Complete Proximate Analysis for Animal Feed.pdf

Proximate Analysis: Moisture     

Principle and Scope Sample preparation Procedure Calculation Possible errors

Principle and Scope

The moisture and low volatile materials are removed by heating at 95-100°C under partial vacuum. It is applicable to fish and fish products, including frozen scallops, both IQF and block frozen, and fish by products. Sample preparation

1. Sample preparation should take into account the type of product and how it is used and prepared  by the consumer consumer forproducts that are packed in water, brine or similar medium that is normally discarded o  by the consumer: consumer: open open the package package and drain the the product on on an appropriate appropriate size size sieve for 1 to 12 minutes. Comminute the part of the sample retained by the screen until a homogeneous blend is obtained. forproducts that are packed in a medium that may be or is normally used by the o consumer, e.g., fish canned in its own juice or oil: transfer the entire contents of the  package into a homogenizer homogenizer and and blend for one minute minute or until until a homogeneous homogeneous mix mix is obtained. forsmoked fish and smoked fish products: remove the sample from the container with o forceps or other suitable instrument, place on a coarse screen and allow to drain for ca 5 minutes. Place the sample on a paper towel or other clean absorbent paper and blot. Repeat blotting using a second and third piece of towel. Comminute the sample until a homogeneous blend is obtained. If the sample texture is too tough for homogenization,  pass the sample through through a grinder grinder a sufficient sufficient number number of times times to obtain a uniform mix. mix. A food chopper may suffice as an initial step if the sample size is large. for brined or salt-cured fish: scrape off as many loose salt crystals as possible with a o spatula, if any are present, then wipe off any remaining salt crystals or moisture with a  paper towel. towel. Comminute Comminute the sample until until a homogeneous homogeneous blend blend is obtained. obtained. forraw fish, fresh or thawed: pass the sample through a grinder a sufficient number of o times to obtain a homogeneous blend. forprocessed products containing no separable liquid: thaw in the package (if frozen) and o  pass the sample through through a grinder grinder a sufficient sufficient number number of times times to obtain a uniform mix. for frozen scallops: obtain five sample units, remove surface glaze from the scallops o under running water until no ice can be felt on the surface of the scallops but it is evident that the ice crystals remain within the product; the interior of the product remains frozen. Block frozen product should be gently broken up to individual scallops or scallop pieces and ice within the block should be removed until the surface of the product is free of ice. Place the scallops on a sieve of appropriate size and drain for 1 to 12 minutes. Obtain a total of approximately 100 g of scallop meat from the five sample units. Comminute the 100 g sample until a homogenous blend is attained. 2. Collect the homogenized sample into a clean, sealable plastic cup or glass bottle.

3. Store the sample in a refrigerator or freezer until required. 4. Ensure that the prepared sample is still homogeneous prior to weighing. If liquid separates from the sample, reblend before use. 5. Since salted samples have a tendency to foam, care should be taken to see that the dish is not over-filled. Procedure

1. Accurately weigh a moisture dish of appropriate size. 2. Add approximately 10 g of the comminuted sample and reweigh. 3. Place the container in a vacuum oven at 100 oC and less than 100 mm Hg for approximately 5 hours. 4. Remove dish from the oven, cover, cool in desiccator, and weigh. 5. Redry 1 hr and repeat process until constant weight has been achieved, i.e., change in weight  between successive dryings at 1 hour intervals is < 5 mg. Calculation

Calculate the percentage moisture (wet weight basis) as follows: 100 (P-a)  percent moisture =

% P

P = weight in g of sample a = weight in g of dried sample Possible errors

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Loss of VOLATILE FATTY ACIDS (VFA's), and LACTIC ACID in SILAGE or any other FERMENTED PRODUCTS Most likely UNDERESTIMATE DRY MATTER This error is relatively small Maillard product formation (protein:CHO complex)

Proximate analysis: Ash     

Principle and Scope Sample preparation Procedure Calculation Possible errors

Principle and Scope

The ash fraction contains all the mineral elements jumbled together. It would be more useful to know the amounts of different individual elements. But it allows calculation of Nitrogen-Free-Extract (by difference) from dry matter (see below) and provides an estimate of contamination (too much soil or too much salt added. This method consists of oxidizing all organic matter in a weighed sample of the material by incineration and determining the weight of the ash remaining. It is applicable to fish, fish products, and other materials with a low carbohydrate content.  Note that the high temperature may cause the volatilization of certain elements (particularly K, Na, Cl, and P) and may also cause the mineral matter to melt and fuse. Sample preparation

