SOP No : QS-LS 15Effective Date : 01/08/2010Rev No. 00 Revision
Date : 01/08/2012Doc. Status : Controlled Standard Operating
Procedure (SOP)
Title: Feed Chemistry at Laboratory Services
1. PURPOSETo test proximate analysis of raw material finished
feed, sales feed and incoming samples to monitor and ensure high
quality raw material and feed quality is done at Laboratory
Services department as per end user department requirements.
2. SCOPEProximate analysis (Moisture, crude protein, crude fat,
crude ash, calcium, phosphorus, crude fibre, soluble protein,
nitrogen, physical test, particle size, water durability) of raw
material (as per SOP MMPD-FM-03), finished feed & sales feed
(as per SOP FM-010) and incoming samples to monitor and ensure high
quality raw material and feed quality is done.3. DEFINITIONS The
wordings in this SOP are defined as follows3.1. Analyte: The
chemical substance or physical property being measured in a
sample.3.2. Stock Standard Solution - A concentrated solution
containing one analyte prepared in the laboratory using an assayed
reference compound or purchased from a reputable commercial
source.3.3. Kjeldahl Method: Kjeldahl digestion in analytical
chemistry is a method for the quantitative determination of
nitrogen in chemical substances. 3.4. Soxhlet Method: The method
described by Soxhlet in 1879 is the most commonly used example of a
semi-continuous method applied to extraction of lipids from
foods.3.5. Parameter: A variable, measurable property whose value
is a determinant of the characteristics of a system.4.
REFFERENCES.NoName of analysisMethod of AnalysisReferences
4.1Moisture Oven Drying methodAOAC Official method 930.15AOAC
Official methods of analysis 17th Edn (Chapter 4, p 2, Clause
4.1.06)
4.2Crude Protein Kjeldahl method- FOSS automated analyzer AOAC
Official method 976.05AOAC Official methods of analysis 17th Edn.
(Chapter 4, p 23, Clause 4.2.05)
4.3Crude Fat Soxhlet extraction methodAOAC Official Method
920.39AOAC Official methods of analysis 17th Edn.(2000) (Chapter 4,
p 33, Clause 4.5.01)
4.4Crude Ash Furnace method AOAC Official method 942.05AOAC
Official methods of analysis 17th Edn. (2000) (Chapter 4, p 5,
Clause 4.1.10)
4.5Crude Fiber Acid & Base digestion method AOAC Official
method 962.09 AOAC Official methods of analysis 17th Edn.(2000)
(Chapter 4, p 34, Clause 4.6.01)
4.6Nitrogen Free extract By difference methodFood Energy -
methods of analysis and conversion factors. Food and Agriculture
Association of the United Nations,Feeds and Feeding, Handbook for
the Student and Stockman", 21st Ed., by Frank B. Morrison, The
Morrison Publishing Co., Ithaca, NY, 1950.
4.7Free Fatty Acid Neutral Alcohol solvent method AOCS Official
Method Ca 5a-40
4.8Acid Value Neutral Alcohol solvent method AOCS Official
Method Ca 5a-40
4.9Acetone Insolubility Acetone solvent method
4.10Calcium Gravimetric methodAOAC Official method 927.02AOAC
Official methods of analysis 17th Edn.(2000) (Chapter 4, p 40,
Clause 4.8.03)
4.11Magnesium Complexometric method / spectrophotometricAOAC
Official method 917.04AOAC Official methods of analysis 17
Edn(2000)(Chapter 4, p 43, Clause 4.8.12)
4.12Salt as NaCl Argentometric AOAC Official method 943.01AOAC
Official methods of analysis 17th Edn. (2000) (Chapter 4, p 41,
Clause 4.8.05)
4.13TVBN Kjeldhls MethodOfficial Journals of European
Commission
4.14Urease Activities Indicator method American Soybean
Association
4.15Sieve test Mechanical Sieving Official Method of Analysis of
the Association of Analytical Chemist;17th Ed.2000
4.16Bulk Density By Weight Official Method of Analysis of the
Association of Analytical Chemist;17th Ed.2000
4.17Peroxide Value Iodometric methodAOAC official method
965.33AOCS official method Cd 8-53
4.18Rancidity Iodometric methodAOAC official method 965.33AOCS
official method Cd 8-53
RESPONSIBILITY4.1. Office of the Managing Director Has the sole
responsibility of authorizing the preparation of this Standard
Operating Procedure, the approval of this SOP and subsequent
revision and amendments.4.2. Office of the Manager, Laboratory
Services is responsible for preparation of this SOP, revise /
review based on the needs of NPC and its implementation.4.3. Office
of the General Manager, Quality & Sustainability is responsible
for identifying and recommending the needs of NPC core business
units; review the SOP & recommends to the Office of the General
Manager for any amendments.4.4. Office of the Head, Quality System
Department- is responsible for the inspection of the procedures,
applications for the above said operations and shall coordinate
closely with the Central Laboratory on various issues related to
monitoring of this SOP, may from time to time give recommendations
and suggestions for the improvement of this SOP.4.5. Technical
Committee - Following people shall form the technical committee who
will review and recommend for any amendment to the General Manager
of NPC. 4.5.1. Manager- Laboratory Services4.5.2. Head Quality
System4.5.3. Head Corporate Systems & Certification,
Q&S4.5.4. Head Feed Mill4.5.5. Residential Nutritionist6.
PROCEDURE 6.1.Estimation of moisture
a. EquipmentHot air oven Instant moisture analyzer
b. Procedure
Regulate air oven to 135 + 2 C. Using low, covered glass dishes
weigh about 10 15 g sample into each dish and shake until the
contents are evenly distributed. With covers removed, place dishes
and covers in hot air oven as quickly as possible and dry samples
for 2 hrs. Place covers on dishes and transfer to desiccators and
cool. Take the weight. Calculate loss in weight as % moisture.
Alternatively use instant moisture analyzer calibrated against
samples tested in air oven at 135 + 2 C.
6.2.Estimation of crude protein
a. Reagentsb. Conc. Sulphuric acidi. Kjeltabs or equivalent (3.5
g K2SO4 + 0.4 CuSO4)ii. Alkali40% sodium hydroxide (LR) solution.
