Reference:
Fiber (Acid Detergent) and Protein (Crude) in Animal Feed and
Forages: Near-infrared Reflectance Spectroscopic Method. (989.03)
Official Methods of Analysis. 1990. Association of Official
Analytical Chemists. 15th Edition.
Martin, G.C., J.S. Shenk, and F.E. Barton II. 1989. Near Infrared
Reflectance Spectroscopy (NIRS): Analysis of Forage Quality.
United States Department of Agriculture, Agricultural Research
Service. Agricultural Handbook No. 643.
Scope:
This procedure is applicable for determining acid detergent fiber
of ground, air-dry or partially dried (90 to 95% dry matter)
forage. Samples must be ground through cyclone grinder with 1 mm
screen and be 90 to 95% dry matter.
Basic Principle:
Random portions of a sample are loaded into an NIR sample holder
and reflected light from the ground sample is measured in the
infrared region (generally 1100 to 2500 nm). Instrument is part
of a system that has been calibrated using representative samples
from population to be tested. Equations selected based on
calibration statistics, which have been validated, are used to
calculate acid detergent fiber content of feed and forage
samples.
Equipment:
Near infrared reflectance spectrophotometer, wavelength range at
least 1100 to 2500 nm Sample holders with infrared transmitting
quartz window Computer with software for collecting, storing and
processing spectra
Reagents:
None.
Safety Precautions:
* Follow manufacturer's recommendation for safe operation of
instrument.
Procedure:
- Prepare samples by the same method as the calibration
samples were prepared.
- For best results run instrument (but not lamp)
continuously. If instrument is cold, warm-up time should
be 1 hr.
- Clean sample holder with a camel hair brush or vacuum.
Additional cleaning may be done with soft tissue or
lint-free cloth. Glass should be free of finger prints
and foreign material.
- Load NIR sample cup placing one scoop of forage
(previously dried to 90-95% dry matter or greater, ground
with cyclone mill to pass 1 mm screen, thoroughly mixed)
on each third of the glass surface to ensure that
portions of different subsamples are scanned. Overfill
the sample holder and scrape off any excess.
- Press back into holder so that sample is firmly pressed
against window.
- If any abnormality appears in window, remove back and
repeat process. Consistency in sample handling,
preparation, and cell packing is crucial to success.
- Scan sample, collect spectra, and process data.
Comments:
- NIR instrument should be maintained in a dust-free
environment. Clean or change filter monthly. Clean
ceramic standard and drawer assembly monthly.
- Never touch or clean grating.
- NIR instrument should be maintained in a
stable-temperature (25 ±3oC) room with controlled
humidity (60 ±5%). Room should be free from vibration
and have stable and dedicated electrical current.
- Sample must be dried and ground by the same methods as
those samples used to develop the calibration equation.
- Spectrophotometer reads only a fraction of 1 mm depth of
material touching glass of sample container. Therefore
container must be loaded carefully with different
subsample in each quadrant to make spectral reading more
representative of entire sample.
Calculations:
Prediction is made by direct comparison to calibration. No
additional calculations. If calibrations were made using
reference values on a dry matter basis, NIR results will be
expressed on a dry matter basis.
Quality Control:
Include at least one set of duplicates in each run. These
duplicates must be two subsamples each packed in a separate
holder. Scanning the same sample twice is not a true replicated
analysis using NIR. An acceptable average standard deviation
among replicated analyses for acid detergent fiber using
reference methods ranges from about ±0.20 for samples with 20%
ADF to ±0.35 for samples with 40% ADF, which results in warning
limits (2s) ranging from ±0.40 to 0.70 and control limits (3s)
ranging from ±0.60 to 1.05. Quality control for NIR analysis
involves monitoring the accuracy of both the instrument and the
calibration equation. Instrument diagnostics should be run and
recorded weekly, after instrument warm-up, to insure that maximum
response, wavelength accuracy and repeatability are within
acceptable manufacturer's tolerances. For example, for NIRSystems
model 6250 and 6500 instruments, maximum response should be
between 55000 and below 51000), suggested wavelength error should
be less than0.5 times currently observed error (restandardize if
observed error is too large) and root mean square corrected
(RMSC) repeatability of multiple scans (32 revolution and 30
scans) should be less than 20 to 30 (correct problems if
greater). Each manufacturer should provide acceptable performance
specifications for their instrument, and often software is
provided to monitor instrument accuracy. However, it is the
responsibility of the operator to run the diagnostic software
routinely and record the results a minimum of once a week. In
addition to meeting specifications, instrument operation should
be monitored by scanning a check sample (that has been sealed in
a sample holder) each day and storing spectral data weekly. Daily
results for each analysis (DM, CP, ADF, NDF, and minerals) should
be plotted on an X-control chart and the chart should be examined
for trends. Standard deviation (s) for the check sample can be
established after 10 scans and should be substantially lower than
acceptable standard deviations of duplicate reference method
analyses (.10, .20, .35 and .60 for DM, CP, ADF, and NDF,
respectively) because the same sample is being scanned. Results
outside of ±2s upper and lower warning limits are evidence of
problems with the analytical system. Results outside of ±3s
upper and lower control limits are evidence of loss of control
and no NIR analyses should be done without detecting and
correcting the problem. Two consecutive analyses falling on one
side of the mean between warning limits and control limits also
indicate a loss of control.
Monitoring the calibration equation consists of two tests that
determine the existence of bias and unacceptable increases in
standard error of prediction corrected for bias [SEP(C)]. Every
20 to 25th sample predicted by the NIR should be analyzed by the
reference method used to develop the calibration equation (Note:
Any bias or increased SEP(C) can be due to inaccuracies in NIR or
differences in reference method analytical procedure). A
continuous time chart of observed bias and SEP(C) should be
plotted to observe trends. After nine (N) samples have been
accumulated, analyze these samples by the reference method (A
sample size of nine is a good compromise between the number of
analyses required and the statistical accuracy desired for the
confidence limits given below). Calculate the observed bias and
SEP(C) using the equations given below. Determine the confidence
limits for bias and SEP(C) based on the standard error of the
calibration equation (SEC). If the SEP(C) of the nine samples is
less than the SEP(C) limit and absolute value of the bias is less
than the bias limit, the calibration equation is acceptable. If
the SEP(C) of the nine samples is less than the SEP(C) limit but
the absolute value of the bias exceeds the bias limit, the
calibration equation may be corrected by adding the bias to the
intercept of the calibration equation or to each value (bias
adjustment) although recalibration is recommended. If both SEP(C)
and bias exceed their limits, add samples to the calibration data
set and recalibrate.
NIR Quality Control Calculations:
Di = Xi - Yi
Bias = Di / N
Bias Confidence Limit = ±0.55 X (SEC)
SEP(C) Confidence Limit = 1.29 X (SEC)
- Di = difference
- Xi = reference method value
- Yi = NIR value for ith sample
- N = 9 (number of samples)
- Bias = average difference between reference and NIR
values
- SEP(C) = standard error for prediction corrected for bias
- SEC = standard error of the calibration equation.
This monitoring procedure can be used with any NIR instrument
and some manufacturers have incorporated this approach into their
quality control monitoring software.
|