1. Sample preparation should take into account the type of product and how it is used and prepared  by the consumer. For fish and fish products that contains no free liquid: comminute the sample until o homogeneous. For products that are packed in water, brine or similar medium that is normally discarded o  by the consumer: open the package and drain the product on an appropriate size sieve for 1 to 1½ minutes. Comminute the part of the sample retained by the screen until a homogeneous blend is obtained. For products that are packed in a medium that may be or is normally used by the o consumer, e.g. fish canned in its own juice or oil: transfer the entire contents of the  package into a homogenizer and blend for one minute or until a homogeneous mix is obtained. For fish meal: grind the sample in a mill or other suitable apparatus until it will pass o through a no. 20 sieve. 2. Collect the homogenized sample into a thoroughly cleaned, sealable plastic cup or glass bottle. 3. Store the sample in a refrigerator or freezer until required. 4. Ensure that the prepared sample is still homogeneous prior to weighing. If liquid separates from the sample, thoroughly reblend before use. Procedure

1. Accurately weight ca 5 g of sample in a crucible wich has been ignited and tared (use 2.5 g of sample in the case of products which have a tendency to swell). 2. Place crucible in drying oven at 100 oC for 24 hours. 3. Transfer to cool muffle furnace and increase the temperature step wise to 550 oC ± 5 oC. 4. Maintain temperature for 8 hours or until a white ash is obtained.

5. If white ash is not obtained after 8 hours, moisten ash with distilled water, slowly dry on a hot  plate, and re-ash at 550 oC to constant weight. Repeat if necessary. 6. Remove crucible to a desiccator and weight soon after cool. Calculation

Calculate the percentage ash content (wet weight basis) as follows: (wt. crucible and ash - wt. crucible) % ASH (wet)=

x 100 (wt. crucible and sample - wt. crucible)

Calculation of ash content on dry basis (when moisture content is known) as follows: % ash (wet) % ASH (dry)=

x 100 (100 - % moisture)

Possible errors

High temperature may volatile some elements such as chloride, zinc, selenium, iodine, etc. Consequently, ash determination tends to underestimate mineral contents This error is small

Proximate analysis: Crude protein      

Principle and Scope Sample preparation Reagents Procedure Calculation Possible errors

Principle and Scope

Estimated by a process developed by a Danish chemist/brewer, Johan Kjeldahl. He discovered that "all  protein" contains about the same amount of nitrogen (16%). He analyzed for nitrogen, which is relatively easy, and calculated crude protein on the basis: 100/16 = 6.25, therefore: NITROGEN x 6.25 = CRUDE PROTEIN In the presence of sulfuric acid, sodium sulphate and a catalyst, the amino nitrogen of many organic materials is converted to ammonium sulphate. The ammonia is distilled from an alkaline medium and absorbed in standardized mineral acid. The ammonia is determined by back titration with a standardized mineral base. This method is applicable to fish, fish products, and fish by-products. Certain species of fish such as dogfish contain non-protein nitrogen; therefore, when analysing these species use AOAC procedure 7.024 (12th Edition) for non-protein nitrogen to correct the results.

Sample preparation

1. Sample preparation should take into account the type of product and how it is used and prepared  by the consumer: forfish and fish products that contain no free liquid: comminute the sample until o homogeneous. forproducts that are packed in water, brine or similar medium that is normally discarded o  by the consumer: open the package and drain the product on an appropriate size sieve for 1 to 1½ minutes. Comminute the part of the sample retained by the screen until a homogeneous blend is obtained. forproducts that are packed in a medium that may be or is normally used by the o consumer, e.g., fish canned in its own juice or oil: transfer the entire contents of the  package into a homogenizer and blend for one minute or until a homogeneous mix is obtained. forfish meal: grind the sample in a mill or other suitable apparatus until it will pass o through a no. 20 sieve. 2. Collect the homogenized sample into a thoroughly cleaned, sealable plastic cup or glass bottle. 3. Store the sample in a refrigerator or freezer until required. 4. Ensure that the prepared sample is still homogeneous prior to weighing. If liquid separates from the sample, thoroughly reblend before use. Reagents