Use 400 g NaOH per liter of solution. Commercially available in
concentrations up to 50%. Do not use concentrations above 40% as
this will lead to crystal formation impairing the function of the
pumps.
iii. Receiver solution Boric acid 1% with bromocresol green /
methyl red indicator solution. Dissolve 100 g boric acid in 10-lit
de-ionized water, followed by addition of 100 ml of bromocresol
green solution (100 mg in 100 ml methanol) and 70 ml of methyl red
solution (100 mg in100 ml of methanol). In order to obtain accurate
results the receiver solution is adjusted[footnoteRef:1] so that a
small (0.05-0.15ml) positive blank is obtained when running a blank
sample. [1: Adjustment of the boric acid solution is made by the
following procedure:Transfer 25 ml boric acid solution to a
receiver flask and add 100 ml of distilled water. If the solution
in the flask is still red, titrate with 0.1 M sodium hydroxide
solution until a neutral gray color is obtained. Calculate the
amount of sodium hydroxide solution necessary to adjust the boric
acid solution in the 10 lit flask with the formulaMl 1.0 M alkali =
ml titrant x 40Add the calculated amount of 1.0 M alkali solution
to the boric acid solution. Mix. For boric acid 1%, addition of 30
ml 0.1 M NaOH usually gives a good adjustment.To check proceed as
follows using 25 ml of the boric acid solution; Run a blank. If the
value of this blank is high (0.5 ml of 0.2 M HCl) the boric acid is
incorrectly adjusted. This might create irregular blanks. For
correction add HCl directly into the boric acid tank, mix it
carefully and repeat until a reading of 0.05-0.15 ml HCl is
obtained. If a positive blank is not achieved, add further small
quantities of 1M NaOH and repeat the check until a satisfactory
value is achieved. The addition of alkali is to achieve a positive
blank value. This should, however, be kept between 0.05 0.15 ml
titrant, to obtain good repeatability when testing blanks.]
iv. Titrant solution0.2 M standard HCl. Standardization is done
using the following method
Weigh approximately 10 g of anhydrous sodium carbonate (Na2CO3).
Use mortar to make fine powder. Dry it for 1 hr at 265 C or 2 hrs
at 200 C. After cooling in a desiccator, transfer the sodium
carbonate to a beaker with a tight lid. Store it in a
desiccator.
Dissolve 0.1 g methyl red in 100 ml ethanol.
Weigh approximately 0.4 g of the standard substance, using an
analytical balance; note the weight (W1). Transfer the sodium
carbonate to a receiver flask and add 40 ml of H20 (distilled or
de-ionized). Add 10 drops of the indicator solution. Titrate to
pink. Note the amount in ml used (A1). Boil this solution for a few
minutes. The solution will turn green. Cool rapidly to room
temperature under running water. Continue the titration until the
next pink color change occurs. Note also this volume (A2). Boil the
solution for a few minutes. Cool rapidly to room temperature under
running water. Continue the titration until the next pink color
occurs. Note also this volume (A3).The color change of this
official procedure (AOAC 936.15) may be difficult to see, therefore
a pH meter to a mixed indicator (e.g. 0.1 g methyl red and 0.1 g
bromocresol green in 100 ml ethanol) will make it much easier to
perform.
Calculation18.870 x W1Molarity (M)= ----------------(A1 + A2
+A3)
b. ApparatusDigester DS 20 with exhaust manipulator and Kjeltec
2300 Analyzer
c. Procedure
i. Sample preparationAnalytical sample from the laboratory
sample of the bulk product may involve one or more treatments such
as:
Physical treatments: shaking, stirring, mortaring, riffling,
coning and quartering
Mechanical treatments: grinding, blending, homogenizing or
milling. As a recommendation the particle size should be equal or
less than 1 mm.
ii. Sample size
Actual weight of the sample required is dependant on Nitrogen
content and homogeneity.
Homogeneous samples (excluding water) 0.1 1.0 gNon-homogeneous
samples1.0 3.0 g or moreWater samples (dependant on the N
content)1.0 100 ml
Using a titrant concentration of 0.2 N the analytical sample
should ideally contain 10 100 mg N and the appropriate sample
weight can be calculated as follows.
1000Minimum weight in mg = ------- XWhere x = approximate % N
anticipated
iii. Digestion
During digestion the nitrogen or protein in the sample is
converted to ammonium sulphate according to the formula Salt
K2SO4Protein + H2SO4 -------------(NH2)2 SO4 Catalyst
Prepare a representative sample[footnoteRef:2] and weigh based
on the nitrogen content to 0.1 mg accuracy into a digestion tube.
Add one kjeltab Cu 3.5 (alternatively 3.5 g K2SO4 and 0.4 g CuSO4 x
5 H2O). Carefully add 10 ml of conc. sulphuric acid and gently
shake to wet the sample with the acid. [2: For samples containing
high fat or carbohydrate levels use 15 ml H2SO4.]
Place the samples in the preheated (420 C) digester with the
exhaust manifold on the top and heat shields in place. The water
aspirator must operate at full flow the first five minutes of the
digestion. This is done to evacuate moisture. After five minutes it
is essential to decrease the aspirating effect to a minimum until
the acid fumes are just contained within the exhaust head to
prevent acid losses.
Continue digestion until all samples are clear with a blue /
green solution. This will normally be after 30 60 minutes depending
on the sample type. Remove the rack of he tubes with exhaust still
in place and put in the stand to cool for 10 20 mins. iv.
