurate and have two spectra per
sample if wet chemistry is expensive.
5. Submit samples for wet chemistry.
6. Combine actual values from wet chemistry with spectra.
7. Run statistical analysis (YOU CAN NOT TELL FROM LOOKING AT SPECTRA!)
1. Examine derivatives not raw spectrum (usually)
2. Multivariate modeling for multiple wavelengths
3. Partial least-squares regression, principal component analysis (PCA) or other
calibration techniques.
8. Use best calibration to select new samples to include (return to step 3).
9. Include validation samples that are not used to develop the calibration to determine
the fit and usefulness of the calibration.

All Samples:
Stem (cellulose, hemi-cellulose, lignin, protein)
Leaf (cellulose, hemi-cellulose, lignin, protein)
Grain (starch, oil, protein)
Grind to 1mil with UDYcyclone

High-Throughput Phenotyping:
NIRS Analysis for Composition
Wet Chemistry performed on ~
10% of Samples
www.dairyone.com

Develop calibration
equations and test fit
ANKOM A200 Filter Bag Technique
Forage Method
Acid Detergent Fiber (ADF)
Neutral Detergent Fiber (NDF)
Acid Detergent Lignin (ADL)

www.udyone.com

FOSS Feed and Forage system

(1100 - 2500nm)
www.foss.dk

Cellulose = ADF - ADL

Hemi-Cellulose= NDF - ADF
Lignin = ADL

Find samples to use to develop

calibration equations

Stem Type

Fit - R2

%ADF

0.94

%NDF

0.95

%Cellulose

0.82

%Hemi Cellulose

0.46

%Lignin

0.77

%Crude
Protein

0.94

Find outliers

Thermo FT-NIRS on Ground Grain

Thermo Ground Grain FT-NIRS Results Rsq. = 0.887

Using 300 calibration samples and 150 validation samples

Consistently under predicts higher levels

FOSS Whole Grain NIRS Results Rsq. = 0.807

Equation built using

toxigenic type only
with a red. (3/4) and
val. (1/4) sets
2,5,5,1 SCVD both
vis and NIR (Rsq =
0.8071)
Plot predicted vs.
Full non-AG
Small sample/
limited cultivars

NIRS Results - Aflaguard (atoxigenic strain) Samples

NIRS over-predicts
contamination
levels on atoxigenic
samples
Suggests that there
is a molecular
signature that NIRS
is picking up not
necessarily aflatoxin
per se.
Help to develop A.
flavus resistant
germplasm

What Else Do I Need to Know?

Particle size:
NIR reflectance is influenced by the particle size properties of ground or powdered
materials. Thus using proper procedures and calibration, NIR reflectance spectroscopy
can be used for particle size analysis - Pasikatan, M.C. , Steele, J.L. , Spillman, C.K. and Haque, E. (2001) Near infrared reflectance
spectroscopy for online particle size analysis of powders and ground materials. Journal of Near Infrared Spectroscopy 9 , pp. 153-164.

Conversely, changing particle size can then disturb your calibration!

Sample number for wet chemistry:
General rule of thumb that I have always heard:
- 100 samples minimum
- >10% total samples
BUT
- Studies are routinely published with 50 or 60 samples total for developing a
calibration.
- Can only predict within the diversity of samples that you have.
Sample detection limitations:
General rule of thumb is constituents can only be detected at 0.1% or 1% of sample
composition.
Need adequate variance / variability in samples for best calibration.

What Else Do I Need to Know?

Signal to noise ratio:
The most important measurement according to NIRS sales people is signal to noise
ratio, each salesperson says their instrument is the best.
The longer the pathlength the more light is lost (usually). The lower the signal the
higher the noise.
More scans per sample will average out noise
- Noise adds up as the square root of the number of scans
- Signal adds up linearly

Scanning Monochrometers vs. Fourier Transformed

Scanning monochrometers
- Measure NIR wavelengths with a mirror and prism that scans over the
spectrum of interest one at a time.
http://www.chemwiki.ucdavis.edu/

http://www.foss.dk/Solutions/ProductsDirect/Fo
odScanMeatAnalyser/Technology.aspx#

Fourier Transformed NIRS

- No diagram form and I am unqualified to explain.