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Sulfuric acid (H2SO4), nitrogen-free. Cupric Sulphate (CuSO4), nitrogen-free, anhydrous. Sodium Sulphate (Na2SO4), nitrogen-free, anhydrous. Sodium Hydroxide (NaOH).  NaOH solution (50% w/v). o  NaOH standard solution (0.1 or 0.2 N). o Boiling granules, selenized. Hengar granules are suitable. Hydrochloric acid (HCl). HCl standard solution (0.1 N). Standardize against 0.1 or 0.2 N NaOH standard solution. Conway indicator. Stock solution. Mix 200 mL of 0.1% Methyl Red solution (in 50% ethanol) with 50 mL o of 0.1% Methylene Blue solution (in 50% ethanol). Working solution. Dilute 1 volume of stock with 1 volume of absolute ethanol and 2 o volumes of distilled water. (pH change 5.4: Acid - Purple, Alakline - Green).

Procedure

1. Accurately weigh a suitable quantity of fine-grained material (ca 1.2 g for fishmeal, ca 2.5 g for solubles or homogenized fish) and place in digestion flask. 2. Add sequentially 15 g Na2SO4, 1 g CuSO4, one or two selenized boiling granules and 25 mL of conc H2SO4 to the flask. 3. Digest until solution is almost colourless or light green (2 hrs for inorganic material) and then at least a further 30 minutes. Do not heat any part of the Kjeldahl flask above the level of the digestion mixture. 4. Cool (do not allow to solidify), and cautiously add 200 mL water. Add additional boiling granules (if necessary) to prevent bumping.

5. Pipette 100 mL 0.1 N HCl into a 500 mL erlenmeyer flask, add 1 mL Conway's indicator and  place the flask under the condenser ensuring that the condenser tip is immersed in the acid solution. (volume of standardized HCl used in distillation may be varied according to the expected nitrogen content of the sample). 6. Tilt the Kjeldahl flask containing the digested sample and add 100 mL of 50% NaOH solution slowly down the side of the Kjeldahl flask so that it forms a layer underneath the digestion mixture.Immediately connect the flask to the distilling bulb of the distillation apparatus. Rotate flask to thoroughly mix contents. 7. Heat until all ammonia has passed over into the standard acid. Collect approximately 150 mL. Caution, flask will bump. Remove immediately (prolonged boiling and too rapid distillation of acid during digestion should be avoided as loss of ammonia may occur). 8. Wash tip of condenser and titrate excess standard HCl in distillate with NaOH standard solution (detailed titration procedure) .

Figure: Soxhlet apparatus and diagram

Provide adequate ventilation for the removal of fumes during digestion. Calculation

Calculate the percentage nitrogen (wet weight basis) as follows: (A - B) x 1.4007 % Nitrogen (wet) =

x 100

weight (g) of sample where:  

A = vol. (mL) std. HCl x normality of std. HCl B = vol. (mL) std. NaOH x normality of std. NaOH

Calculate nitrogen content on dry basis (when moisture content is known) as follows: % Nitrogen (wet) % Nitrogen (dry)=

x 100 (100 - % moisture)

Calculate the percentage protein (wet or dry basis) as follows: 

% PROTEIN = % nitrogen x 6.25

where 6.25 is the protein-nitrogen conversion factor for fish and fish by-products. Possible errors & disadvantages 

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This procedure assumes ALL nitrogen present in the sample are in PROTEIN form. This assumption is NOT necessarily true. Nitrogen could be in nucleic acid (RNA, DNA), urea... Different proteins need different correction factors because they have different amino acid sequences The use of concentrated sulfuric acid at high temperatures poses a considerable hazard, as does the use of some of the possible catalysts The technique is time consuming to carry-out.