Distillation
All samples after digestion will form ammonium sulphate
((NH4)2SO4). The distillation principle is to convert ammonium
(NH4) into ammonia (NH3) by using alkali (NaOH) and thereafter
steam distil it into a receiver flask containing boric acid and
titrate with standard acid solution using colorimetric end point
detection.Place the digestion tube in the distillation unit and
close the safety door[footnoteRef:3]. Dispense 50 ml of 40% NaOH in
to the tube[footnoteRef:4]. Then the distillation cycle is
controlled automatically. [3: Dilution water is automatically added
on the Kjeltec 2300 if the option is selected.] [4: In general 4
times the amount of the acid = the amount of alkali to use with the
concentration of 40%. When 20 ml concentrated H2SO4 is used during
digestion the amount of alkali used (40%) should be 20 x 4 = 80 ml
of 40% NaOH. It is very important that alkali is present in excess;
otherwise hydrogen sulphide (H2S) will be formed. Hydrogen sulphide
is a strongly acidic gas that will make the indicator turn red and
no result will be achieved.]
v.Blank determination
Full chemical blanks should be run before each batch of analyses
to compensate for any contribution from the reagents used. Blanks
should be treated identically to samples to be meaningful.
vi.Chemical check Use ammonium sulphate (NH4)2SO4 >
99.5%Molecular weight = 132.14 g/mol.Dry ammonium sulphate for 4
hrs at 102 C, and store in a desiccator.% Nitrogen in ammonium
sulphate (99.5%) = 21.09Weigh 0.15 g Ammonium Sulphate into a
tubeAdd 75 ml distilled water and 50 ml 40% NaOH and distil
(ml blank) x N x 1.401% Nitrogen =
------------------------------ g sample
N = Normality of titrant to 4 places of decimal. Actual %
Nitrogen% Recovery = ------------------------ x 10021.09
Use ammonium iron (II) sulphate (NH4)2 Fe(SO4)2 x 6 H2O
Molecular weight = 392.14 g/mol% Nitrogen in ammonium iron (II)
sulphate = 7.145Weight 0.5 g ammonium iron (II) sulphate into a
tube.Add about 75 ml distilled water, and 50 ml 40% NaOH. (ml
blank) x N x 1.401% Nitrogen = ---------------------------------- g
sample N = Normality of titrant to 4 places of decimal. Actual %
Nitrogen% Recovery = ------------------------ x 100 7.145
Please note that the above calculations must be adjusted if
other purity levels of ammonium salts are used. For a full check of
your chemistry a substance like glycine can be digested and used as
a test sample.d.Calculation of results
The most common ways of reporting Kjeldhal results are as %
Nitrogen, % protein, mg N/liter (ppm), g N/liter and mg N/100 ml.
The calculations are as follows:
(T B) x N x 14.007 x 100%N =
----------------------------------Weight of the sample in mg%
Protein = % N x F
(T B) x N x 14.007 x 1000mg N / liter
=-----------------------------------Volume of the sample in ml
(T B) x N x 14.007g N/liter =
------------------------------------- Volume of the sample in
ml
(T B) x N x 14.007 x 100mg N/100 ml =
-------------------------------------- Volume of the sample in
ml
WhereT = Titration volume for the sample (ml)B = Titration
volume for blank (ml)N = Normality of the acid to 4 places of
decimal.F = Conversion factor for Nitrogen to protein e.g. 6.25,
5.7, 6.38 depending on sample.
6.3. Estimation of crude fat
a. Regents Hexane, petroleum ether or any suitable reagent.b.
EquipmentSoxtec Avanti 2055 systemc. Sample preparationSamples
prepared using physical treatments such as shaking, stirring,
mortaring, riffling, coning and quartering or mechanical treatments
such as grinding, blending, homogenizing, or milling is placed into
extraction thimble to allow for maximum solvent contact.
Recommended particle size is 20 25%0.5 1 g + 0.1 mg
e. Instrument settings Control unit / service unit is set at
temperatures to get an even boiling of the solvent with a reflux
rate of about 3 5 drops / sec. This setting varies with the solvent
used and the type of extraction cups used (whether glass or
aluminum). If too high temperature is used the boiling of the
solvent can become too vigorous which for some samples increases
the risk for foaming. The risk of oxidation of the extractable
matter during the evaporation phase is also increased.f.
ProcedurePress the mains button (switch lamp should light up). Set
temperature to achieve a reflux of 3 5 drops of solvent per second.
(See application sub note to achieve desired reflux of solvent).
Select the proper program and check time settings for boiling /
rinsing / evaporation / pre-drying on the control unit. Open the
cold-water tap for the reflux condensers. With cooling water at
approximately 15 C the flow should be adjusted to 2 l/min to
prevent solvent evaporation from the condensers. Prepare the
thimbles and attach them to adapters. Prepare and weigh sample into
thimbles with a precision of + 0.1 mg . Use the thimble support
with the balance. Move the thimbles to the thimble stand. Use
thimble handler. Put a thin layer of de-fatted cotton on the top of
the sample and place the thimbles into thimble stand. Use the
thimble handler and move the thimbles to the thimble supports.
Insert the thimbles in to the extraction unit. Attach them to
magnets. Remove the thimble supports.
Use the cup holder to insert the pre-dried extraction cups,
tared with 5 10 glass pellets (5 6 mm) to prevent shock boiling,
loaded with the solvent to be used. See ASN for recommended volume.
The buzzer signal will inform you when the temperature on the hot
plate is sufficient for lowering the thimbles with the handle. Boil
the thimbles immersed according to boiling time in the ASN. Make
sure that the condenser valves are open. Move the thimbles to
rinsing position. Rinse according to rinsing time in the ASN. Also
note that the condenser valves are open. After rinsing, close the
condenser valves by turning a quarter turn. Move the handle to
recovery position. When almost all the remaining solvent is
collected, press the air button. The last traces of solvent will
now be collected in the condenser /solvent collection vessel.
Remove the cups. Dry the cups at 103 C for 120 150 mins. Cool the
cups in the desiccator and weigh them. g. CalculationW3 W2Crude fat
% = ------------- x 100 W1 WhereW1 = Sample weight (g)W2=
Extraction cup weight (g) W3= Extraction cup + residue weight
(g)6.4.Estimation of crude asha. EquipmentMuffle furnaceb.
ProcedureWeigh 3g sample into silica crucible and place in
temperature controlled furnace preheated to 600 C. Hold at this
temperature for 2 hrs. Transfer crucible directly to desiccator,
cool and weigh immediately. Calculate the % of crude ash. Weight of
the ash% Crude Ash= --------------------------- X 100Weight of the
sample
6.5.Estimation of acid insoluble ash
a. Equipment Muffle furnace
b.ProcedureMoisten the contents with 5-10 ml of conc. HCl, after
ashing as described in the procedure for estimation of crude ash.
