Proximate Analysis

MUHAMMAD ASIF 2K8-VAS-32LUAWMS UTHAL BALOCHISTAN

Setting of Analytical Laboratory

• Oven

• Weighing Balance

• Furnace

• Suction Pump

• Racks

• Dessicator

• Fiber Extraction Apparatus

• Soxbelt Apparatus (Fat Digestion)

• kjeldahl Apparatus

Following things are required

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• Balance and oven are placed together so as to avoid the moisture loss and less time is consumed.

• Dessicator is moisture absorbent having Ca and Silicate salts.

• Never place furnace near balance because its air current disturb reading.

• Organic Substances - - - - - - - - 70 - 80oC

• Inorganic Substances - - - - - - - - - 100oC

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• For protein and fat digestion, fume hood with sliding roller used.

• Distillation apparatus should be placed near water supply and titration flask.

• Acids and alkalis should be placed on lower side of rack.

• Fiber extraction apparatus and suction pump should be placed on same shelf.

• Soxbelt apparatus and solvent should be on the same shelf.

Proximate Analysis

In 1865, for describing various feeds and fodders, Hennebrg and Stobman at Weende Expi Station Germany proposed a scheme of chemical analysis, also called Weende Analysis Scheme/Weende’s System of Analysis.

There are following analytical procedures used under Proximate Analysis

Fraction Component

Moisture Water (VA and bases if present)

Crude protein Proteins, Amino acids, Amines, Amides, Nitrates

Ether extract Fat, Oil, Waxes, Pigments, Sterols

Crude fiber Cellulose, Hemicellulose, Lignin

Ash Minerals, Silica

Nitrogen free extract Sugar, Starch, Protein

Food

Dry Matter

Inorganic

Minerals

Organic

Carbohydrates

Nucleic Acid

Organic Acid

Lipid

Protein

Vitamins

Water

Birth750 –

800gm/Kg

Mature500gm/Kg

Moisture

Moisture or water content is most important in a feed. It gives information about the characteristics of feed whether it is succulent or dry (bullkey feed stuff).

Moisture

• Moisture ↑ than 15% → promote growth of fungus and mold when

stored .

• Fermentation may also take place resulting in combustion.

Silage Moisture = 65 – 70%

Hay Moisture = 15%

• 1 gm of carbohydrate, protein & fat yield approximately 0.6, 0.4, 0.1 ml metabolic water on oxidation.

• Water content in feed also determine water requirement of an animal.

Procedure

Take china dish / petri dish(no water absorbent), then wash it and keep it in oven at 105oC, then weigh it(W1). Add sample(gm) and then put it in the oven(60 - 70oC) overnight(at least for 12hrs.), after that take it out and put in dessicator for upto 10min until it reaches at room temperature. Then put it out and weigh it(W2)

DM %

Moisture

Dry Matter

Dry matter is the constant wt. of sample when heated at 100oC but it is suitable for inert materials, not for organic substances.

All organic materials are dried at 70 – 75oC through Three methods.

• Low temperature drying

• High temperature drying

• Freeze drying

Dry Matter

In low temperature drying, some labs adopted this method , vacuum drying oven 30oC temps along with 16mm Hg of pressure. This will reduce losses of variable substances. In high temperature materials are dried at 100oC for DM estimation and 70 - 75oC for analysis of organic substances. Freeze drying is adopted to have minimum change In chemical composition but it can not be used for DM.

Determine the CP% of given sample through Proximate Analysis

Crude Protein:

Crude mean that may contain things other than protein e.g. NPN, urea, amide, ammonium sulphate. In it we measure N % in feed stuffs are we sold according to protein.

Principal:

Sample + conc. H2SO4→ (NH4)2.SO4

(NH4)2SO4 + Alkali → Ammonia is liberated and collected in boric acid → Ammonium Borate complex.

Titrate it against N/10 or N/100 H2SO4 → N% is obtained

N% 6.25 → CP%

Determine the CP% of given sample through Proximate Analysis

Detergents:

Mixed indicator for ammonia titration. Mix 10 mL of Bromocresol green (0.1%) + 2 mL of methyl red (0.1%). 

• Sulfuric acid solutions, 0.1 N (0.25%) and Concentrated sulfuric acid, 98%.  

• Digestion mixture :K2SO4 + CuSO4. + FeSO4

• Sodium hydroxide solution:(4%) Dissolve 40 gm sodium hydroxide in 80 mL of distilled water and dilute it to 100 mL with distilled water.  

• Boric acid solution: (2%)

Dissolve 20 gm boric acid per liter of distilled water.

Determine the CP% of given sample through Proximate Analysis

Procedure:There are three steps.• Digestion• Distillation• Titration

1. Digestion

• Take specific volume (1 – 2g) of sample, then add 25 – 30ml conc. H2SO4 and 5gm digestion mixture. Then put it in heater(macro kjeldahl) for 5 – 6hrs. With occasional stearing till light green or colourless solution appear. Then let it cool and make its volume upto 250ml in the volumetric flask.(kjeldahl flask)

Digestion Mixture:

FeSO4 : CuSO4 : K2SO4

1 : 2 : 20

• FeSO4 is used to avoid bumping.

• CuSO4 is used as catalyst.

• K2SO4 is used to minimize the boiling point.

