David L.

Wetzel
Department of Grain Science and industry
Kansas State University
Report
Manhattan, Kan. 66506
Downloaded via UNIV FED DO RECONCAVO DA BAHIA on February 1, 2022 at 14:48:22 (UTC).
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Sleeper Among Spectroscopic Techniques

Near-infrared reflectance analysis direct separation are necessary for in- Quantitative diffuse reflectance in
(NIRA), which relies effectively upon dividual determinations of compo- the near-infrared region has probably
chemometrics, is a “sleeper” because nents. Since the 1978 work of Peter not yet received the attention it de-
it is unknown, illogical, or presumed Griffiths (1), diffuse-reflectance in- serves. In the case where components,
a priori to be illegitimate by spectros- frared Fourier transform (DRIFT) present in the amount of 1% or more,
copists and analytical chemists. Ex- spectroscopy has received consider- of weakly absorbing samples require
amination of the facts concerning able attention. This mid-infrared quantitation, near-infrared (diffuse)
near-infrared reflectance in Table I technique uses spectral subtraction to reflectance at multiple wavelengths
will acquaint the reader with the po- uncover hidden features within mix- coupled with multivariate statistics
tential of this technique. tures and has been successfully ap- might be applied as the method of
Traditionally applied quantitative plied to analysis of pharmaceuticals, choice. The convenience of sample
infrared spectroscopy of mixtures in highly absorbing samples such as coal, handling, computer assistance, and
powder form could hardly be de- and trace quantities such as catalysis additivity of the near-infrared re-
scribed as a user-friendly technique studies of adsorbed chemicals. To date sponse makes this possible.
since extensive sample workup is re- over 200 diffuse reflectance attach- Pioneering work in near-infrared
quired. Thus, for such analytical prob- ments have been sold for FT-TR in- transmission spectroscopy was done
lems, chromatographic methods or in- struments. by Kaye (2, 3) of Beckman Instru-
ments in the 1950s. Classical spectros-
copy in this region prior to 1969 has
Table I. Facts Concerning Near-Infrared Reflectance been reviewed by Whetsel (4). Al-
Industrial applications of NIRA include: though commercial single-purpose
• molecular weight of propylene and ethylene glycol polymers dual-wavelength (near-infrared) dif-
• moisture content of coal and hematite fuse-reflectance production line mois-
• textile blends of cotton-polyester and of rayon-poiyester ture monitors had existed previously,
• finishes on textile fibers it was Norris (5) of the Beltsville
• amount of cross linkage in chemically modified starch USDA laboratory who recognized the
•
hydrogenation of unsaturated fatly acid esters potential of diffuse-reflectance mea-
surement in the near-infrared for rou-
•
hydrocarbon mixture of n-hexane, benzene, cyclobenzene, and iso-octane tine quantitative analysis of major
• volatiles {loss on drying) and moisture in cosmetics
components in agricultural commodi-
• total alkaloids, nicotine, and reducing sugar in tobacco processing ties. This latter work caused the ap-
• moisture in pharmaceutical excipients and in detergent powders pearance of commercial muitiwave-
Agricultural commodities in worldwide commerce are analyzed by NIRA for their content length filter instruments in the 1970s
of water, protein, lipid, etc. by Dickey-John, Neotec (Pacific Sci-
Pharmaceuticals have been identified by applying previously determined canonical
entific), and Technicon. European in-
variables resulting from multi wavelength measurements. struments have subsequently ap-
peared in the 1980s.
Rapid and timely analysis for process control in various processing industries is based
upon NIRA methods. What the Instrument Sees
Sample preparation prior to spectroscopic measurement is minimal even in the case Most filter instruments in routine
of powders. use generate raw optical data for only
a few wavelengths. However, the spec-

0003-2700/83/ A351-1165$01.50/0 ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983 . 1165 A
1983 American Chemical Society
©
 1.0
Wavelength (nm)
Figure 1. (a) Mustard seed; (b) millet; (c) durum; (d) soybean
Data taken from Reterence 6
Table II. Composition of Cereal Grains and Oilseeds
Starch
Product (%>
Mustard seed —
Millet 60-65
Durum 60
Soybean —
tral features of commodities scanned
over the entire near-infrared region by
use of a grating monochromator per-
mit one to observe the available ana-
lytical information. Figure 1 shows the
reflectance value expressed as
log 1/reflectance in the near-infrared
region for four agricultural commodi-
ties composed primarily of starch, pro-
tein, water, lipid, cellulose, and other
fibrous polysaccharides.
A look at the region of 2100 nm in-
dicates the presence of structures
common to starch and cellulose. Data
in Table II indicate high levels of
starch in those commodities exhib-
iting this peak. On either side of the
band at 2100 nm are absorptions at
2055 nm and 2180 nm corresponding
to amide structures present in protein.
In both of the oilseed (soy and mus-
tard) samples the protein peaks are
prominent because the band of carbo-
hydrate is essentially absent. Relative
prominence of the central carbohy-
drate peak to the side amide peaks
(i.e., shoulders) provides a pattern
typical of the other higher starch com-
modities. Such preliminary qualitative
information is useful as an aid in
quantitative work, since high-starch
samples may require that measure-
ments be made at the 2100-nm wave-
length and that this term be included
in the quantitation equation to com-
1166 A • ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
confirm the high lipid content of these
samples. Also, a subtle difference due
to CH bands present in cellulose but
not in starch allows one to explain the
greater absorption in the 2336-nm and
2352-nm regions for low-fat samples
containing high fiber.
Figure 2 shows how the reflectance
value is subject to differences of scat-
tering and sample penetration due to
differences in particle size. The data
shown are for a coarse wheat-milling
fraction (first mids) from the Kansas
State University pilot flour mill. Note
that the same material reground on a
sample mill (UD-Cyclotec with 1-mm
screen, Boulder, Colo.) and by ball
mill for 28 h gave respectively higher
reflectance (lower log 1/R), and less
contrast between the “absorption”
maxima and baseline of the reflec-
tance spectrum.
It can also be noted from the ab-
sorption peak at 1940 nm that water
was partially evaporated during the
regrinding. Thus, relative granulation
and relative water content of the sam-
plesare made obvious by selective
Protein Lipid Fiber
<%i <%> <%> near-infrared reflectance measure-
ment.
35.0 25-40 12.0 It is readily apparent that a visual
10.5 3.9 2.9 look at the spectrum is inadequate to
15.5 2.0 3.5 observe many of the spectral features
40.0 21.0 5.5 discussed. For this reason, a computer
is used to accumulate multiple digi-
tized readings at selected wavelengths.
Comparison of time-integrated signals
pensate for the effect of starch on the at analyte (indicator) wavelengths
background of the analyte (protein) with reference wavelength readings al-
absorption at 2180 nm. lows dual-wavelength spectrometry
In the areas of 2310 nm and techniques to be applied. The re-
2348 nm prominent peaks appear for sulting dual-wavelength measure-
the oilseed samples, indicating the ments are then related to differences
presence of lipid. Data in Table II in chemical composition. Differences
Wavelength (nm)
Figure 2. Kansas State University pilot mill first mids wheat flour
(a) Coarse; (b) reground on Cyclotec mill (1-mm screen); (c) reground on ball mill for 28 h.
Data taken from Reference 7
 pplications Report:
New Column Technology