Boil for 2 min, evaporate to dryness and heat on steam bath for 3
hrs to render SiO2insoluble. Moisten residue with 5 ml conc. HCl,
boil for 2 min, add about 50 ml distilled water. Heat on a water
bath for 5 min, filter through Whatman filter paper No. 1 and wash
thoroughly with distilled water. Then ignite at 500 550 C, cool and
weigh to calculate acid insoluble ash % by difference
Weight of acid insoluble ashAcid insoluble ash % =
-------------------------------------- X 100 Weight of the
sample6.6.Estimation of crude fibre
a. ReagentsSulphuric acid: 1.25 % (12.5 g of H2SO4 diluted to 1
lt and mixed)Sodium hydroxide (NaOH) or potassium hydroxide (KOH)
1.25% (12.5 g of NaOH or KOH dissolved in deionized water and
diluted to 1 lit.n-OctanolAcetoneb. EquipmentMuffle furnaceHot air
ovenc. ProcedureGrind the sample to pass through 1 mm screen in a
grinder. Weigh and transfer 2 g (Wo) of defatted sample in to a
beaker. Add 200 ml preheated 1.2% sulphuric acid and a few drops of
octanol to prevent foaming. And heat to boiling. Adjust heat and
boil for 30 mins. Filter through a 200 micron cloath and wash with
hot de-ionized water three times. Transfer again to the same beaker
and add 200 ml hot 1.25 % NaOH or KOH solution and boil for 30 min.
Filter and then wash with de-ionized hot water three times and then
finally with acetone (25 ml each) three times. Collect the residue,
dry it in hot air oven at 100 C over night or at 130 C for 2 hrs.
Cool and weigh (W1) the material. Ash the sample in the crucibles
at 550 C for 2 hrs. Cool the crucibles to room temperature and
weigh again (W2). Calculate the fibre content from the formula W1
W2Crude fibre % = -------------- x 100 Wo
6.7. Estimation of water durability
1Objective:To assess the water durability of shrimp feed
2Parameters of the judgment
Swelling:Indicates the degree of water absorption. The higher
the water absorption by the pellets the higher the risk for
disintegration
Cracks:Cracks are the result of the pelletizing process. They
are fine as hairs and are visible under the microscope or when put
into water. Cracks are the entrance for penetration of water into
pellets.
Disintegration:Due to absorption of water, the particles of the
pellets fall off. Falling off particles from the pellet is the
beginning of the disintegration
3Assay
Drop ~5 g feed sample, just enough to form one layer on the
bottom of a 250 ml glass beaker filled with water. This is stirred
automatically and continuously so as to simulate the pond water
movement during aeration.
4Procedure of judgment
Rating (judgment) is done at the end of 1 hour and 2 hours
Rating of the water durability is done by applying 1 to 10
points of fractions thereof, preferably by three persons
Feed has to be observed carefully after putting into water for
any quick changes with reference to swelling, cracks and
disintegration, especially at the end of the intervals given above.
Testing may be stopped if the samples get a failure rating as per
judgment chart given below.
5Judgment chart
Chart for the judgment of the water durability of shrimp
feed
Swelling:Cracks 1 (in pellets)DisintegrationPoints ( 1 to 10
)
Little to normalNoneNone10
NormalLess than 1/5 th None9
Normal1/5 th to 1/3 rdVery less8.5
StandardNormal1/3 rd to 1/2~ 15% 28
Normal1/2 to 2/3 rd~ 20 %7.5
Normal to full2/3 rd to 3/4 th ~ 25%7
Normal to full> 3/4 th~ 50%6
FullTotal~ 75%5
FullTotalNearly total ( ~90%)4
Full Total Total 3
Full (within 5 min)-Total (within 5 min)2
Full (immediate) -Total (immediate)1
1Means number of pellets actually developed cracks but have not
fallen apart
2Visual estimation of fallen off particles in comparison to the
volume of the feed present
6.8.Estimation of calcium
a. Reagents
i) Ammonium oxalate solution: Boil 45g Ammonium oxalate in 1L of
distilled water, cool and filter.
ii) 0.1N Potassium permanganate (Std. KMnO4 soln.): Weigh
accurately about 3.2g of KMnO4 crystals (AR Grade) into a beaker.
Add cold water and stir thoroughly with a glass rod. Break up the
crystals with glass rod and dissolve maximum crystals. Decant the
solution through a plug of glass wool supported in a funnel. Leave
the undisclosed residue in the beaker and dissolve this in fresh
water. Continue this until all the crystals dissolve. Dilute the
solution to 1L, shake well and keep for 24 hrs in an amber coloured
bottle with stopper.
Standardization of KMnO4 solution: Weigh accurately about 0.3g
Sodium Oxalate (Na2C2O4) AR (preheated for 2 hrs at 1050C) into a
500ml beaker, add 250ml distilled water and 10ml conc.H2SO4. Warm
on a hot plate up to 700C and titrate against KMnO4 while hot till
a pink colour persists.
Normality of KMnO4 = W x 1000/V x 67
Where W = Weight in g of Na2C2O4 takenV= Titre volume of
KMnO4
iii) Acetic acid Glacial
iv) Phenol Red indicator solution: 0.1g dissolved in 30ml 0.01N
NaOH solution and diluted to 250 ml with distilled water.
b.ProcedureAsh 1g of feed in a crucible as per the standard
procedure for determination of %Crude Ash. Cool and transfer
quantitatively all the contents in the crucible into a beaker.
Rinse the crucible with a few drops distilled water followed by a
few drops of (1+1) HCl Soln and these washings also transferred to
the same beaker. Add 10ml (1+1) HCl Soln, cover the beaker with a
watch glass and heat just below boiling point on a hot plate for 20
minutes. Filter through Whatman 541 9cm filter paper in to a 250ml
volumetric flask. Wash the filter paper well with distilled water
and make up the volume to 250ml.
