Appraising the Proximate
analysis system
Dr Gbola Adesogan
Proximate Analysis Summary
FEED SAMPLE
105oC
overnight
MOISTURE
DM
Kjeldahl Nitrogen x 6.25
muffle furnace at
600 OC overnight
CRUDE
PROTEIN (CP)
Ether
ASH
(minerals)
Boil in acid
& then alkali
ETHER EXTRACT(EE)
CRUDE FIBER
(+ ASH)
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
Oven drying 80oC
Oven drying 100oC
Microwave drying
Freeze-drying
Toluene distillation
Karl fischer titration
Saponification
Mean DM of 14 silages
304.5
299.0
311.2
301.0
305.8
325.5
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
(Bissel, 2002)

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
Conversion Factors
Protein
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

Nitrogenous
compounds in plants
include:
(Bissel, 2002)
Nucleic Acids (DNA/RNA)
Cell Wall Proteins
Nitrogenous Lipids
Ammonium Salts
Hence
the true
protein in the plant is
overestimated by the
Crude Protein value.
the name
‘crude’ protein
Secondary Compounds
Free Amino Acids
Peptides
Protein
Hence
Maynard and Loosli, 1979
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