New tools
Peak Identity Molecular Weight Co'umn Bio-Si! TSK-250
1 Thryroglobulm 670.000 300* 7 5 mm
2 IgG 150.000 Flow Rate 1 ml-min
3 Ovalbumin 45.000 Buffer OiMNaSO*
4 Myoglobm
5. Cyanocobalamn
17,500
1,355
0 02M NaHjPOi pH
Bio-Rad Gel
Filtration Standard
6 8

for protein
separations.
New HPLC column technology from Bio-Rad
gives researchers a number of worthwhile
alternatives for protein separations.
For example, here are three separations
of the same sample, using three different
approaches
—

high performance gel filtration,

MINUTES our new high performance ion exchange
column, and our new Hi-Pore™ reverse
phase column.
Column: Bio-Rad TSK-1EX-540 DEAE
Sample: Bio-Rad Gel Filtration Standard, Looking at those separations, it's appar-
10^1 injection ent that these HPLC columns provide
Gradient
Solvent A. '_5V
" ‘

3.*% » ph
'

fast separations with excellent resolution.

Solvent B: 0 5M NaCI in 0 05 M Tris Buffer, pH 7 2
Linear gradient programmed Not only that, certain compounds appear
from 0% to 100% B for 45 minutes in the ion exchange and reverse phase
Flow Rate-. u »r r

Detector v -r 28C. j i -
separations that don't reveal themselves
Peak identity- in the gel filtration separation. HPLC lets
1. IgG in solvent front 5 Ovalbumin related peptide
2 Myogfobm 6 Ovalbumin ,
you "read between the peaks" with both
3 Cyanocobaiamin 7 Ovatbumm related peptide
4 Ovalbumin related peptide speed and precision.
These new columns complement rather
than compete with each other. Our HPLC
y ion exchange column for analysis of ioniz-
able biochemical compounds is an excellent
complement to our Bio-Sil gel filtration
MINUTES column. By using all three columns, better
characterization of proteins (including large
ones) may be obtained.
Column: Bio-Rad Reverse Phase Gradient
(RP-304) Hi-PoTe™ Solvent A i
"

- =* i
As always Bio-Rad is prepared to assist you
Sample: *
Filtration Standard. 101% TFA/ in plotting an effective strategy for your pro-
IOmI imection Solvent B3: /. 25% IPA i i_,rku
,n H0H tein separations, using any or all of these
I 01'= TFA /
100% A for four
techniques. Give our applications staff a call,
1
o 25
minutes
2 Lmear gradient from w or contact us at
0% to 100% B
0 20 -

over 4 minutes
Flow Rate C fi -** -

Detector UV a:

Peak identity
1
280 0 nm

Cyanocobaiamin
2 Ovalbumm
015-
BIO-RAD
related impurity
3.. 4. Myoglobm
5 Protein impurity Bio-Rad Laboratories —Chemical Division
6 Ovatbumm 2200 Wright Avenue
Richmond, CA 94804
(415) 234-4130
Also in Rockville Centre, N.Y., Australia, Austria,
Canada, England, Germany, Italy, Japan, The
Netherlands, and Switzerland,
CIRCLE 29 ON READER SERVICE CARD
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983 • 1167 A
in near-infrared optical response of
samples with different, compositions
are very small compared to typical ul-
traviolet, visible, or mid-infrared ana-
lytical curves. However, they are re-
producibly measurable and are the
basis of the success of NIRA as a
quantitative technique.
Overcoming Apparent
Shortcomings
It is the nature of absorptions in the
near-infrared to be weak since they
consist of overtones or combinations
of fundamentals. The subtle differ-
ences among samples previously re-
ferred to require a careful measure-
Table III. Assumptions for Analytical Diffuse Reflectance
• Light incident on a powder or granular material changes directions as it encounters
each individual boundary of powder granule. Reflection, refraction, and diffraction take
place.
• Diffuse reflection occurs when a portion of the light entering the sample is scattered
by boundaries within the sample and exits the body of the sample from the surface of
entry.
• In the absence of absorption and with sufficient (effectively infinite) thickness and
multiple scattering-induced direction changes, a maximum of light is scattered back out
of the powder.
•
Absorption may occur as the scattered light is transmitted between the scattering
boundaries within the sample before being scattered back.
• The amount of transmittance back and forth between the scattering boundaries affects
the opportunity for absorption or the effective thickness (cell path length) of the
sample.
• The size and shape of the sample particles, the voids between them, and the amount

ment of the signal. NIRA is concerned
with observing differences between
two samples in milliabsorbance units.
For differences in this range to be
meaningful, the noise levels must be
kept to only a few microabsorbance
units. Such instrumental require-
ments are more stringent than those
of compacting affects the amount (concentration) of material through which the scattered
light must be transmitted between individual scattering boundaries.
• Some of the preceding factors also affect the mean free path between scattering
boundaries.
• The transmission path length (thickness) is shorter for strong scattering (opaque)
materials and longer for translucent materials.
• Reduction of particle size increases the scattering of transparent materials.
• Specular reflection, though geometrically well defined, comes off individual grains