Pipette 25ml of the sample solution to a 300ml flask, add 50ml
distilled water and 5 drops Phenol red indicator solution (Solution
turns to yellow colour). Then add drops of (1+1) ammonia solution
with stirring till the colour of the solution turns to purplish red
(pH =8.5). Bring back the colour to just yellow by adding drops of
Acetic acid (Glacial) carefully. Boil the solution on a hot plate.
At the same time, boil 25ml of ammonium oxalate solution in another
beaker. When both the solutions are started to boil, add ammonium
oxalate solution portion by portion into the solution with
stirring. Allow to cool and keep overnight for complete
precipitation.
Filter the solution through Whatman No: 1 filter paper, wash the
precipitate and beaker with hot water until the washings are free
from oxalate (non-cloudiness with CaCl2 solution). Take 100ml
distilled water in the beaker, add 4ml Conc.H2SO4 into it and heat
to boiling. Pour this through the filter paper into a fresh conical
flask with punching the filter paper, wash the filter paper
thoroughly with hot water and collect all washings in the conical
flask. Also put the filter paper into the conical flask and heat
the flask to 700C. Then titrate while hot against Std.KMnO4
solution till a pale pink colour persists.
Calculation
% Calcium = N x V x 20/ W
Where N= Normality of Std. KMnO4 V = Titre Volume of Std. KMnO4
W = Weight of feed sample in g taken for ashing
6.9.Estimation of phosphorus (Gravimetric method)
a. Reagents
Concentrated Nitric acid Nitric acid 1:1- Mixture of equal
volumes of concentrated nitric acid and water Ammonium molybdate
stock solution Take 200g of powdered ammonium molybdate (AR grade)
in a stoppered graduated cylinder of 1000 ml capacity .Add to it
800 ml of distilled water and shake well for 25 minutes to dissolve
the ammonium molybdate. Add gradually 25 % (m/v) ammonium hydroxide
solution till the solution is clear (about 100 to 140 ml of
ammonium hydroxide may be required.) Avoid adding excess of
ammonia. Make up the volume to one liter, if necessary, filter the
solution through a filter paper and stock this solution. Nitric
acid solution 2 % Potassium nitrate solution 3% Standard sodium
hydroxide 0.1 N Phenolphthalein indicator solution Dissolve 0.1g of
phenolphthalein in 100 ml of 60% alcohol.
b. ProcedurePrecipitation Take 10 ml aliquot of the prepared
solution (general solution as prepared for cal) In a 150ml beaker.
In a dry baker, prepare ammonium molydbdate solution by pouring
into it, quickly and simultaneously. 10 ml of conc. nitric acid or
take 10 ml of conc. nitric acid solution in the beaker and into
thispour quickly 10 ml of ammonium molybdate solution, whirling the
beaker during addition. Pour this freshly prepared clear liquid in
to the beaker containing the aliquol and stir. But much better, if
the above analysis is done fast, so that the temp raised due to
nitric acid will be enough and if it is done slow , care should be
taken .
Note: The temperature developed in the molydbdate solution is
sufficient to precipitate all the phosphorus present in the
aliquant. Under no circumstances the phosphomolybdate precipitate
should be heated either on a water bath or directly over a burner
so as to avoid precipitation of molybdie anhydrate.
Filtration and Washing Allow the precipitate to stand overnight
and then filter through whatman filter paper no 42 over an ordinary
funnel. As far as possible only the supernatant liquid is passed
thru the filter paper, retaining the precipitate in the beaker.
When the supernatant liquid is decanted off the precipitate is
washed twice with dilute nitric acid and then with potassium
nitrate solution until the washings are free from acid. Freedom
from acidity may be tested by collecting sufficient filtrate in
test tube to which as few drops of phenolphthalein indicator
solution and one drop of the standard sodium hydroxide solution are
added. If the pink color appears with one drop of the standard
alkali, the precipitate is free from acid.
Titration Transfer the precipitate with the filter paper back to
the beaker in which precipitation was carried out. Add sufficient
quantity of the standard solution hydroxide solution from a burette
just to sufficient to dissolve the ppt and then add 5ml in excess.
See that no yellow ppt sticks to the filter paper. Note the total
volume of the standard sodium hydroxide solution added. Add about
2-3 drops of phenolphthalein indicator solution and filtrate the
excess of alkali with the standard nitric acid.
Calculation: phosphorous = 336.75(AN1- BN2) --------------- M x
100 Where,
A - Volume in ml of the standard sodium hydroxide solution used
N1- Normality of the standard sodium hydroxide solution B - Volume
in ml of the standard nitric acid used into neutralize the excess
alkali.N2- Normality of the standard nitric acid. M Mass in gram of
the material taken for the test (it means that total weight of
sample which is taken for ash)
6.10.Estimation of salt (Chlorine as sodium chloride)a.Reagents
Ferric sulphate solution: dissolve 60 g of ferric sulphate
[Fe2(SO4)3] in one liter of water.Ammonium hydroxide solution:
Prepared by mixing one volume of ammonium hydroxide with 12 volumes
of water.Concentrated nitric acid.Ferric sulphate indicator
solution: Prepare as 25 % (m/v) solution. Filter and add equal
volume of nitric acid.Standard silver nitrate solution: 0.1 N
(follow procedure as described later).Standard potassium
thiocyanate solution: 0.1 n (follow procedure as described
later).b.Procedure
Weigh accurately about 1 g of the dried material and transfer to
a 250 ml volumetric flask. Add 50 ml of the ferric sulphate
solution with a pipette, gently swirling the flask. Add 100 ml of
the ammonium hydroxide solution to bring it to the mark. Swirl the
flask enough to ensure through mixing but avoid vigorous shaking.
Allow to settle for 10 min. filter through 11 cm Whatman filter
paper # 41 or its equivalent. Transfer from the filtrate, an
aliquot of 25 ml to a 250 ml beaker. Add 10 ml of nitric acid and
10 ml of the ferric sulphate indicator solution. Then add with
constant stirring, known quantity of the standard silver nitrate
solution in slight excess. Heat the solution to the boil and cool
to room temperature and stir to coagulate the precipitate. Titrate
the excess of silver nitrate with the standard potassium
thiocyanate solution. The end point is indicated by the first
appearance of reddish tint that persists for 15 seconds.