Why Should I Care Which NIRS Instrument I Use?

Assumptions:
- You will spend a lot of time and effort scanning samples.
- You will spend a lot of money collecting wet chemistry data.
Challenges:
- Can you bring it to the field?
- How available is it?
- Use (Dr. Rooneys Foss is in constant use)
- Sharing atmosphere / charges for use?
- How accurate is it?
- How sensitive is it to moisture and vibrations?
Why cant I transfer calibrations?
- In theory you CAN however:
- Each make and model has a different pathlength
- Each instrument has its own quirks (many are custom built)
- Need standards that have been scanned on both machines
- Transferring calibrations is painful on the agricultural side, maybe not so
much if you are looking at pure or nearly pure compounds.

NIRS Available Equipment in SCSC / at TAMU

ASDI AgriSpec
Advantages:
- Portable and rugged
- Many scanning accessories (hand held probe and mug lamp)
- Provides own white light source
- 350-2500 nm spectra range (VisNIR)
- high signal to noise ratio for soils compared to other specs
Disadvantages:
- 3 detectors for whole spectral range make it expensive
- no spining disk for homogenizing samples
- long pathlength of probe may decrease resolution/ accuracy
Software used:
- Unscrambler ($900)
- R (free)
Approximate cost:
- $45,000
Currently being used for:
- Lab Soil characterization (clay, fine clay, organic carbon, inorganic carbon, most
soil properties related to clay fraction)
- In situ mapping of soil profiles (In R&D Phase)
Owner/ responsible person:
Cristine Morgan (SCSC College Station) cmorgan@ag.tamu.edu

NIRS Available Equipment in SCSC / at TAMU

ASD LabSpec Model 5000

Advantages:
http://www.asdi.com/news/asd- Portable
releases-labspec-5000-5100spectrometer
- Rugged
- Diffuse Reflectance / transmission probe and Muglight Diffuse Reflectance
- NIRS and visible spectral ranges 250 25000 nm
Disadvantages:
- long pathlength of probe may decrease resolution/ accuracy
Software used:
- Indico Pro and Unscrambler
Approximate cost:
- $57,000
Currently being used for: Model Development: Wet chemistry complete for large range of land covers
in watersheds in Scotland, Puerto Rico, Czech Republic, New Hampshire, Maine
- Soil CO2 evolution NIR picking up labile C fractions
- Soil Organic Carbon and Total Nitrogen analysis done, scanning soil for model
- Free and organically-bound Iron, Aluminum and Manganese
- Water Extractable soil DOC and DON
Model Development: Texas
- Stream water signatures and non-point source inputs to fresh waters
- Grave soil: Identification of grave soil signature

Owner/ responsible person:

Jacqueline Ann Aitkenhead-Peterson (SCSC College Station) jpeterson@ag.tamu.edu

NIRS Available Equipment in SCSC / at TAMU

Unity Model SpectraStar 2400
Advantages:
- NIRS and visible spectral ranges
- Accuracy
- Calibrations already developed and available for soil and forage
- No splits over spectral range
- Can take different sample cups
- Easy to use software
Disadvantages:
- No probe
Software used:
- Scanstar, new software coming soon.
Approximate cost:
- $47,000
Currently being used for:
http://www.unityscientific.com/products/NI
- Soil and forage analysis in service laboratory
R-at-line/spectrastar-rtw.asp

Owner/ responsible person:

Tony Provin (SCSC College Station) t-provin@tamu.edu

NIRS Available Equipment in SCSC / at TAMU

Thermo
Advantages:
- NIRS and visible spectral ranges
- Accuracy
- Calibrations already developed and available for soil and forage
Disadvantages:
- no probe
Software used:
- ????
Approximate cost:
- $$$$$
Currently being used for:
- Peanut Analysis