for most other routine spectroscopic with surfaces of random slope in any direction and is a factor in the measurement of
quantitation. The character of diffuse diffuse reflectance.
reflectance requires special consider- • Weakly absorbing samples allow a simple specular reflectance correction (the constant

ation in the areas of optical design, op-
eration sequence, sample handling,
data accumulation, and statistical
treatment.
Near-Infrared. A few simple fun-
damental vibrational bands in the
mid-infrared region of the spectrum of
a particular compound will produce
multiple overtones yielding many
higher frequency bands in the near-
infrared region that overlap and that
are difficult to interpret. Unlike the
mid-infrared region, which is valuable
as a tool for obtaining structural infor-
mation, in the near-infrared region
such structural information is ob-
scured. When working in the near-in-
frared region there is a lack of refer-
ence spectra to help one predict what
one will see. Knowledge of the mid-
infrared absorption wavelengths of
certain functional groups is of some
help in knowing where to look in the
near-infrared for overtones. Analytical
information may also be obtained by
observing a shift in the frequency of
the bands due to influence of neigh-
boring molecules. A salt that does not
absorb in the near-infrared may be de-
tected and quantitated by its effect on
the water absorption. Such shifts
often explain the success of determin-
ing nonabsorbing materials because
these materials do affect the absorp-
tion spectra of other molecules
present.
Weak absorptions in the near-in-
frared have been cited as a shortcom-
ing. Instrumentally this is true, but
relative absorption strength also
serves as a convenient self-limiting
factor to restrict which vibrations are
observed. For example, a fundamental
vibration occurring at 15 ^m, with over-
background primarily dependent on refractive index effect can be subtracted out to move
the specular component) handled by regression.
• A constant specular background results at any wavelength when the refractive index
does not change and the effect of absorption is negligible.
tones at 7.5, 5.0, 3.75, and 3.0 pm is
not likely to be observed in the near-
infrared because the fifth or sixth over-
tones lack intensity. Weak absorp-
tions in the near-infrared provide se-
lectivity. The commonly used region is
from 1000 to 2500 nm where over-
tones of fundamental vibrations no
higher than 5-8 /xm, depending on
their intensity, appear. The long
wavelength end of the mid-infrared
beyond 8/um does not contribute to
the near-infrared. This means that the
overlapping bands in the near-in-
frared produced by many combina-
tions and overtones, although spectro-
scopically complicated, are from only
a few molecular groups.
In the near-infrared we predomi-
nantly see the result of vibrations of
light atoms that have strong molecular
bonds. If the chemical bond is weak,
or the atoms are heavy, the vibrational
frequency is low and its overtone will
not be detectable in the near-infrared.
Therefore, we primarily see chemical
bonds containing hydrogen attached
to atoms such as nitrogen, oxygen, or
carbon, thereby limiting the chemical
structures that are observable to fairly
simple ones that are common in many
organic compounds. These weak over-
tone bands are more subject to their
environment than is the fundamental
of the same vibration. A slight pertur-
bation in the bonding scheme causes
small changes in the fundamental, but
drastic frequency shifts and amplitude
changes in the near-infrared. Since
these weak bands are broad and over-
lapping, resolution is not a problem,
but reproduction of the same wave-
length is essential. The practical result
of this is that there are numerous re-
gions in the near-infrared where if
wavelength reproducibility is high,
signals maximized, and noise mini-
mized, then the optical responses are
sensitive to the environment of the ab-
sorbing molecules and the number of
the molecules present. Thus quantita-
tive measurement can be made and
successfully correlated to chemical
data obtained by other means. From
these data and application of a suit-
able statistical relationship with ap-
propriate constants, determination of
analyte concentrations of unknowns
can be made with surprising success.
Diffuse Reflectance. Scattering is
greater at the shorter wavelengths of
the near-infrared than in the mid-in-
frared region. In diffuse reflectance,
scattering is an important factor. Dif-
fuse reflectance characteristics are
summarized in Table III.
A volley of photons fired into a non-
absorbing, scattering sample will not
interact identically. Not all will be
scattered back the same way since
some willbe transmitted through
more of the sample and undergo many
more scattering boundaries before
exiting the sample. They will have dif-
ferent penetration depths, a different
number of scattering events (encoun-

1168 A • ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983

New Dimensions in Surface Analysis
ESCA 200A
250// r
Scanning Auger ^
Multiarray Detector
VG Instruments adds important new dimensions
to the analysis of surfaces—the flexibility of
ESCALAB will greatly expand the range and in-
crease the efficiency of your surface-analysis in-
vestigations. Now, without moving or changing the
sample, you can utilize ESCA, Auger, SAM, SIMS,
and other surface methods.
How will ESCALAB enhance the results of your
analyses? VG’s incorporation of a new high-
transmission analyser, a multiarray detector, and
the capability ol selective-area analysis offer you:
faster analysis time; improved spatial resolu-
tion; lower limits of detection—improved
sensitivity; and, cleaner, less cluttered spectra
for more detailed surface studies.
What are the new dimensions that will add these
significant new perspectives to your surface-
analysis investigations?
* Small
Spot ESCA (250^)
•
200A Scanning Auger
Multiarray Detector
r

2000A SIMS
-

For information on how VG can add new dimen-

sions to your analyses of surfaces, circle fhe
inquire number or VG instruments.