5.845 (AN1 BN2)Chlorine as sodium chloride percent by mass =
---------------------m
WhereA =Volume in ml of the standard silver nitrate solution
used.N1 =Normality of the standard silver nitrate solutionB =Volume
in ml of the standard potassium thiocyanate solution used up by the
excess silver nitrate solutionN2 =Normality of the standard
potassium thiocyanate solutionm =mass in g of the material taken
for the test.
6.11.Estimation of magnesiuma.Reagents Buffer solution pH 10:
Dissolve 67.5 ammonium chloride in 200 ml distilled water. Add 570
ml ammonium hydroxide and dilute to 1 liter. Potassium hydroxide
(KOH) potassium cyanide (KCN) solution: dissolve 280 g KOH and 66 g
KCN in 1 liter distilled water. Potassium cyanide solution 2 % :
Dissolve 2 g KCN in 100 ml distilled water. Erichrome black T
indicator: Mix 1 g indicator with 99 g potassium nitrate. A
quantity of 30 to 40 mg is to be used. Magnesium standard solution:
0.25 and 1.0 mg / ml : Dissolve 0.25 and 1.00 g magnesium turnings
in HCL (1+10) and dilute each to 1 liter with distilled water.
Calcium standard solution 1mg / ml: Dissolve 2.4973 g calcium
carbonate, AR grade, previously dried for 2 hrs at 285 C, in HCL
(1+10). Dilute to 1 liter with distilled water. Pattons and reeders
reagent: 1% indicator mixed with 99%sodium sulphate. Disodium
dihydrogen EDTA (Na2H2EDTA) standard solution: 0.4 % : dissolve 4 g
Na2H2EDTA in liter distilled water. Standardize against standard Ca
and Mg solutions. 0.1%: Prepare as above (i), using 1 g Na2H2EDTA
and standardize against 0.25 mg / ml Mg standard
solution.b.Standardization: For Calcium: Pipet 10 ml standard Ca
solution into 250 ml conical flask and add 10 ml distilled water.
Add 10 ml KOH-KCN solution and about 35 g pattons and reeders
reagent. Titr with 0.4% EDTA standard solution till end point. Titr
3 aliquots and use average to calculate titr Ca solution = 10 / ml
EDTA solution.
For Magnesium: Pipet 10 ml 0.25 and 1.00 mg / ml Mg standard
solutions into 250 ml conical flasks and add 150 ml distilled
water. Add 7 ml pH 10 buffer, 2 ml 2% KCN solution, and 30 to 40 mg
erichrome black T indicator. Titr. with 0.1 and 0.4% EDTA standard
solutions respectively, until color changes permanently from wine
red to pure blue. Titr 3 aliquots and use average to calculate
titer Mg solution = 2.5. ml EDTA solution, or 10 / ml EDTA
solution, respectively.c.DeterminationDry sample at 110 C to
constant weight and cool to room temperature. Grind to pass # 60 or
80 ASTM sieve and mix thoroughly. Accurately weigh about 0.5 g into
250ml beaker, add 20 ml HCL (1+1), and evaporate to dryness on hot
plate. Dissolve residue in 5 ml HCl (1+10), dilute to about 100 ml
wit distilled water and digest over water bath for 1 hr. Coll,
transfer to 250 ml volumetric flask, dilute to volume, mix and let
settle or filter. For Calcium: Pipette 10 ml aliquot into 250 ml
conical flask and titer. As in II a, observing end point thru
solution and away from light. % Ca = (titer EDTA standard solution
for Ca x ml EDTA standard solution x 2 / g sample).
For Magnesium (for samples containing 4 % Mg) : For Ca + Mg,
pipet 10 ml aliquot into 250 ml conical flask and titr. with 0.4%
EDTA solution as in II b. % Mg = (Titer EDTA standard solution for
Mg ) x [(ml EDTA standard solution in Ca + Mg titrn) - (ml EDTA
standard solution in Ca titrn)] x 2 / g sample.
For Magnesium (for samples containing 2 4% Mg): Pipette 10 ml
aliquot (0.5 1.0 mg Mg) into 250 ml conical flask and add exact
volume of 0.4% EDTA solution required for Ca determination. Titrate
with 0.1% EDTA solution as in II b. % Mg = (Titer EDTA standard
solution for Mg) x ml EDTA standard 0.1% solution x 2 / g
sample.
6.12.Estimation of urease activity in soybean meala.
ReagentsUrea crystalsPhenol red indicator (0.62 % solution in
distilled water)b.ProcedureWeigh 0.2 g soybean meal into a test
tube, add 0.02 g urea crystals and 2 drops of phenol red indicator.
Finally add 2 3 ml distilled water into the test tube. Shake the
test tube for 10 seconds and observe for the appearance of pink
color in the test tube. Record the time span for color development.
c.Identification of the strength of the urease activityIf color
appears within 1 minute -Very strongIf color appears between 1 to 5
minutes-strongIf color appears between 5 to 15 minutes-Some
activityIf color appears between 15 to 30 minutes -No
activityd.ConclusionIf soybean meal sample did not have pink or red
color appearance within 10 minutes, means this soybean meal is
acceptable for feed production.
6.13.Estimation of Acid Value[footnoteRef:5] [5: mg potassium
hydroxide necessary to neutralize acids present in 1 g sample.
]
a. ReagentsSodium hydroxide (NaOH) 0.1 N Ethyl alcohol 95%Add
little phenolphthalein and neutralize with alkali to a faint but
permanent pink color just before using. Phenolphthalein indicator
1% in 95% ethyl alcohol.Petroleum ether or hexane.b. Preparation of
sampleWarm the sample to soften the product (do not heat over 60 C)
and then mix thoroughly.c. ProcedureWeigh about 2 g of sample into
a 250 ml conical flask. Dissolve in 50 ml of petroleum ether or
hexane by mixing gently. Then add 50 ml of neutral alcohol and mix.