Proximate analysis: Crude lipid      

Principle and Scope Sample preparation Reagents Procedure Calculation Possible errors

Principle and Scope

A dried, ground sample is extracted with diethyl ether which dissolves fats, oils, pigments and other fat soluble substances. The ether is then evaporated from the fat solution. The resulting residue is weighed and referred to as ether extract or crude fat. Both the ether and the samples must be free of moisture to avoid coextraction of water-soluble components in the sample such as carbohydrates, urea, lactic acid,

glycerol, etc. If water-soluble components are present in large amounts in the sample, they are washed out of the sample prior to drying. Low temperatures are used to evaporate the ether and remove residual moisture to prevent oxidation of the fat. Petroleum ether does not dissolve all of the plant lipid material, and therefore it cannot be substituted for diethyl ether. This method is applicable for the determination of crude fat in dried forages and mixed feeds. It is not applicable for oilseeds, baked and/or expanded products (pet foods), liquid feeds, sugar products, and feeds containing dairy products. Sample preparation

1. A) Weigh 1.5 to 2 g of ground sample into a thimble recording the weight to nearest 0.1 mg (W1). Weigh a second subsample for dry matter determination. - OR B) If the sample contains large amounts of carbohydrates, urea, glycerol, lactic acid or watersoluble components, weigh 2 g sample to nearest 0.1 mg (W1) into a small filter cone. Extract with five 20 mL portions of deionized water allowing each portion to drain, then insert the paper and sample into thimble. 2. Dry for 5 hr at 100oC. 3. Dry beakers to be used for fat determination for at least 1 hr at 100oC. Cool the appropriate number of fat beakers in a desiccator. Weigh and record the weight to the nearest 0.1 mg (W2). 4. When the drying period is over, remove the samples from the oven to a desiccator. (This is a convenient stopping point. The samples should be stored in a desiccator if not immediately extracted.) Reagents

Anhydrous Diethyl Ether, purified for fat extraction. To prevent ether from absorbing water, purchase it in small containers and keep containers tightly closed.

SAFETY MEASURES Ether has an extremely low flash point:      

Have no open flames nearby.  Avoid inhaling ether vapors. Store ether in metal containers. Handle open containers (reagent cans and fat beakers) in a hood. Conduct the extractions in a well ventilated area. Make sure all ether is evaporated from the beakers before placing them in the oven to avoid a fire or explosion.

Procedure

Extraction: 1. Line the fat beakers up in front of the extractor and match the thimbles with their corresponding fat beakers. 2. Slip the thimble into a thimble holder and clip the holder into position on the extractor.

3. Add about 40 mL of diethyl ether (one glass reclaiming tube full) to each fat beaker. 4. Wearing white gloves, slip the beaker into the ring clamp and tightly clamp the beaker onto the extractor. If the clamp is too loose, insert another gasket inside the ri ng. 5. Raise the heaters into position. Leave about a 1/4 inch gap between the beaker and the heating element. 6. Turn on the heater switch, the main power switch, and the condenser water. 7. After the ether has begun to boil, check for ether leakage. This can be detected by sniffing around the ring clamp. If there is leakage, check the tightness of the clamp and if necessary replace the gasket(s). 8. Extract for minimum of 4 hr on a Hi setting (condensation rate of 5 to 6 drops per second), or for 16 hr on a Low setting (condensation rate of 2 to 3 drops per sec). 9. After extraction, lower the heaters, shut off the power and water, and allow the ether to drain out of the thimbles (about 30 min). This is a good stopping point.

Figure: Set-up for fat extraction

Destillation and weighing 1. Remove the thimble from the holder, and rinse the holder with a small portion of diethyl ether from the washbottle. Clip an ether reclaiming tube in place and reattach the fat beaker. 2. Reposition the heaters and turn on the electricity and water. Proceed to distill the ether using a Hi setting. Watch Closely. 3. Distill until a thin layer of ether remains in the bottom of the beaker, and then lower the heater. Do not allow beakers to boil dry. Overheating will oxidize the fat. When the last beaker has finished, shut off the power and water. 4. Wipe the exterior of the beaker clean with a Kimwipe as it is being removed from the extractor. 5. Empty the reclaiming tubes into the "USED" diethyl ether container. 6. Place the tray of beakers in an operating hood to finish evaporating the ether. If there is no hurry, air moving through the hood will be sufficient without heat. A steambath may be used to speed up the evaporation. Beakers should remain in the hood until all traces of ether are gone. Carefully sniff each beaker to determine if any ether remains. 7. Place the beakers in a 102°C gravity convection oven. Warning: If a beaker containing ether is  placed in the oven an explosion may occur. 8. Dry for 1/2 hr. No longer. Excessive drying may oxidize the fat and give high results. 9. Cool in a desiccator and weigh and record weight to the nearest 0.1 mg (W2). The fat beakers are  best cleaned by warming on a steambath or on a hot plate on a low setting. Add some used ether to dissolve the fat. The use of a rubber policeman is helpful. 10. After soaking the beakers in Alconox detergent, wash them using hot water and vigorous  brushing. The thimbles are best cleaned by blowing out with air. If doing a proximate analysis, the residue left in the thimble may be used to determine crude fiber. Calculation