VG INSTRUMENTS
VG Instruments Inc. Mass Spectrometers
300 Broad Street Molecular Beam Epitaxy
Stamford. CT 06901 ESCA • Auger * SIMS * ISS
(203) 322-4546 Electron Microscopy
CIRCLE 226 ON READER SERVICE CARD
ter a different number of scattering
boundaries), and a different length
transmission path through the sample.
Individual photons of a volley fired
into an absorbing, scattering medium
will have different probabilities of
being absorbed since the effective
thickness of the path within the sam-
ple will vary with multiple scattering
events and penetration depth (Figure
3). Consideration of these facts makes
it necessary to accumulate and aver-
age data to develop empirical con-
stants and to treat the data statisti-
cally.
From the detailed list of assump-
tions concerning the nature of diffuse
reflectance as found in Table III, we
see that the following sample treat-
ments are necessary. In practice, the
variable of particle size difference
from sample to sample and from sam-
ple to standards must be minimized.
In the development of any NIRA
method it is prudent to establish the
limits within which one can deviate
between mean particle size within an
experiment and between different
particle size distributions within a
given type of sample preparation.
Since the wavelength is on the order of
1-2 pm, the lower limit of particle size
should be no smaller than a few mi-
crometers in diameter. The upper
limit is based on the need to get a
large number of particles in the beam
to adequately represent the sample
and the need to optimize scatter. This
is particularly true in the case of het-
erogeneous materials. In general, the
median diameter is on the order of
100 pm, and it is desirable to avoid too
broad a distribution. The overriding
factor on particle size, however, is to Figure 3. Various pathways of diffuse reflectance from weakly absorbing scatter-
ing particles
reproduce a mean particle size and
particle size distribution for each type Fortunately, in the near-infrared the signal-to-noise ratio. A high-intensity
of material and standards of that ma- refractive index effect is essentially stable source and a low-noise highly
terial. constant. Also, the absorptions are sensitive detector are required. Also
The nature of diffuse reflectance in- usually weak, and thus constancy of the ability to capture the maximum
volves a change in the direction of the the specular background is common amount of the diffuse component of
light which comes off randomly in all for many organic samples. The right the reflected radiation at multiple an-
directions but varies with the angle of combination of high scatter, weak ab- gles and to reject the specular compo-
observation from the normal. As a de- sorption, and nearly constant index of nent is needed. The mechanism and
tector is moved away from the angle of refraction allows scattered back trans- frequency of optical referencing may
incidence, the intensity will decrease. mission within the sample to be mea- contribute to favorable noise and drift
From this consideration, we observe sured in the presence of a steady-state characteristics.
that the instrument design is impor- specular reflectance background. Intensity of the light scattered back
tant. There is radiation coming off Under optimum conditions the re- varies with angle from the normal, di-
360° around the sample and coming sponse is sufficiently linear over the minishing at larger angles. In practice,
out in a solid angle around the inci- measurement range. Much of the past the angle of observation is fixed by the
dent beam. It is advantageous to col- success with plant materials, for ex- design of the instrument. Since a
lect a maximum amount of this radia- ample, may be due to the constancy slight vertical difference in positioning
tion for three reasons: first, to maxi- found in nature. of the sample would result in a differ-
mize the signal; second, to average all Consideration of the nature of near- ence in the angle observed by the fixed
directions; and third, to represent all infrared absorption and diffuse reflec- optical components, a mechanism for
desirable angles from the normal. tance measurement allows one to real- reproducible positioning of the sample
For samples of interest in certain ize both the quantitation possibilities is necessary.
analyses, peculiar circumstances favor and practical limitations and the ne- Packing a granular sample against a
a simplification to correct for the cessity for empirical statistical input. window regiments the orientation of
specular effect referred to in Table III. Instrumental Considerations. the sample so that the orientation is
The specular contribution involves The primary instrumental consider- no longer random. The degree of regi-
both refractive index and absorption. ations are those that provide a high mentation affects the intensity mea-

1170 A • ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983

THERE ARE STILL A FEW THINGS
AS PORTABLE AS AN OHAUS BALANCE.
It’s getting harder every day to stay one hop with any other balance? Send for our new
ahead of everyone else. Now Ohaus makes it catalog today.
easier with our Port-O-Gram electronic balances.
They not only go anywhere to do almost any Ohaus Scale Corporation
weighing job. They do it for as little as $395. 29 Hanover Road,
They’re small enough to fit in the palm of Fiorham Park,
your hand (or pouch). Inside, microprocessor NJ 07932
electronics compute weights instantly and pop (201) 377-9000
up bright, bold rock-steady readings. Just touch Telex: 136518
the pushbutton controls and you're ready to
weigh in grams, ounces or pennyweight. Some
models even allow select parts counting or your
own customized weighing units.
Long battery life lets you take it with you
on the line, in the labs, indoors or out.
When you're staying put, an AC adapter’s
included.
For over 75 years, Ohaus has
been making dependable balances of
rare quality. The Port-O-Gram balances
are no exception. Now why would you go