Add 4 drops of indicator and while mixing titrate with 0.1 N NaOH
to the first pink color, which persists for 5 sec. Adding the bulk
of the alkali solution rapidly until near the end point, and then
slowing down to about 4, then 1 2 drops fairly easily ascertain the
end point, at a time. ml. titrant x N x 56.1Acid value (mg KOH / g
) of sample[footnoteRef:6] = ------------------------ [6: Although
sodium hydroxide is used in the procedure, results are expressed
based on mg KOH per gram. The factor used in calculation is based
on the fact that 1.0 ml of 1N NaOH solution is equivalent to 56.1
mg KOH.]
Weight of the sample
Where N = Exact normality of the standard NaOH solution6.14.
Estimation of Acetone insolubility
a.Reagents: Acetone
b.Procedure
Weigh 5 g lecithin sample accurate to 0.001 g into the beaker.
Weigh filter. When convinced, by means of thermometer, that the
acetone is at 0 C, add about 20 ml thereof to the lecithin and
start to knead with glass rod. Allow the solids to settle for a
short period and filter off the oil-containing acetone through the
filter into the conical flask. Repeat the kneading step at least 5
times using in each case 20 ml acetone at 0 C. The initially greasy
tacky material must finally have disintegrated to a fine powder.
Transfer as much as possible of the kneaded lecithin to the filter
and wash with cold acetone at 0 C until a drop of the filtrate
evaporates on a glass plate without leaving a residue. Then dry the
filter together with the remainder of the kneaded lecithin for 30
min in a hot air oven at 105 C. when the material is dried, cool it
in a desiccator, weigh it.
Acetone Insolubility % = [(F-E) x 100] W
Where F = Final weight of filter with sample in g
E = Empty weight of filter in g
W= Original sample weight in g
6.15.Determination of pepsin digestibility[footnoteRef:7]
(Filtration method) [7: Defatted sample is digested for hrs in the
presence of warm acid solution of pepsin under constant agitation.
Insoluble residue is isolated by filtering, washed, dried and
weighed to determine % residue. Residue is analyzed for
protein.]
a.ReagentsPepsin solution - % pepsin (activity 1:10,000) in
0.075N HCl, freshly prepared. Do not use pepsin NF or pepsin of
activity other than 1:10,000. Prepare just before use by diluting
6.1 ml HCl to 1 liter and heating to 42 45 C. Add pepsin and stir
gently until dissolved. Do not heat pepsin solution on hot plate or
over heat.b.EquipmentAgitator continuous, slow speed (15 rpm),
end-over-end type, to operate inside incubator at 45 + 2 C and
carry 250 ml screw cap prescription bottles, or equivalent.
Filtering device Buchner funnel or equivalentGlass fiber filter
paper, for indigestible residues.45 angle settling rack
c.ProcedureGrind sample to pass through # 20 ASTM sieve. Weigh
accurately 1.000 g of material into the thimble and extract with
hexane as in procedure for determination of crude fat. Transfer the
defatted sample to 250 ml sample bottle. Add 150 ml freshly
prepared 0.2% pepsin HCl solution and heat to 42 to 45 C. Be sure
sample is completely wetted by pepsin solution. Stopper bottle,
clamp in agitator, and incubate with constant agitation for 16 hrs
at 45 + 2 C remove bottles from agitator. Place in 45 angle
settling rack and loosen the caps. Let residue settle for 15 min or
more.
Place weighed filter paper in funnel. Filter the sample and
quantitatively transfer residue on to filter washing properly with
hot water. If filtration rate is slow it may be accelerated by
adding acetone coupled with suction. Add 15 ml acetone into the
bottle. Hold thumb over bottleneck and shake vigorously. Release
pressure, replace thumb over bottleneck and shake bottle in
inverted position over filter. Repeat rinse two or three times till
the bottle is free from residue. Dry the residue in the filter
paper in a hot air oven as in the procedure of determination of
moisture. Calculate the % indigestible residue. Determine
indigestible protein by transferring filter-containing residue
directly to Kjeldahl flask and determine crude protein by Kjeldahl
method as described previously.Make blank determination on 1 sheet
of glass filter and subtract from each sample determination if
necessary.Calculate % of protein based on original sample weight.
Result represents % indigestible protein in sample.
% Indigestible crude protein in sample% Protein indigestible =
-------------------------------------------- x100 % Crude
protein
% Pepsin digestibility = 100 - % protein indigestible.
6.17. Estimation of wet gluten index in wheat flour
Weigh 20 g wheat flour in to a bowl and add 12 ml tap water, mix
to form a firm paste by thumb (only). Let the dough stand still for
5 min. Wash the dough in the bowl several times (app. 20 min) till
the water is clear. Each washing has to be passed through 100 ASTM
mesh to prevent gluten pieces from escaping. Gluten pieces, if any,
found to be sticking to the mesh, are to be collected back.
Test the completeness of washing by squeezing out water from the
gluten ball, which should not form a ring or thick starch cloud in
a beaker full of water. Remove excess water adhering to the gluten
ball by rolling over a cotton cloth and weigh it.
Weight of wet glutenWet gluten index % =
--------------------------- X 100Weight of wheat flour
6.18.Estimation of wet gluten index in wheat gluten
Take 10 g sample of wheat gluten, add 40 ml tap water and then
soak it for 15 min in a bowl. Then the contents are transferred
into a filter paper in a funnel to let the excess water filter
out.
Wet gluten content in the filter paper is made into a ball and
thoroughly washed over a mesh (100 ASTM) for 20 30 min to remove
excess of starch. Pure wet gluten ball is then made free of
adhering water over a cotton cloth and weighed for calculating the
percent gluten index.Weight of the wet glutenWet gluten index %
=-------------------------------- X 100Weight of the dry gluten
6.19.Estimation of dry matter retention
a.EquipmentSmaller pore size meshAquariumAeratorHot air oven
b.Procedure Take 20 g pelletized feed in to a mesh of smeller
pores than feed and place it in slowly circulating fresh water in
an aquarium. Water can be circulated with the help of aerator.