Owner/ responsible person:

Mark Burow(SCSC Lubbock) mburow@tamu.edu

NIRS Available Equipment in SCSC / at TAMU

Unity Model SpectraStar RTW
Advantages:
- NIRS spectral ranges
- Accuracy
- Calibrations already developed and available for soil and forage
- Open cup
Disadvantages:
- no probe
Software used:
- ????
Approximate cost:
- $$$$$
Currently being used for:
- Sugarcane biomass
http://www.unityscientific.com/products/NI
R-at-line/spectrastar-rtw.asp
- Sugarcane agronomy

Owner/ responsible person:

Nael El-Hout (Formally SCSC Weslaco) I do not know who is now responsible.
Try Robert McGee, Operations Manager r-mcgee@tamu.edu

NIRS Available Equipment in SCSC / at TAMU

Infratec 1226 Grain Analyzer (bought out by FOSS)
Advantages:
- NIRS spectral ranges
- Calibrations already developed and available for cornand soybeans
- Ease of use
Disadvantages:
- Old technology no longer supported
- Few options
- Poor resolution and accuracy
Software used:
- Integrated into machine no computer needed
Approximate cost:
http://agronomyday.cropsci.illinois.edu/2002/extractable- $? , free donated from companies
starch/index.html
Currently being used for:
- Corn and wheat composition

Owner/ responsible person:

Lloyd Rooney (SCSC College Station) lrooney@tamu.edu

NIRS Available Equipment in SCSC / AgriLife

Foss Model 6500
Advantages:
- NIRS and visible spectral ranges
- Calibrations already developed and available for many products
Disadvantages:
http://dubuque.ebayclassifieds.com/busines
- No probe
s-industrial/dyersville/foss-5000-nirinstrument/?ad=5653757
- Slow
- Expensive given value
Software used:
- WinISI, ISI scan
Approximate cost:
- ~$65,000
Currently being used for:
- Botanical composition for ruminants
- Fecal analysis of ruminant diets
Owner/ responsible person:
John Walker (Rangeland San Angelo) jwalker@ag.tamu.edu

NIRS Available Equipment in SCSC / at TAMU

Foss Model XDS
Advantages:
- NIRS and visible spectral ranges
- Calibrations already developed and available for many products
- Liquids module
FOSS XDS
Disadvantages:
Scanning Monochrometer
- No probe
- Slow
- Expensive given value
Software used:
- WinISI, ISI scan
Approximate cost:
- ~$75,000 + $25,000 for liquids module
Currently being used for:
- Sorghum biomass composition

Owner/ responsible person:

William "Bill" Rooney (SCSC College Station) wlr@tamu.edu
Thomas Stefaniak (SCSC College Station) tstefaniak@ag.tamu.edu

NIRS Available Equipment in SCSC / at TAMU

Thermo Anteris II Fourier Transformed NIRS
Advantages:
- NIRS spectral ranges
- Integrating Sphere, liquids module, probe
- Fast, accurate and precise (FT)
Disadvantages:
- Expensive
- Heavy and bulky
Software used:
- TQanalyst, Omnic
Approximate cost:
- ~$62,000 + $8,000 for probe
Currently being used for:
- Corn aflatoxin and composition analysis
- Epicuticular wax analysis
- Cotton gossypol analysis
- Wheat composition
Owner/ responsible person:
Seth Murray (SCSC College Station) sethmurray@tamu.edu

Thermo Antaris II
FT-Interferometer

Combining NIRS with High Speed Seed Sorting

- Removing corn kernels infected by
mycotoxins would be useful for food
safety and research reasons.
- Certain NIR bands are correlated with
infection.
- A high speed seed sorter can separate
contaminated kernels from clean kernels.

T.C. Pearson, D.L. Brabec and S. Haley, Color image based sorter for
separating red and white wheat, Sensing and Instrumentation for Food
Quality and Safety 2008 (2) (2008), pp. 280288.