Appraising the Proximate

analysis system

Dr Gbola Adesogan

Proximate Analysis Summary

FEED SAMPLE

105oC overnight

MOISTURE

Ether

ETHER EXTRACT(EE)

Kjeldahl Nitrogen x 6.25

CRUDE

PROTEIN (CP)

muffle furnace at

600 OC overnight

ASH

(minerals)Boil in acid

& then alkali

CRUDE FIBER (+ ASH)

DM

NFE = 100 – (CP + EE + CF + Ash + Moisture)

Proximate analysis benefits

Pros

– Basic overview, useful for many years

– Cheap

– Rapid

– Easily understood

Cons

Inaccurate quantification of nutritional entities

Oven drying methods

AOAC (1990)

– 135o C for 2 hr (feeds)

– 105o C for 16 h

MAFF

– 100oC for 18 hr

Note that these methods measure DM concentration

rather than moisture concentration

– Reflects importance of DM for expressing results

& formulating rations

Moisture Assay problems

Destructive

VFAs, NH3 & alcohols lost during oven drying at 105oC

– Mainly a problem with fermented feeds –silage,

brewers grains

Oven-drying only measures physically bound water

– Even at 130O C leaves some chemically bound water

(water of hydration, chemically bound to minerals)

Drying above 60o C can create artifacts that hinder

lignin, fiber & ADF analysis

Proportion of volatiles lost from

grass silage during drying

Oven drying Toluene distillation

Formic acid 0.67 0.77

Acetic acid 0.89 0.94

Ethanol 0.99 0.96

Lactic acid 0.41 0.10

Ammonia 1.00 -

Alternative DM methods

Correction equations

– Corrected DM = ODM + 0.67 (formic acid) + 0.89 (total

VFA) + 0.41 (lactic acid) + 0.99 ( Ethanol) + NH3

– Species-specific

Freeze drying (lyophilizing )

– Cooling rapidly to -40°C under vacuum

+ ice removal by sublimation

Slow, low temperature drying e.g. 55oC for 48 h

Alternative moisture methods

Toluene distillation

– Water distilled from sample is trapped under toluene.

– May not remove all the water

– Assumes water left = volatiles lost, - overestimates DM

– Accurate when combined with correction equations

– Hazardous, flammable, carcinogenic vapours

Alternative moisture methods

Karl Fischer Titration (The reference method)

I2 + SO2 + 2 H2O 2HI + H2SO4

– V. accurate (suitable for forages with volatiles)

– Hazardous & time-consuming, costly

– Sampling errors due to small sample size

Alternative moisture methods

Gas chromatography

– Based on ratio of water:alcohol in standard & in

sample analyzed

– Accurate but requires special equipment

Saponification

– Lipid + alkali soap + H2O

– Time consuming

Effect of method on DM content

Mean DM of 14 silages

Oven drying 80oC 304.5

Oven drying 100oC 299.0

Microwave drying 311.2

Freeze-drying 301.0

Toluene distillation 305.8

Karl fischer titration 325.5

Saponification 326.8

(Kaiser et al., 1995)

EE assay problems

Assumed to represent ‘high energy’ components i.e. true fat & oil content (2.5 x energy of carbs)

Includes complex lipids that are low in energy

– E.g. waxes, pigments, fat-soluble vitamins, sterols

Some are not digestible e.g. waxes

May include protein & other ether-soluble, non fat compounds e.g. chlorophyll, resins etc.

Ash assay problems

Excludes volatile minerals eg. I & Se, Cl & Zn

May include sand & other inorganic elements

of organic origin e.g. P & S from proteins

Doesn’t identify individual minerals

Use atomic absorption spectrophotometer

to accurately quantify minerals

CF Assay problems

Contains some lignin, cellulose & hemicellulose

Excludes some fiber fractions

– (included in NFE fraction)

– Cellulose, lignin

Doesn’t reflect the different types of fiber e.g.

cellulose, hemicellulose, lignin

% of feed lignin & cellulose

recovered in CF fraction

Lignin Cellulose Pentosans

Legumes 30 28 63

Grasses 82 21 76

Others 52 22 64

(Van Soest, 1977)

CP assay problems

Determined by Kjeldahl analysis which:

– Ignores nitrates

– Does not indicate unavailable fiber-bound / heat

damaged protein

– Is based on some questionable assumptions

Assumptions of Kjeldahl analysis

1. All proteins contain 16% N

Hence uses constant‘6.25’ to convert N to protein

2. All N in the food comes from true protein

Problems with assumption 1

All amino acids are not created equal

Amino Acid % Nitrogen

Alanine 15.72

Arginine 32.16

Aspartate 10.52

Cystine 11.66

Glutamate 9.52

Therefore, all proteins are not created equal

Protein % Nitrogen

Casein 15.9

Glycinin 17.5

Hemoglobin 16.8

Ovalbumin 15.5

Serum globulin 16.2

Jones, 1931(Bissel, 2002)

Conversion FactorsProtein Kjeldahl Conversion Factor

Corn 6.25

Eggs 6.25

Milk 6.38

Wheat 5.70

Soy 5.71

Whole Cottonseed 5.90

Ammonia 1.21

(6.25 gives 515% CP)

Urea 2.22

(6. 25 gives 281% CP)

Jones, 1931

Problems with assumption 2

Nucleic Acids (DNA/RNA)

Cell Wall Proteins

Nitrogenous Lipids

Ammonium Salts

Secondary Compounds

Free Amino Acids

Peptides

Protein

Nitrogenous

compounds in plants

include:

Hence the true

protein in the plant is

overestimated by the

Crude Protein value.

Hence the name

‘crude’ protein Maynard and Loosli, 1979

(Bissel, 2002)

Alternative CP methods

1. Combustion methods (e.g. LECO analyzers)

– Based on the Dumas (1831) Combustion method

– Sample N burnt to elemental N; measured by thermal conductivity

Pros

– More repeatable/ precise, (error =0.9 vs 0.14 for kjeldahl)

– No reagents; less hazardous ,

– 1.5-2% better estimates of sample N

– Quicker; easier

Cons

– Small sample size problems (now solved)

– May need dried sample (now solved)

True protein methods

2. Ninhydrin analysis

– Involves hydrolyzing protein to aas &

– Adding ninhydrin which:

– reacts with a-amino acids to produce a purple color.

– Color intensity reflects amino acid concentration.

– The test is sensitive but the reagent is difficult to prepare

3. Trichloroacetic acid (TCA) precipitation

– TCA precipitates protein in a liquid e.g. milk

– Protein is filtered off, NPN is retained in filtrate

– Maceration may be required to free fiber- bound protein

True protein methods

4. Colorimetric/ dye binding techniques

– Lowry method, Bio-rad, Bradford’s assay etc.

– Involve reactions between peptide N, or acidic or

basic aas with a dye

– Measure soluble/available N

– Appropriateness for forages with fiber / tannin

bound N

– Results may depend on particle size and peptide

size, presence of reagents that interfere with the

reaction

NFE calculation problems

Not directly measured, calculated by difference

NFE is a very vague measure of several constituents

It should estimate non-fiber carbohydrates but does

not do this accurately

Often includes cell wall components

May be less digestible than CF

Transfer errors

Proximate analysis summary

In addition to all the problems previously mentioned,

Proximate analysis:

– doesn’t account for fecal, urine and gaseous losses.

– Ignores palatability, digestibility, toxicity etc

Hence, most labs have replaced aspects of the

Proximate analysis system with modern analytical

techniques

References (Prox. Anal. Lecture)

D.J.R. Cherney Characterization of Forages by Chemical Analysis. Forage Evaluation

in ruminant Nutrition. Eds Givens, Owens & Ohmed. CABI

Windham WR, Robertson JA, Leffler RG A comparison of methods for moisture

determination of forages for near-infrared reflectance spectroscopy calibration and

validation CROP SCI 27 (4): 777-783 JUL-AUG 1987

Cole, E.R., 1969. Alternative methods to the Kjeldahl estimation of protein nitrogen.

Review of pure and applied chemistry, 19: 109-130.

Galleti, G.C. and Piccaglia, R., 1988. Water determination in silages by Karl Fischer

titration. J. Sci. Food Agric., 43: 1-7.

D J. Levey, Heidi A. Bissell, and Sean F. O’keefe. Conversion of nitrogen to protein and

amino acids in wild fruits. Journal of Chemical Ecology, Vol. 26, No. 7, 2000

Kaiser, A.J, Mailer, R J. and Vonarx, MM. A comparison of Karl Fischer titration with

alternative methods for the analysis of silage dry matter content. J Sci Food Agric.

1995. 69:51-59