® 1983 Ohaus Scale Corporation. Ohaus and Port-O-Gram are registered trademarks of Ohaus Scale Corporation. Prices and specifications are subject to change without notice.
CIRCLE 156 ON READER SERVICE CARD
sured at any angle. The amount of reg-
imentation varies with the compacting
pressure and with particle size and
shape. The direction of observation
can have an effect on the intensity
measured at a given angle depending
on whether the observation is against
the grain or with the grain of a
textured sample. Such is the case with
naturally textured samples as in meat.
Surfaces may be oriented preferential-
ly in one direction by polishing, slicing
a section of sample, or by striking off
as in the case of an uncovered powder
or granular surface.
Sample observation variables ad-
dressed by the instrument design, in-
cluding the median angle of observa-
tion, angular boundaries of obser-
vance, and the direction of observa-
tion, are summed up in the designa-
tion of solid angle. The observation
may be simultaneous for different an-
gles and directions, or sampling of a
particular angular segment may be in
one direction continuously or in dif-
ferent directions intermittently.
The signal intensity depends on the
fraction of reflected light hitting the
detector (solid angle of observation)
and on the total flux from the source
that strikes the sample. Wavelength
selection by interference filters or in-
terferometry does not limit optical
throughput; however, in using a grat-
ing monochromator this is a major
consideration and optics with f/2 or
less are desirable.
The instrument operator is respon-
sible for sample preparation and must
ensure that procedures for unknowns
and calibration standards are kept the
same. This includes grinding (homog-
enization of liquid suspensions), stor-
ing of the sample, and packing and po-
sitioning of the sample cell. Any
change requires experimental verifica-
tion that analytical results have not
been affected.
Instrumentation
The typical operating sequence of a
routine instrument for NIRA involves
measurement of reflected intensity off
the sample surface at a number of
wavelengths and off a standard refer-
ence reflecting surface at those same
wavelengths. The reflectance mea-
surement in practice is thus a relative
measurement to a standard reflector.
The term I0 (incident intensity) does
not appear in the practical relation-
ship. Reflectance I(sample)/I(refer-
=
ence). Such a relationship exists at
every wavelength measured. The tra-
ditional Kubelka-Munk function with
appropriate scattering and absorption
constants has generally given way
among NIRA practitioners.to logarith-
mic reflectance terms used with em-
pirical coefficients that accommodate
the scattering effects as part of the
1172 A • ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
calibration. The general analytical ex-
pression is of the type:
% = z + a log 1/Rj + b log I/R2
+ c log I/R3 +
A commonly used term involves a
coefficient multiplied by log 1/R as
shown above. The equation has been
modified in some instances to allow
for a transformation of R that may
combine data from a number of wave-
lengths into one term. The result of
this is that there are fewer terms in
the equation but each of these terms
becomes considerably more compli-
cated.
Modern Hardware Features.
Commercial instruments commonly
use tungsten-quartz-halogen sources
and lead sulfide detectors, but the
mechanisms for referencing to a stan-
dard reflector and collecting the dif-
fusely reflected radiation differ con-
siderably (8).
Intermittent referencing of reflect-
ed radiation intensity from sample to
maximum reflected intensity from a
standard reflector allows each mea-
surement to be expressed as a ratio. In
the most elaborate referencing system,
this is done at each wavelength read-
ing before indexing to the next sam-
ple. In other instances, sample mea-
surements are made at all selected
wavelengths followed by measurement
of the standard at those same wave-
lengths.
Multidirectional (360°) reflectance
sampling allows simultaneous collec-
tion of radiation in all directions with
an integrating sphere, time-averaging
with rotating sample, or use of multi-
ple detectors. A maximum solid-angle
(approximately 2 r steradians) simul-
taneous reflectance collection is ob-
tained with an integrating sphere.
Available commercial NIRA instru-
ments include interference filter and
grating monochromator systems.
Commercial grating monochromator
instruments designed specifically for
NIRA are specialized systems with ap-
propriate computer software and
high-throughput monochromators.
The Technicon InfraAlyzer 500 per-
forms incremental step scanning. The
Pacific Scientific 6350 accumulates
rapid scan data. Diffuse reflectance
accessories are commercially available
for use with existing FT-IR instru-
ments.
In filter instruments two types of
mountings are the continuously mov-
ing type and the turret wavelength se-
lection of discrete narrow band inter-
ference filters. In the second type
(Figure 4), radiation intensity is mea-
sured at one wavelength and stored in
the computer and, after rotation of the
filter mount to the next preselected
filter, the process is repeated. In the
continuously moving filter mount,
multiple wavelengths are obtained
from the same filter depending on the
angle of tilt at the instant of transmis-
sion through this filter. There are
...
three levels of filter instruments (min-
imal, basic, enhanced) from which to
choose for analysis by multichannel
discrete wavelength measurement and
computer data treatment.
Enhanced systems have auxiliary
computer hardware and software to
allow rapid in-house custom calibra-
tion and method development. Other
channels or wavelengths may be
added to, or substituted for, those
commonly used on the basic system.
The system may allow direct raw data
transfer to a computer equipped with
statistical software suitable for identi-
fying the channels best correlated to
the quantity of analyte of interest by
the “t” test or other statistical func-
tions. Statistical terms as well as cali-
bration coefficients typically result
from multiple linear regression of data
from a suitable training set. Such a set
is selected to include a suitable range
of analyte concentration in a matrix
that contains other constituents in a
typical variation cross section for that
product. Good reference classical labo-
ratory analytical data are a prerequi-
site for success.
Developing a Method
In NIRA the principle of multiple-
wavelength spectroscopy is used. At
least one wavelength is responding to
the amount of a particular analyte in
the sample matrix. The measurement
of at least one other wavelength is
used as a reference of the overall re-
flectivity of that particular sample
matrix. In some instances a third,
fourth, fifth, etc., wavelength may be
used. These may also perform a refer-
ence function or may give either a neg-
ative or positive correlation to the an-
alyte. To obtain the most transferable
(robust) calibration, one should mini-
mize the use of data at wavelengths
that are not essential to the calibra-
tion. When simultaneous analyses are
performed, data are collected at the
wavelengths required for each of the
analyses, and a different equation is
solved for each different analyte. In
each equation the corresponding cor-
rect wavelengths appear, and their ap-
propriate coefficients are used. We
have thus the capability for simulta-
neous multicomponent analysis. For
example, one set of reflectance mea-
surements for milk could be used to
determine protein, fat, total solids,
and lactose by the solution of four dif-
ferent equations using the reflectance
data at the appropriate wavelength in
each equation, and the proper regres-
sion coefficients for each wavelength.
Experimental. To perform a cali-
bration, it is necessary to assemble a
 Microbore HPLC
Compromise Isn’t
Good Enough.

LDC/MM

LDC/Milton Roy

Higher sensitivity and more resolution at very low flow
rates —that's the promise of microbore HPLC. Now that
promise is met in full by LDC/Milton Roy's new components,
offering you the first system truly designed for microbore
HPLC —u'it/iont compromise! No adaptation of existing
components.
LDC/Milton Roy’s new microMetric,u Metering Pump
offers true pulse'free flow from 1-999 ju,L/min with better than
1% of thumbwheel setting accuracies. A 5 mL syringe capacity
permits multiple chromatograms. Refill is accomplished in
one minute. High pressure capability to 10,000 psi for both
today's packed columns and tomorrow’s capillary columns,
New spectroMonitor* D with microCeU™ offers crossflo\
optics to minimize thermal and flow noise without a bulky
heat exchanger. A 1 mm tubular quartz fused-silica cell pert
direct coupling to microhore columns for maximum efficier
Unique fiber optics and high performance mirror collect
and deflect light to the integral preamplifier circuit board,
Learn more about the marvels of microbore HPLC, and
about how LDC/Milton Roy has packaged all the benefits
one new, uncompromising set of components. Write or ca
for further information.