2. Distillation

Take 10ml sample (ariquotes)(Digested Diluted Material) and put it in distillation assembly, then add 10ml 40% NaOH and close it. Put 10ml of 2% boric acid in conical flask. Put it under the tip of cond- ensor of distillation assembly. Put 1 – 2 drops of phenolphthaein a methyl red shocking.

Pink → Brown/ Yellow

3. Titration

In titration we use N/10 or N/100 H2SO4. After NH3 and boric acid reaction .find the volume of sulphuric used at end point of titration

Calculation

Nitrogen % =

End Point

•  00000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000JJLJJKHJJJ000000000

Determine the E.E% of given sample through Proximate Analysis

• Crude fat/ E.ECrude fat ether extract contains all type of fats volatile and none volatile fatty acids containing fats like lipids, oils, waxes phospholipids etc.

principle

• Ether extract of the feed/food sample is carried out in Soxtherm extraction unit. It facilitates that always fresh ether repeatedly dissolves the fat from the sample placed in extractor tube. In this unit fat is dissolved in ether. The excess of the ether is then recovered leaving the extracted fat in the beakers. The beakers are placed in the oven at 1030C for 30-40 minutes.

Determine the E.E% of given sample through Proximate Analysis

• REAGENT 

a) Diethyl ether or

b) Ether solvent (Benzene ether) or

c) Mixture of (CHCl3 + Acetone)

Weight of ether extractEther Extract (%) = ------------------------------- X 100

Weight of sample

Procedure

• Weigh 1.5 to 4.0 gm sample (into a thimble) or on a piece of filter paper 10 x 10 cm. Fold it to form a packet. Staple it such that no feed particles can escape from it.

 • Place the packet containing sample in the thimble and fix it under the condenser.

Add 100 ml of diethyl ether to the solvent beaker and place it under condenser with screw ring, which is tightened as much as possible by hand. Turn on the water that cools the condenser. Raise the hot plates until they are in contact with the beakers and turn on the heaters. Check for ether leaks after the ether starts to evaporate and condense. Allow the extraction process for 40 minutes.

 • After the completion of extraction, lower the heater and allow the thimble to drain

empty. Remove the sample and place the reclaiming tubes under the condenser. Raise hot plate and distill ether into the reclaiming tube. Remove beaker, pour ether from reclaiming tubes into a container for reuse.

 • Dry the ether extract in a 1050C explosion proof oven for 30 minutes. Cool in

desiccator to room temperature and weigh.

Calculations

 

Weight of ether extract

Ether Extract (%) = ------------------------------- X 100

Weight of sample

Determine the C.F% of given sample through Proximate Analysis

PRINCIPLE

 

Plant material is treated with dilute acid and alkali solutions under boiling condition. This treatment removes the soluble substances leaving the insoluble in the form of residue as crude fiber.

Reagents

• Sulfuric acid solution:

• Dissolve 1.25 gm H2SO4 in 100 ml distilled water. Check normality by titration and adjust if necessary to 0.255 N.  

• Sodium hydroxide solution:

• Dissolve 1.25 gm NaOH in 100 ml distilled water (free from Na2CO3). Check normality by titration and adjust if necessary to 0.313 N.  

• Ethyl alcohol

Procedure

 • The residue left after the extraction of crude fat is used for

determination of crude fiber (see method of ether extraction on previous page). 

• Transfer the extracted residue to the digestion container.  • Add 200 mL of 1.25% sulfuric acid solution into the digestion container

and heat it after covering with condenser.  • After boiling for 30 minutes cool the mixture and filter it with a linen

cloth in a filtration flask. Use vacuum pump if necessary. Wash the residues thoroughly with boiling water.  

• Put the residue back into the container and add 200 ml of 1.25% sodium hydroxide solution and boil it for 30 minutes.  

• After boiling cool the mixture and filter it with linen cloth as before. Wash the sample with boiling water.  

• Give final washing with ethyl alcohol (5-10 mL). 

Procedure cont.

• Transfer the residue to a tared crucible.  • Dry the sample in oven at 1050C overnight.  • Cool the dried sample to room temperature in a dessicator and weigh

it.  • Ignite the contents of crucible on oxidizing flame, till no fumes are

evolved.  • Ignite the contents of crucible in a muffle furnace at 5000C for 1 hour.  • After ignition transfer the crucible into a dessicator to cool and then

weigh • Note the loss of weight due to ignition as crude fiber.  

Calculations

 

Loss of weight due to ignition

Crude fiber (%) = ----------------------------------------- X 100

Weight of sample before extraction of fat

Determine the Crude Ash of given sample through Proximate Analysis

PRINCIPLE

On oxidation of feed/food material at higher temperature it oxidizes all the organic matter to CO2, H2O and NO2 leaving mineral matter as oxides of the metals known as ash.

Procedure

 • Place a clean crucible in a muffle furnace at 6000C for one hour. Transfer

crucible from furnace to a dessicator and cool to room temperature.  • Weigh by difference 1.5 to 2 gm of sample into a pre-weighed porcelain

crucible.  • Heat the crucible on oxidizing flame till no smoke is evolved.  • Place the crucible in a muffle furnace at 5500C for four hours.  • After ashing transfer the crucible to a dessicator and cool to room

temperature.  • Weigh the crucible containing ash as quickly as possible to prevent

moisture absorption. • Calculate the weight of ash by subtracting the weight of empty crucible

from the weight noted in step 6. • Save the ash sample if mineral determination is to be done.

Calculations

 

Weight of ash

Ash (%) = --------------------------- X 100

Weight of sample

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