6. Acid Detergent Insoluble Nitrogen and Acid Detergent Fiber Crude Protein
References:
Goering, H.K. and P.J. Van Soest. 1970. Forage fiber analysis
(apparatus, reagents, procedures, and some applications). USDA
Agricultural Research Service. Agriculture Handbook No. 379.
Goering, H.K., C.H. Gordon, R.W. Hemken, D.R. Waldo, P.J. Van
Soest, and L.W. Smith. 1972. Analytical estimates of nitrogen
digestibility in heat damaged forages. Journal of Dairy Science.
55(9): 1275-1280.
Scope:
This method is applicable for the determination of acid detergent
insoluble nitrogen in all forages.
Basic Principle:
Acid detergent insoluble nitrogen (ADIN) is the nitrogen
remaining in the acid detergent fiber residue and, while some
occurs naturally in all plant material, is generally considered
to be an estimate of heat damage occurring during storage or
processing. Nitrogen in excessively heated samples is usually
unavailable to animals. ADIN is determined as the nitrogen in ADF
residue. The two options used to determine ADIN differ in the
amount of the ADF residue that is analyzed for nitrogen. If the
total ADF residue is collected on filter paper and analyzed for
nitrogen, ADIN (DM basis) is determined by measuring the nitrogen
(corrected for a filter paper blank) in the total ADF residue and
dividing by the original dry sample weight. It is difficult, if
not impossible to collect all ADF residue from fritted glass
(Gooch) crucibles, therefore only a subsample of the total ADF
residue is analyzed for nitrogen. When only a part of the ADF
residue is analyzed, by sampling the ADF residue from a fritted
glass crucible (or from filter paper), the nitrogen content of
the ADF residue must be determined by dividing the nitrogen in
the ADF sample by the ADF sample weight. Then ADIN (DM basis) is
calculated by multiplying the nitrogen content of ADF by the ADF
content in the dry matter. When sampling ADF residues from
fritted glass crucibles, be careful not to scrape glass particles
into the partial ADF residue that is analyzed for nitrogen.
Acid detergent fiber crude protein (ADFCP) is ADIN expressed
as crude protein on a dry matter basis.
Equipment:
See method 3.1,
3.2, or
3.3
for equipment for appropriate nitrogen determination method
See method 4.1 for equipment
for acid detergent fiber procedure Filter paper, acid hardened,
Whatman 54 or equivalent
Reagents:
See method 3.1,
3.2, or
3.3
for reagents for appropriate nitrogen determination method
See method 4.1 for reagents
for acid detergent fiber procedure.
Safety Precautions:
See method 3.1,
3.2, or
3.3
for safety precautions for appropriate nitrogen determination
method
See method 4.1 for safety
precautions for acid detergent fiber procedure.
Procedure:
Option A: Determination of ADIN using total ADF residue (filter
paper)
- Dry 6 papers overnight at 100oC to determine average
filter paper DM. Weigh filter papers to be used to
collect ADF residues to nearest 0.1 mg (W1).
- Thoroughly mix and weigh sample (W2) (approximately
0.9-1.1 g, record weight accurate to 0.1 mg) into
Berzelius beaker. Weigh a second subsample for laboratory
dry mattter determination.
- Add 100 mL acid-detergent solution at room temperature.
Place beaker on heater under the cold water condenser.
- Heat to boiling in 5-10 min; reduce heat to avoid foaming
as boiling begins. Reflux 60 min from onset of boil,
adjusting boiling to slow, even level.
- After about 30 min, wash down sides of beaker with
minimal amount of acid detergent solution. A wash bottle
is convenient for dispensing solution.
- Remove beaker, swirl, and filter onto filter papers using
Buchner funnels with a retainer ring to hold the filter
papers in place during filtering. Use minimal vacuum.
Include 2 blanks (filter paper washed with ADF solution)
to estimate nitrogen in paper and reagents.
- Police and rinse the Berzelius beaker with boiling water
while inverted over the funnel to insure quantitative
transfer of all fiber particles into the filter paper.
- Soak twice with boiling (90-100oC) water by breaking up
mat and filling funnel each time with vacuum off and
allowing to soak a minimum of 15 to 30 sec (2 min
recommended). While filling the Buchner funnel with hot
water or acetone, rinse the top edge and sides to remove
residual acid detergent.
- Rinse twice with 30 to 40 mL acetone, filling funnel each
time with vacuum off, allowing a minimum of 15 to 30 sec
(2 min recommended) before vacuuming dry.
- Fold filter paper to retain sample, dry 3 hr in
forced-air oven (100oC) and weigh hot, recording weight
(W3) to nearest 0.1 mg.
- Insert filter paper and sample into Kjeldahl flasks, add
5 mL additional acid to digest the filter paper and
determine nitrogen by method 3.1
or 3.2.