LDC/Milton Hoy-
U.S. HEADQUARTERS, P.O. Box 10235, Riviera Beach, FL 33404 Toll-free: 800-327-6182, In Florida: (305) 844-5241 International Telex: 441098, Domestic Telex: 513479
SUNNYVALE OPERATION, 560 Oakmead Parkway, Sunnyvale, CA 94086 Toll-free: 800-538-1595, In California: (408) 736-0300
UNITED KINGDOM, LDC UK, Milton Roy House, 52 High Street, Stone, Staffordshire ST15 8AR England Phone: 785-81-3542 Telex: 36623
WEST GERMANY, Milton Roy (Deutschland) GMBH Jahnstrasse 22-24 6467 Hasselroth 2, West Germany Phone: 6055-2031 (3366) Telex: 4184357
JAPAN, Milton Roy K.K., Takanawa Dai-lchi Building 21-41, Takanawa 2-Chome, Minato-Ku. Tokyo 108, Japan Phone: (03) 473-3181 Telex: 242-4296

127 ON READER SERVICE CARD

 Source

Figure 4. Typical turret-mounted dis-
crete filter instrument (Technicon In-
fraAlyzer 400)
Mirror position 1, reflectance off the sample; mir-
ror position 2, reflectance off ihe standard refer-
ence reflecting surface. Both detectors collect ra-
diation in the sphere with the mirror in either posi-
tion
collection of preanalyzed samples of
the product of interest. This collection
should contain the full range of the
analyte. For example, if you are ana-
lyzing protein content in wheat you
should have a range of perhaps 9-19%
protein. The learning set should also
he representative in terms of the ma-
trix material. If you plan to analyze
wheat of a number of varieties, differ-
ent varieties should then be used to
assemble the learning set. With this
set of samples (minimum of 30), and
classical laboratory analysis data such
as the Kjeldahl protein value, optical
data are collected for each of the sam-
ples. While this occurs the Kjeldahl
value of each sample is entered into
the computer. Correlation transforma-
tion, typically multiple linear regres-
sion, is then performed. The result of
this statistical procedure is the best
set of coefficients by which each re-
between the classical values and those
determined spectroscopically gives the
operator an idea of how good his cali-
bration is. Standard errors of differ-
ence for routinely performed commod-
ity analyses are expected to be on the
order of 1 or 2% relative.
Computation. The analyte concen-
tration is correlated to the spectro-
scopic response by individual wave-
lengths (log 1/R), by difference be-
tween pairs (log R2-log Ri), and by
trios (2 log R2-log R]-log R3) where
the reflectance of the central wave-
length is mathematically weighted in
comparison to the contribution of re-
flectance at wavelengths incremental-
ly spaced on either side. Plots or ta-
bles of correlation by wavelength re-
sult from single, pair, and trio reflec-
tance measurement subjected to sim-
ple regression.
Correlation is also determined by
direct application of multivariate sta-
tistical techniques. Multiple linear re-
gression, for example, is commonly ap-
plied to groups of wavelengths prese-
lected by filter choice. For example,
reflectance readings taken at 12 dis-
crete wavelengths, not necessarily re-
lated to each other, would appear as
12 separate terms in the multivariate
treatment (Table IV, Equation a).
Reverse stepwise multiple linear re-
gression beginning with 12 individual
reflectance terms is commonly used to
select a simplified expression contain-
ing two to five of the original 12 terms
which in use will be more robust. Sim-
ilarly the best combination of three or
of two, found by successive multiple
Table IV. Typical Computational Algorithms
(a) % = z + a log 1/R,
linear regression of all different com-
binations, is used as a starting point.
Wavelength terms may then be added
by forward stepwise multiple linear
regression. Ultimately, by either pro-
cess, a usable expression is produced
that contains the optimum number of
wavelength terms, the optimim wave-
length choice, and good regression
coefficients for each of the terms in
the expression.
An alternative approach to generate
an expression for analytical use based
upon the measurement at 12 wave-
lengths is to composite them by pairs
or trios. (Table IV, Equations b and
d). In this way a restriction is imposed
on the data prior to statistical treat-
ment. Twelve measurements compos-
ited by trios produce an expression
containing four complex reflectance
terms. Regression or other multivar-
iate treatments are subsequently used
to generate coefficients or eliminate
unneeded reflectance terms. Even the
composite reflectance terms may be
composited (Table IV, Equations c
and e). Such is the case when 12 re-
flectance measurements are composit-
' ed
by trios and subsequently divided.
Both the individual reflectance
term and the composited reflectance
term calculation methods have been
used successfully with the instruments
that they complement. Software needs
are dictated by instrumental hard-
ware, and the final test is performance
of any analysis system as a whole.
Developing a calibration for an ana-
lyte in a particular matrix involves 1)
choosing the wavelengths for incorpo-
+ b log 1/R2 + c tog 1/R3 + . . .

spective logarithmic reflectance re- (individual)

sponse is multiplied to solve the ana- z + a (log R2
(b) % log R,) + b (log R4 +
= -

log R3)
-

lytical equation and spectroscopically

.. .

(pair; first difference)

determine the percent protein. Once
/log log R,\
+ b /log R6
-

this is done, the coefficients for each R2 log R5)\

-

(C) % + a +
wavelength are programmed into the \log R4 log R3/
-

\ log
-

R8 log R7 j

built-in computer of the NIR analyz- (pair; ratio first difference)

er. Another set of samples, also prean-
(d) % = z + a (2 log R2 log R,
— -

log R3) + b (2 log R5

—

log R4
—

log R6) +
alyzed, is then analyzed by NIR. To
. . .

