METHODOLOGY
A. MOISTURE
Principle:
The loss in weight of the feed sample after oven drying could be accounted to
volatization of water. This is the moisture content of the sample.
Procedure:
1. Place crucible(s) in the oven and dry to constant weight (overnight at 105ºC or at
least 2 hours at 135ºC).
2. Take out crucible(s) from the oven and place immediately in dessicator.(a) Let it
cool for 20-30 minutes depending on the number of crucibles.(b)
3. Weigh cooled crucible(a)
4. Add 0.5 - 1.0 gm sample(c)
S. Place weighed sample in the oven at 105ºC overnight or at least 2 hours at 135ºC.
6. mace dried sample in dessicator, allow to cool (20 to 30 minutes) and weigh. (a)
wt.before drying − wt.after drying
% moisture = 𝑥100
𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡
% dry matter = 100 - % moisture
B. ASH
Principle:
Igniting sample in the furnace burns off all organic matter to carbon dioxide and
water. The residue (ash) represents the inorganic constituent of the sample.
Procedure:
1. Weigh 0.5 - 1.0 g sample or use the dried sample from A6 and place in a muffle
furnace.(d)
2. Ignite to 550ºC for about least 4 hours.
3. Let it cool for at least 2 hours.
4. Place the ignited sample in a dessicator.
5. Cool to room temperature and weigh.(a)
wt.of crucible+ash − wt.of crucible
% ash = 𝑥100
𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡
C. CRUDE PROTEIN
Principle:
The sample is digested with concentrated H2SO4 in the presence of a catalyst (salt
mixture). Upon oxidation of the carbonaceous material, the total nitrogen of the sample is
converted to (NH4)2 SO4. Addition of NaOH to the resulting solution liberates ammonia
(NH3) which is passed into a solution of boric acid. Amount of NH3 liberated is
determined by titration with standard acid.
Reagents:
H2SO4, concentrated
Salt mixture [0.5 part anhydrous CuSO4 and 10.0 parts anhydrous Na2SO4 or
K2SO4]. Mix 5 gm of anhydrous CuSO4 to 100 gms of anhydrous Na2SO4
or K2SO4.
45% NaOH solution. Dissolve 450 gm of NaOH in 1 liter of distilled water.
2% Boric Acid. Dissolve 20 gm boric acid in 500 ml distilled water. Dilute to 1
liter with distilled water.
Methyl red - bromcresol green indicator. Mix 1 part 0.2% alcoholic solution of
methyl red and 5 parts 0.2% alcoholic solution of bromcresol green.
a. 0.2% Methyl red - Dissolve 0.2 gm methyl red powder to 100 ml
ethanol.
b. 0.2% Bromcresol green - Dissolve 0.2 gm bromcresol green
powder to 100 ml ethanol.
2% methyl red - dissolve 2 gm methyl red powder to 100 ml ethanol.
0.1 N Standard HCl. Dilute 8.9 ml HCl to 1 liter of distilled water.
Standardization Procedure for 0.1 N HCl:
1. Weigh 0.1 g Tris[hydroxymethyl]amino methane(e) and dissolve in 75 ml distilled
water.
2. Add 3 drops of 2% Methyl red
3. Titrate the solution with the prepared 0.1 N HCl until the orange endpoint is
reached.
Calculations:
wt. of Tris[hydroxymethyl]amino methane
Normality of HCl = 𝑚𝑙 𝐻𝐶𝑙 𝑥 𝑚𝑒𝑞.𝑜𝑓 Tris[hydroxymethyl]amino methane
where:
meq Tris[hydroxymethyl]amino methane = 121.10 / 1000
Procedure:
1. Digestion
a. Weigh 0.10 - 0.30 gm sample and wrap in a small pj«e of filter paper.
b. Place weighed sample in a 300 ml kjeldahl flask.(f)
c. Add approximately 0.5 gm salt mixture and 5 ml concentrated H2SO4.
d. Digest for about 45 to 60 minutes or until the solution clears to a bluish or
colorless solution.
e. Cool.
2. Distillation
a. Immerse the end of the condenser (glass tubing) in a receiver (125 ml
Erlenmeyer flask) containing 10 ml 2% boric acid and 2 drops indicator.
b. Add approximately 125 to 150 ml distilled water to Kjeldahl flask (le).
Swirl gently to dissolve digested sample.
c. Add 30 ml 45% NaOH (Do not shake) and immediately. Connect flask to
spray trap (Kjeldahl bulb).
d. Swirl the flask gently to mix the solution thoroughly.
e. Collect enough distillate (75 ml).
f. Run a blank, using filter paper and the same amount of reagents to correct
for any nitrogen present.