After 2 hrs, collect the sample and dry it in hot air oven at 135 C
till it is fully dried. Weigh dried feed pellets and calculate the
dry matter % retained in the mesh.6.20.Abrasion test
a.Equipment : Slowly rotating mechanized tumbler b.Procedure
:
Place 500g pelletized feed in to the tumbler and rotate at slow
speed ( about 30 to 50 rpm) for15 min. Collect the sample and sieve
it through smaller pore size mesh .The dust and fines generated the
period of tumbling are calculated as percent by weight .
6.21.ESTIMATION OF COARSENESSa.Equipment : Test sieves ASTM-40,
60 and 80
b.Procedure
10-20 g of sample is weighed and then sieved through any of the
sieves in order to find out the quality of coarse material.
Depending on the type of material, particular sieve has to be used.
Quantity of coarse material retained over the sieve is weighed and
calculated as percent.
6.22.Preparation of standard potassium permanganate solution
Dissolve3.2g of potassium permanganate (KMnO4) in one litter
distilled water to prepare 0.1N solution. Boil the solution for 1
hr. Protect from dust and let stand overnight. Standardization: For
0.1N solution, transfer 0.3g dried (1hr at 150c) Sodium oxalate
(Na2C2O4) to 500ml beaker. Add 250ml sulphuric acid (5+95) ,
previously boiled 10-15 min and then cooled to 27-30C . Stir until
sodium oxalates dissolved. Add 39-40ml KMnO4 solutions. If too
concentrated, discard and begin again, adding few ml less of KMnO4
solution. Heat to 55-60c and complete titrn by adding KMnO4
solution until faint pink persists 30sec. Add last 0.5 1ml drop
wise, letting each drop decolorize before adding next. Determine
excess of KMnO4 solution required to turn soln pink by matching
with color obtained by adding KMn O4 soln to same vol, of boiled
and cooled dil. sulphuric acid at 55- 60c. This correction is
usually 0.03 0.05ml. From net vol, KMnO4, calculated
normality.Normality= g Sodium oxalate X 1000 / ml KMnO4
X66.9996.23.Standardization of 0.1N nitric acid solution
Titrate dilute nitric acid solution against 20 ml standard NaOH
solution using phenolphthalein indicator. Note down the volume of
dilute nitric acid required to neutralize 20 ml standard NaOH.
Determine the normality of the nitric acid using the formula: N2
=N1 V1 / V2
N1= Normality of standard NaOH
V1= Volume standard NaOH used
N2= Normality of standard nitric acid to be obtained
V2 = Volume of nitric acid used.
6.24.Preparation of standard silver nitrate solution
a.Preparation of standard solution
Dissolve slightly more than theoretical weight of silver nitrate
(AgNO3) in distilled water and dilute to volume. Thoroughly clean
glassware, avoid contact with dust and keep prepared solution in
amber glass stopper bottles away from light.
b. ReagentsPotassium chloride Dry KCl at 110c and then heat at
about 500 C to constant weight Potassium chromate solution 5%
solution of K2Cr2O4 in distilled water. c.Standardization
Accurately weigh enough KCl to yield titration of about 40ml
(approximately 0.03g for 0.01Nsolution) and titrate with AgNO3
solution until first perceptible pale red brown appears. From
titration volume subtract ml of the AgNO3 solution required to
produce end point color in 75 ml distilled water containing 1ml
K2CrO4 solution. From net volume AgNO3, calculate normality.
Normality = g KCl x 1000/ml AgNO3 x 74.555.
6.25. Preparation of standard potassium thiocyanate solution
Preparation of about 0.1N solution using 9.718 g potassium
thiocyanate (KSCN) / L.
Standardization:
Accurately weigh enough purified AgNO3 to give titration of
about 40ml (about 0.7g for 0.1N solution) and transfer with
distilled water through glass funnel to 250ml conical flask.
Dissolve in about 75ml distilled water and add 5ml HNO3 (1+1 ) and
2ml Fe alum ( Ammonium Iron 111 sulphate or Ferric salt ) solution
.Titrate with thiocyanate solution until titrated solution is
reddish brown .which remains after shaking vigorously 1 min. Record
burette reading and set the flask aside 5min ,shaking occasionally
and maintaining end point color by addition of thiocyanate solution
as required .Then add additional thiocyanate solution , If
necessary , to produce permanent end point color, matching with
color of reference solution . From total volume thiocyanate
solution used in titration, subtract volume, contained in the
reference solution. Normality = g AgNO3 X 1000/ml titer X
169.87
6.26. Determination of the concentration of volatile nitrogenous
bases (TVB-N)
10g 0.1g of sample is homogenized/blended with 90mL of
Perchloricacid (PCA) 6% and filtered by 150mm filter (Whatman No.
1) The extract thereby obtained can be kept for at least seven days
at a temperature between approximately 2 6 0C 10mL of the extract
is estimated in automated 2300 Kjeltec distillation unit (Foss
Tecator AB). Silicone antifoaming agent is added if foaming starts.
Blank test is carried out with 10mL aqueous solution (6.0%) of PCA.
10 ml of filtrates (6.1 clause ) taken in to digestion tube and set
the Kjeltec analyzer as mg N/100 gm. Enter the weight of sample (
filtrated solution ) taken for test. TVB- N value is expressed in
mg N/100g sample. Duplicate analysis is required for each analysis.
After one week, the same filtrate is rechecked for TVB-N Value
6.27. The feed analysis is being carried out by laboratory
services according to feed mill requirements.
6.28. Apart from the chemical analysis physical examination of
feed sample will be done by visual observation.
7.0 DOCUMENTATION
7.1 Annexure - I Raw materials Analysis report Format -
(RMI-05)7.2 Annexure II Sales feed analysis Test Report Format
(LS-FA-01)7.3 Annexure - III Finished feed analysis report Format
FAN-05
All the document formats quoted here are given in Annexure I -
III
8.0 REVISION HISTORY
Issue No. DateRevision No. & DateAuthor(s)Change
ReferenceReason for Change
Issue 1, 01.08.2010----
9.0 APPROVALS
Prepared by:
Reviewed by:
Process Control, LSRecommended by:
Department Manager, Laboratory Service Verified for
Endorsement:
Department Head, Quality Systems
Endorsed by:
General Manager Quality & Sustainability Approved by:
General Manager Quality & Sustainability