Percent Crude Fat (Ether Extract), DM basis: (AWres - Wta) % Crude fat (wet) =

x DM (%) weight (g) of sample

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Wta = tare weight of beaker in grams Wres = weight of beaker and fat residue in grams

Possible errors

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This process assumes ALL substances soluble in ether are fats This assumption is NOT TRUE. Plant pigments, wax which are also soluble in ether, but do NOT have the same nutritional values of fats However, this error is generally small.

Proximate analysis: Crude fiber     

Principle and Scope Sample preparation Reagents Procedure Calculation

Principle and Scope

Two boiling processes simulate the pH conditions of the digestive tract, acidic in the stomach and alkaline in the small intestine, but nothing more. However, the enzymatic digestion is not simulated. Reagents

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Sulfuric acid (H2SO4) 1.25% - 0.255 ± 0.005 N. 12, 5g, 98% concentrated to 1000 ml with distilled water. Control the concentration by titration. Potassium hydroxide (KOH) 1.25% - 0.223 ± 0.005 N, free from carbonate. 12.5 g to 1000 ml with distilled water. Control the concentration by titration. n-octanol as antifoam. Anhydrous acetone

Procedure

1. Determine separately the sample moisture by heating in an oven at 105 °C to constant weight. Cool in a desiccator. 2. Weight accurately 1 g about of grinded sample (1 mm about) approximately with 1 mg. ==> W1 3. Add 1.25% sulfuric acid up to the 150 ml notch, after preheating by the hot plate in order to reduce the time required for boiling. 4. Add 3-5 drops of n-octanol as antifoam agent. 5. Boil 30 minutes exactly from the onset of boiling.

6. Connect to vacuum for draining sulfuric acid. 7. Wash three times with 30 ml (crucible filled up to the top) of hot deionized water, connecting each time to compressed air for stirring the content of crucible. 8. After draining the last wash, add 150 ml of preheated potassium hydroxide (KOH) 1.25% and 3-5 drops of antifoam. 9. Boil 30 minutes. 10. Filter and wash as point 7. 11. Perform a last washing with cold deionized water aimed to cool the crucibles and then wash three times the crucible content with 25 ml of acetone, stirring each time by compressed air. 12. Remove the crucibles and determine the dry weight after drying in an oven at 105 °C for an hour or up to constant weight. Let cool in a desiccator. This weight (W2)represents the crude fiber plus ash content in comparison to initial weight. Calculation

Calculate the percentage crude fiber (wet weight basis)as follows: (W2 - W1) % Crude fiber (wet) =

x 100 W1

Possible errors

This is the most unsatisfactory principle of the Proximate Analysis. Major problem: 1. acid and base solubilize some of the true fiber (particularly hemicellulose, pectin and lignin). 2. Cellulose too is partially lost. Hence, crude fiber underestimates true fiber. The number or value obtained in this procedure, therefore, is practically meaningless. Most laboratories have phased out the CF term and replaced it with the Van Soest "Detergent Fiber" determination.

Proximate analysis: Nitrogen Free Extract (NFE)   

Principle and Scope Calculation Possible errors

Principle and Scope

A very inaccurate name indeed. This fraction has nothing to do with nitrogen and it's not an extract either.  NFE supposedly represents the soluble carbohydrate of the feed, such as starch and sugar. Crude fiber represents insoluble carbohydrate. Calculation

% NFE = % DM - (% EE + % CP + % ash + % CF) where: NFE = nitrogen free extract DM = dry matter EE = ether extract or crude lipid CP = crude protein CF = crude fiber Possible errors

This is the ONLY component in the Proximate Analysis which is not determined ANALYTICALLY but is calculated by difference. Therefore, NFE accumulates all of the errors that existe in other proximate analysis components (CF is the biggest error)

Proximate analysis: Van Soest fiber analysis  

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Principle and Scope Reagents: 1.  Neutral Detergent Fiber 2. Acid Detergent Fiber Procedure 1.  Neutral Detergent Fiber 2. Acid Detergent Fiber