(trio; second difference)

obtain calculated (NIRA) values, the
equation is solved using the appropri- / 2 log Rs log R7 log R„ \
- -

(e) % = z + a (2logRa"logRi-|og r3| + b

ate coefficients and the spectroscopic log R5
-

log R4
-

log Re/ \2 log R„ -

log R,0
-

log R1SJ
response. When these are compared to (trio; ratio of second differences)
the corresponding classical laboratory Note that R,, R2, R?„ R4 R„ represent reflectances in order by wavelength.
. ..

values a standard error of difference

1174 A • ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983

 For $5890.00 you get Generations of
Quality, Performance, Experience...

The new LDC/Milton Roy spectroMonitor® D variable wavelength detector.

On the outside, the newest variable wavelength HIGHEST EFFICIENCY AND SENSITIVITY -

detector from LDC/Milton Roy looks very much STANDARD

like its predecessors. But on the inside, where it Our new max N™ series high efficiency high ,

counts, the spectroMonitor®D variable wavelength
detector sets new industry standards for quality and
performance,
LOWEST NOISE AND DRIFT
The new spectroMonitor D variable wavelength de-
tector delivers the lowest noise (2 x 10'5 AU peak to
peak) and drift (1 x 10'4 AU/hr.) specifications avail-
able today. Highly efficient dual beam optical system
and state-of-the-art electronics make these specifi-
pressure (1000 psi) fluid cell is a standard feature in
the spectroMonitor D. The competition makes you
pay extra for high efficiency. In addition, the max N
series fluid celt has optimized flow dynamics and a
true 10mm pathlength to maximize sensitivity ac-
cording to Beer's Law. Others have tried to beat our
noise and drift specifications by cutting cell path-
length, but in the process, they decreased sensitivity.
With the spectroMonitor D you get it ail low noise, -

cations possible. low drift and high sensitivity.

UV/VIS MEASUREMENTS
The spectroMonitor D also features absorbance
measurements over the entire UV/VIS spectrum (190-
700nm) without a tamp change. One 35-Watt deute-
rium lamp is all you need to buy or replace.
2.64
DIGITAL DIAGNOSTICS
3.34
New LED diagnostic circuitry, introduced in the
spectroMonitor D, provides you with a switch select-
0.001 AUFS able digital display of absorbance units, reference and
TIME PICO GRAM NAME TIME PICO GRAM
sample energy in mV, lamp current in mA, and zero
2.64 31 BIPHENYL 2.64 3110
offset.
3.34 11 ANTHRACENE 3.34 1138
4.15 26 PYRENE 4.14 2673 A TRADITION OF QUALITY AND PER FORMANCE
SPECTROMONITOR D SPHEHISOHB ODS COLUMN LDC/Milton Roy pioneered HPLC technology
5 UM 4£ 250 MM
'
WAVELENGTH 241 NM over 3 decade ago. We have put all of our techno-
RISE TIME 1.0 SEC ELUENT: 80% CH3CN 20%H2C
RANGE 0.001 A.U.F.S. 20 UL INJECTION logical experience and knowledge into making an
instrument that is worth much more than its $5890.00
Chromatogram illustrating detection limits with the price. Let us show how the spectroMonitor D can
spectroMonitor D, work for you.

For further information call our Technical

EH
V n
Representatives at 800-327-6182.
U.S. HEADQUARTERS. P,0. Bo* 10235, Riviera Beach, FL 33404 Toll-free: 800-327-6182, In Florida: (305) 844-5241 International Telex: 441098, Domestic Telex: 513479
SUNNYVALE OPERATION, 560 Oakmeed Parkway, Sunnyvale, CA 94086 Toll-free: 800-538-1595, in California: (408) 736-0300
UNITED KINGDOM, LDC UK, Milton Roy House, 52 High Street, Stone, Staffordshire ST15 8AR England Phone: 785-81-3542 Telex: 36623
WEST GERMANY, Milton Roy (Deutschland) GMBH Jahnstrasse 22-24 6467 Hasselroth 2. West Germany Phone: 6055-2031 (3366) Telex; 4184357
JAPAN, Milton Roy K.K., Takanawa Dai-lchi Building 21-41, Takanawa 2-Chrome. Minato-Ku, Tokyo 108, Japan Phone: (03) 473-3181 Telex: 242-4296