Calculation: Percent Acid Detergent Insoluble Nitrogen
(ADIN), DM basis using total ADF residue (filter paper)
When determining nitrogen using colorimetric method:
%ADIN (DM basis) = [(mgN in ADF+filter paper
/250 mL) - (mgN in blank filter paper/250 mL)] X 100 W2 X 1000 X
Lab DM / 100
- W2 = sample weight in grams
- 1000 = conversion factor for grams to mg
When determining nitrogen using standard sodium hydroxide
titrant:
%ADIN (DM basis) =[(VHCl x NHCl) - (B x NNaOH)
- (VNaOH x NNaOH)] x 1.4007 / W2 X Lab DM / 100
- VNaOH=mL standard NaOH to titrate ADF + filter paper
- VHCl = mL standard HCl pipetted into titrating flask
- NNaOH = Normality of NaOH
- NHCl = Normality of HCl
- B = mL standard NaOH needed to titrate 1 mL standard HCl
minus VBK
- VBK = mL standard NaOH needed to titrate filter paper
blank carried through method and distilled into 1 mL
standard HCl
- 1.4007 = milliequivalent weight of nitrogen X 100
- W2 = weight of sample in grams
When determining nitrogen using boric acid trapping
solution/standard HCl titrant:
% ADIN (DM basis) =(VA - VB) x NHCl x 1.4007 /
W2 x Lab DM / 100
- VA = Volume, in mL, of standard HCl required for ADF
residue + filter paper
- VB = Volume, in mL, of standard HCl required for filter
paper blank
- NHCl = Normality of standard HCl
- 1.4007 = milliequivalent weight of N X 100
- W2 = sample weight in grams
Calculation: Acid Detergent Insoluble Nitrogen (as
percent of total nitrogen). Also called ADIN to N ratio.
% ADIN (of Total N) = % ADIN (DM basis) X 100%
N (DM basis)
- %ADIN = ADIN (% of Dry matter) calculated above
- %N = % nitrogen of original sample
Calculation: Percent Acid Detergent Fiber Crude Protein
(ADFCP), DM basis
% ADFCP (DM basis) = %ADIN (DM basis) X 6.25
- %ADIN = ADIN (% of Dry matter) calculated above
Option B: Determination of ADIN using partial ADF
residue (from fritted glass crucibles)
- Samples should be microwave dried or oven dried at 55oC
to ³85% dry matter, then ground to pass a 1 mm screen.
- Dry 50 mL fritted glass crucibles overnight at 100oC and
hot weigh (W1), recording weight to nearest 0.1 mg. (Hot
weigh techniques described in method 2.2.2.2.)
- Thoroughly mix and weigh sample (W2) (approximately 0.9
to 1.1 g, record weight accurate to 0.1 mg) into
Berzelius beaker. Weigh a second subsample for laboratory
dry matter determination.
- Add 100 mL acid-detergent solution at room temperature.
Place beaker on heater under the cold water condenser.
- Heat to boiling in 5-10 min; reduce heat to avoid foaming
as boiling begins. Reflux 60 min from onset of boil,
adjusting boiling to slow, even level.
- After about 30 min, wash down sides of beaker with
minimal amount of acid detergent solution. A wash bottle
is convenient for dispensing solution.
- Remove beaker, swirl, and filter through tared (step 2)
fritted glass crucible, using minimal vacuum. Police and
rinse the Berzelius beaker with boiling water while
inverted over the crucible to insure quantitative
transfer of all fiber particles into the crucible.
- Soak twice with boiling (95-100oC) water by breaking up
mat and filling crucible each time with vacuum off and
allowing to soak a minimum of 15 to 30 sec (2 min
recommended) after each wash. While filling the crucible
with hot water or acetone, rinse the top edge and sides
to remove residual acid detergent.
- Rinse twice with 30-40 mL acetone by filling crucible
each time with vacuum off, allowing a minimum of 15 to 30
sec (2 min recommended) before vacuuming dry.
- Dry 3 hr or overnight in forced-air oven (100oC) and
weigh hot, recording weight (W3) to nearest 0.1 mg.
- Sample a portion of the ADF residue from fritted glass
crucible using a teflon or plastic policeman into
Kjeldahl flasks. Do not scrape so hard as to dislodge
glass from the fritted disk.
- Weigh partial ADF residue (W8), recording weight to
nearest 0.1 mg.
- Determine nitrogen content of the ADF residue subsample
using the Kjeldahl (method 3.1
or 3.2) or combustion
methods (method 3.3).
Calculation: Percent Acid Detergent Insoluble Nitrogen
(ADIN), DM basis using partial ADF residues (from fritted glass
crucibles or filter paper)
ADIN (DM basis) = %Ns X %ADF (DM basis) / 100
- %NS = percent nitrogen of ADF residue (using W8)
- W8 = weight of ADF residue used for nitrogen analysis in
grams
- %ADF (DM basis) = percent ADF calculated using W1 to W3
(Method 4.1)
Calculation: Acid Detergent Insoluble Nitrogen (as
percent of total nitrogen). Also called ADIN to N ratio.
% ADIN (of Total N) = % ADIN (DM basis) X 100
% N (DM basis)
- %ADIN = ADIN (% of Dry matter) calculated above
- %N = % nitrogen of original sample
Calculation: Percent Acid Detergent Fiber Crude Protein
(ADFCP), DM basis
% ADFCP (DM basis) = %ADIN (DM basis) X 6.25
- %ADIN = ADIN (% of Dry matter) calculated above
Quality Control:
Include a filter paper blank and one or more quality control (QC)
samples in each run, choosing QC samples by matching analyte
levels and matrices of QC samples to the samples in the run.
Include at least one set of duplicates in each run if single
determinations are being made.
An acceptable average standard deviation among replicated
analyses for acid detergent insoluble nitrogen (expressed as % of
total N) ranges from about ±0.22 to ±0.40, which results in
warning limits (2s) ranging from ±0.44 to 0.80 and control
limits (3s) ranging from ±0.66 to 1.20. Plot the results of the
control sample(s) on an X-control chart and examine the chart for
trends. Results outside of upper or lower warning limits, ±2s
(95 percent confidence limits), are evidence of possible problems
with the analytical system. Results outside of upper or lower
control limits, ±3s (99 percent confidence limits), indicate
loss of control and results of the run should be discarded. Two
consecutive analyses falling on one side of the mean between the
warning limits and the control limits also indicate loss of
control.
|