Principle and Scope

The concept behind the detergent fiber analysis is that plant cells can be divided into less digestible cell walls (contains hemicellulose, cellulose and lignin) and mostly digestible cell contents (contains starch and sugars). Van Soest separated these two components successfully by use of two detergents: a neutral detergent (Na-lauryl sulfate, EDTA, pH =7.0) and an acid detergent (cetyltrimethyl ammonium bromide in 1 N H2SO4). Hemicellulose, cellulose and lignin are indigestible in non-ruminants, while Hemicellulose and Cellulose are partially digestible in ruminants. NDF = Hemicellulose + Cellulose + Lignin ADF = Cellulose + Lignin  Neutral Detergent Fiber is a good indicator of "bulk" and thus feed intake. Acid detergent fiber is a good indicator of digestibility and thus energy intake

Figure: Principle of detergent partitioning of the fiber fraction by Van Soest

Although aquaculture does not deal with ruminants at all, it may be useful to be aware of these analysis. For instance dealing with plant material as imortant feed ingredient or dealing with herbivorous fish. Reagents

Reagents for NDF determination (Neutral detergent fiber) 1.  Neutral detergent solution: Sodium borate decahydrate (Na2B4O7 - 10 H2O) 6.81 g o Disodium ethylenediaminetetraacetate (EDTA, C10H14N2Na2O8) o 18.61 g Sodium lauryl sulfate neutral (C12H25NaO4S) o 30 g 2-ethoxyethanol (Ethylene glycol monoethyl ether, Cellosolve, C4H10O2) o 10 ml Disodium phosphate anhydrous (Na2HPO4) 4.56 g o Distilled water 1000 ml. Pour Sodium borate and disodium EDTA in a beaker and o dissolve with a part of distilled water while heating. Add lauryl sulfate and 2ethoxyethanol. Separately dissolve disodium phosphate in part of distilled water while heating until complete solution is obtained. Mix the two solutions and the remaining distilled water and control the pH which must be between 6.9 and 7.1 2. 2. n-octanol (C8H18O) octilic alcohol 3. 3. Sodium sulfite anhydrous (Na2SO3) 4. Acetone Reagents for ADF determination (Acid detergent fiber) 1. Acid detergent solution: Cetyltrimethylammonium bromide technical grade (C19H42BrN) 20 g o

Sulfuric acid 1 N (H2SO4, 49.04 g/l) 1 l Dissolve tensioactive into acid while stirring to promote dissolution. o 2. n-octanol (C8H18O) octilic alcohol. 3. Acetone. o

Procedure

Procedure for NDF determination (Neutral detergent fiber) 1. Grind the air dried sample to pass 1 mm screen. 2. Weigh in a crucible 1 g of grinded sample with 1 mg approximation. 3. Add 100 ml of neutral detergent solution at room temperature into crucible with 0.5 g of sodium sulfite and some drops of n-octanol. 4. Heat to boiling and reflux 60 minutes from onset of boiling. 5. Filter and wash 3 times with boiling water, then twice with cold acetone. 6. Dry 8 hours at 105 °C and let cool in a desiccator. 7. Weigh. 8. Calculate neutral detergent fiber: NDF % = (weight of crucible + weight of residue) - weight of crucible / weight of sample x 100. Neutral detergent solubles: NDS % = 100 - NDF %. 9. Ash in a muffle at 550 °C 2 hours and let cool in a desiccator. 10. Weigh. 11. Calculate ash insoluble in neutral detergent: loss on ashing / weight of sample x 100. Procedure for ADF determination (Acid detergent fiber) 1. 2. 3. 4. 5. 6. 7. 8.

Grind the air dried sample to pass 1 mm screen. Weigh in a crucible 1 g of grinded sample with 1 mg approximation. Add 100 ml of acid detergent solution at room temperature and some drops of n-octanol. Heat to boiling and reflux 60 minutes from onset of boiling. Filter and wash 3 times with boiling water, then twice with cold acetone. Dry 8 hours at 105 °C and let cool in a desiccator. Weigh. Calculate acid detergent fiber: ADF % = (weight of crucible + weight of residue) - weight of crucible / weight of sample x 100. 9. Ash in a muffle at 550 °C 2 hours and let cool in a desiccator. 10. Weigh. 11. Calculate ash insoluble in acid detergent: loss on ashing / weight of sample x 100.