3. Titration
a. Titrate the distillate with standard 0.1 N HCl until the end-point is
reached. The color of the distillate changes from blue to light yellow or
light brown.
b. Calculate the % CP using the following formula:
(𝑆𝑎𝑚𝑝𝑙𝑒 𝑡𝑖𝑡𝑟𝑎𝑡𝑖𝑜𝑛−𝐵𝑙𝑎𝑛𝑘 𝑡𝑖𝑡𝑟𝑎𝑡𝑖𝑜𝑛) 𝑥 𝑚𝑒𝑞.𝑁 𝑥 0.1 𝑁 𝐻𝐶𝑙
%N= x100
𝑆𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔𝑚)
where:
meq. N = 14 / 1000 = 0.14
% CP = % N x 6.25 (The factor will vary depending on the kind of sample but
6.25 is generally used) .
D. Crude Fiber
Principle:
This method simulates the digestion in the stomach and small intestine. Crude
fiber id determined by digestion of sample with .255 NH2SO4 to hydrolyze the
carbohydrates, protein materials and some minerals. This is followed by boiling with
0.313 N NaOH solutions to saponify lipids. The undigested portion or residues of the
sample is ignited at 650ºC. The lost in weight after ignition is the crude fiber.
Reagents:
0.255 N H2SO4. Add 7.1 ml H2SO4 to 1liter distilled water
0.313 N NaOH. Dissolve 12.5 g NaOH to 1liter distilled water
Asbestos — Gooch grade, medium fiber, acid-washed, and ignited is usually
satisfactory but should be tested for chemical stability and filtering speed
before use.
Procedure:
1. Weigh 0.1- 0.3 gm sample(g) and place in a 600 ml Berzelius beaker.
2. Add 200 ml 0.255 N H2SO4 and boil for 30 minutes While boiling swirl beaker
frequently to prevent samples from adhering on the sides of the beaker.
3. Filter using filtering cloth in fluted runnel or fitted glass (coarse porosity) and
wash the residue with distilled water 3 times or until washings are no longer
acidic.
4. Wash residue back to beaker with 200 ml 0.313 N NaOH.
5. Reflux or boil for 30 minutes.
6. Filter through gooch crucible (h) and thoroughly wash residue with distilled water.
7. Dry gooch crucible in the oven at 105ºC overnight or at 135ºC for 2 hours.
8. Cool in a dessicator for 20-30 minutes and weigh.
9. Ignite in muffle furnace at 550ºC for 4 hours. Cool to room temperature. Weigh.
𝑤𝑡.𝑎𝑓𝑡𝑒𝑟 𝑑𝑟𝑦𝑖𝑛𝑔−𝑤𝑡. 𝑎𝑓𝑡𝑒𝑟 𝑖𝑔𝑛𝑖𝑡𝑖𝑜𝑛
% Fat = 𝑥100
𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔𝑚)
E. Crude Fat or Ether Extract
Principle:
Fats/oils are soluble in ether. The loss in weight after fat extraction is crude fat.
Reagent:
Petroleun ether (B.P. 30-60ºC)
Procedure:
1. Weigh 0.3 - 0.5 gm samples in triplicate.
2. Wrap individually in a small piece of filter paper.
3. Dry to constant weight in the oven (105ºC overnight or at 135ºC for at least 2
hours).
4. Weigh hot dried sample one at a time
5. Place samples in the Soxhlet extractor and extract fat continuously with petroleum
ether for 16 hours
6. Remove samples from extractor and place in watch glass/petri dish to evaporate
solvent.
7. Dry to constant weight at 105ºC overnight or 135ºC for at least 2 hours/
8. Weigh while the sample is hot.
𝑤𝑡. 𝑏𝑒𝑓𝑜𝑟𝑒 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛−𝑤𝑡. 𝑎𝑓𝑡𝑒𝑟 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛
% E.E. = 𝑥100
𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔𝑚)
F. Nitrogen-Free-Extract (NFE)
Includes all soluble carbohydrates and obtained summing up the values for
moisture, crude protein, crude fiber, ether extract and ash and subtracting
the total from 100.
% NFE = 100 – ( % H2O + % CP + % CF + % Crude Fat + % Ash)
NOTE:
a. Do not touch the crucibles. Use tongs instead.
b. Allow 2 min. cooling time per crucible.
c. Add sample while crucible is inside the balance.
d. You may use sample from moisture analysis.
e. Dried at 105ºC for 2 hours.
f. Sample + filter paper.
g. For samples containing crude fat > 4 %, defat sample first.
h. Gooch crucibles need not to be tared.