CIRCLE 128 ON READER SERVICE CARD

ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983 • 1175 A


Electrochemical and
Spectrochemical
Studies of Biological
Redox Components
Advances in Chemistry Series
No. 201
Karl M. Kadish, Editor
University of Houston
Based on a symposium sponsored
by the ACS Division of Analytical
Chemistry of the American Chemical
Society
This in-depth treatment of biological
redox components brings together the
research of renowned scientists from
diverse backgrounds who work on
closely related topics. Both lechnique-
and problem-oriented researchers
contribute to the 29 chapters, which
discuss such subjects as electron
transfer in porphyrins, heme proteins,
cyctochrome oxidase and copper
proteins. The presentations include
reviews as well as reports of original
research not published before. This
volume is useful to both students and
active researchers in the field.
CONTENTS
Thin-Layer Electrochemical Techniques *
Heterogeneous Electron Transfer Kinetic
Parameters • Temperature Dependence of
Reduction Potentials • Surface Enhanced Raman
Spectroscopy • Redox Potentials in Mononuclear
and Dinuclear Copper Cryptates • Binuclear
Copper (I) Complexes as Mimics of Protein Active
Sites • Heterogeneous Electron Transfer
Properties of Cytochrome c • Rapid Electron
Transfer Reactions of Metalloproteins *
Cytochrome C3 of Desulfovibrio vulgaris • Electrode
Reactions of Protein Prosthetic Groups • Energy
Levels of Metallophthalocyanines • Iron and Coball
Porphodimethenes • Redox Tuning of Iron
Porphyrins • Protoporphyrin IX Compared to
Synthetic Models * Iron (I) and Iron (IV) Porphyrins
•
Magnetic Resonance of Oxidized
Metalloporphynns * Models of Cytochromes b 6 *
Aliphatic Thioethers as Models for Biological
Electron Transfer • Reactions of Chlorpromazine
Cation Radical with Physiological Nucleophiles •
Reduced Pterin Cofactors • Spectral and Redox
Characteristics of Chlorophyll Radicals *
Photosynthesis and Electron Transfer in
Bioenergetic Membranes • Enzymatic and
Electrochemical Reduction of Dioxygen •
Superoxide Ion in Aprotic Media • Electrocatalytic
Reduction of Molecular Oxygen • Cytochrome
Oxidase Study * Utility of Proton Hyperfine Shifts •
3,5-Di-rerf-butylcatecholato and o-Semiquinonato
Complexes • Caialytically Active Molybdenum; —
Catechol Complexes
752 pages (1982) Clothbound
US & Canada $89.95 Export $107.95
LC 82-11487 ISBN 0-8412-0661-9
Order from:
Distribution Office 23
—
American Chemical Society
1155 Sixteenth Street, N.W.
Washington, D.C. 20036
or CALL TOLL FREE 800-424-6747
and use your credit card.
1176 A • ANALYTICAL CHEMISTRY, VOL. 55, NO, 12, OCTOBER 1983
ration into the prediction (analytical)
equation, 2) assigning empirical coeffi-
cients, and 3) testing the applicability
(robustness) to the range of samples.
Even though the absorption bands are
broad, for quantitation, wavelength
reproduction is essential. Also, for in-
struments using composited reflec-
tance term calculations, the incre-
ments between close pairs or trios of
wavelengths are a part of the calibra-
tion and must not vary.
In practice, if available, spectro-
scopic information on the analyte and
other major constituents of the sample
is used along with the multivariate
statistical methods to choose the
wavelengths to be used. Prior work
with the same analyte in similar ma-
trices can serve as a starting point in
this selection. However, there are also
cases where the chemical basis of a
sample’s important characteristics is
not defined but that the near-infrared
reflectance analyzer can still be used.
For such cases, we must rely entirely
upon correlation transformation sta-
tistics. Other calculation methods
have included least-squares curve fit-
ting, principle component, latent vari-
ables, and row reduction (9-13).
Role of analytical chemists in NIRA
Since correlation transformation
may uncover a useful relationship that
may not be obvious from known theo-
ry, it may be necessary to put aside
one’s pride and try this approach.
Here is where contributions can be
made. Good solid analytical sense and
methodology are also needed to obtain
highly reliable and appropriate refer-
ence data from which specific NIRA
methods emerge for rapid and routine
application. The analytical reference
method developed for this purpose
need not be rapid and practical but
just accurate. This is a departure from
the usual criteria and motivation for
analytical method development. One
of the most extreme examples of such
an approach is the use of solid state
carbon 13 NMR data by Barton and
coworkers (14) to obtain structural in-
formation concerning natural prod-
ucts. Near-infrared reflectance data
correlated to the quantitative expres-
sion of structure allows subsequent
rapid analyses of large numbers of
similar natural product samples, ap-
propriate quality assessment, and
sorting for particular end use. As
problem solvers we need every avail-
able tool and cannot afford to over-
look a potentially valuable one such as
NIRA. Greater chemical input and in-
sight will undoubtedly aid in the rec-
ognition of its legitimacy.
References
(1) Fuller, M. P.; Griffiths, P. R. Anal.
Chem. 1978,50, 1906.
(2) Kaye, W. Spectrochim. Acta 1954,6,
257.
(3) Kaye, W. Spectrochim. Acta 1955,1,
181.
(4) Whetsel, K. B. Appl. Spectrosc. Rev.
1968,2 (1), 1.
(5) Ben-Gera, I.; Norris, K. H. Israel J.
Agr. Res. 1968,18, 125.
(6) Wetzel, D. L.; Mark, H. “Scanning NIR
of Grains, Oilseeds, and Their Compo-
nents”; ICC Symposium: Use of Near-
Infrared Techniques, 6th World Bread
Congress, Winnipeg, 1978; Paper No.
S2.1.
(7) Wetzel, D. L. “Particle Size as a Vari-
able in Near-Infrared Reflectance Analy-
sis,” American Association of Cereal
Chemists, 62nd Annual Meeting, San
Francisco, Calif., 1977; Paper No. 44.
(8) Stark, E. W. “State of the Art of NIRA
Instruments”; Research Conference on
Diffuse Reflectance Spectroscopy,
Chambersberg, Pa., August 1982.
(9) Hamid, A.; McClure, W. F.; Whitaker,
T. B. Am. Lab. 1981,13 (3), 108.
(10) Hruschka, W. R.; Norris, K. H. Appl.
Spectra. 1982 36 (3), 261.
(11) Hruschka, W. R.; Martens, H. “Prin-
cipal Component Analysis Predicts Pro-
tein and Moisture Content from Near In-
frared Spectra of Ground Wheat,” Pitts-
burgh Conference on Analytical Chemis-
try and Applied Spectroscopy, Atlantic
City, N.J., 1982; Paper No. 375.
(12) Martens, H.; Jensen, S. A. “Proceed-
ings of the 7th World Cereal and Bread
Congress,” Prague Czechoslovakia, 1982,
Paper No. S.45; Elsevier: Amsterdam, in
press.
(13) Honigs, D. E.; Hieftje, G. M.; Hirsch-
feld, T. “Near Infrared Reflectance
Analysis (NIRA) Correlation Methods
and Performance," Pittsburgh Confer-
ence on Analytical Chemistry and Ap-
plied Spectroscopy, Atlantic City, N.J.
1982; Paper No. 380.
(14) Barton, F. E., Ill; Akin, D. E., Him-
mel9bach, D. S.; Windham, W. R. “Ab-
stracts of Papers”; 186th National Meet-
ing of the American Chemical Society,
Washington, D.C., Aug. 28-Sept. 2,1983;
American Chemical Society: Washing-
ton, D.C., 1983; CELL 11.
David L. Wetzel, professor at Kansas
State University, joined the grain
science department there in 1973 to
develop analytical methods for ce-
real-based foods and the grain pro-
cessing industry. His research inter-
ests include HPLC, laser diffractome-
try, and NIRA. His PhD graduate
work was in analytical chemistry, and
he held a number of teaching and re-
search positions prior to joining
KSU. He currently writes a column
on instrumentation for Cereal Foods
World.