Analysis of Carbohydrates, Proteins, Fats, Ash content, Moisture content

1. PROXIMATE ANALYSIS OF MACRONUTRIENTS
Presented by
Y. Divya
M.Pharm I-II Sem
Pharmaceutical Analysis
SPMVV

2. CONTENTS
• Introduction
a)Proximate Analysis
• Analysis of Macronutrients
a) Carbohydrates
b) Proteins
c) Fats
• Determination of
a)Ash Values
b)Moisture Content
c)Niterogen Content
• Conclusion
• References

3. INTRODUCTION
Quantitative estimation of Macronutrients is carried out
by a method known as Proximate Analysis or Weende
Analysis.
This method was developed in 1860 by Henneberg and
Stohmann in Germany.
Proximate analysis partitioned the compounds in feed into
6 categories based on chemical properties of the compound.

4. Proximate
Analysis
Moisture
Ash
Crude
Protein
Crude
Lipid
Crude
Fiber
Nitrogen
free
extract

5. SCHEME OF PROXIMATE ANALYSIS
Sample
Ground to pass seive #20
Dried in oven at 98-100 0C(Analytical Chemist) and 98-130 0C( Cereal Chemist)
Air Dry Sample
ASH Ether Extract KJELDHAL NITROGEN
Residue
Residue
Residue
Inorganic
mineral matter
acid digestion
alkaline digestion
CRUDE FIBER
CRUDE PROTEIN
ASH
CRUDE FAT
Solution Lipids
Nitrogen Free
Extract
X 6.25

6. CARBOHYDRATES
These are one of the important components in many foods,
Present as isolated molecules,
Present associated (or) chemically bound to other molecules.
If they are attached to proteins, they are known as Glycoproteins,
When attached to lipids known as Glycolipids

7. CLASSIFICATION
Based on number of sugar units in the total chain they are
classified as:
Monosaccharide: Single sugar unit
Disaccharide: Two sugar unit
Oligosaccharide: 3-10 sugar unit
Polysaccharide: More than 10 units
Chaining based on bridging on oxygen atoms: Glycoside
bonds

8. Isolation Of
Carbohydrates
Usually a very little preparation is
needed prior to analysis of
carbohydrates
Aqueous solutions
Require only a little preparation
Carbohydrates bound to
other components
Require isolation of carbohydrates
from the rest of food prior to analysis
Like Fruit juices, Syrups, Honey Nuts, Cereals, Fruits, Bread, vegetables

9. Food is
dried
under
vacuum
Ground to
fine
powder
Defatted by
solvent
extraction
Boil
defatted
sample
with 80%
alcoholic
soln
Mono &
Oligo
saccharides
are soluble,
but
polysacchari
des &
dietary fiber
are not
soluble
Filter and
collect the
two
filterate
fractions.
These two fractions are dried and weighed to determine their
concentration
Procedure For Isolation Of Carbohydrates

10. Since various other small molecules like amino acids, organic acids,
pigments, vitamins, minerals are also present in alcoholic extract, they are
removed by following methods:
Treating the solution with Clarifying agents: Like heavy metal salts
(Lead acetate), they form insoluble complexes that can be removed by
filtration.
By passing it through one or more Ion Exchange Resins: Since many
Mono & Oligosaccharides are polar non charges molecules they can be
separated from charged molecules by passing through Ion exchange
columns

11. ANALYSIS OF CARBOHYDRATES
Colored Reactions
Chemical Reactions
Titrimetric- lane Eynon Method
Gravimetric- Munson & Walker Method
Colorimetric- Anthrone Method
Enzymatic Methods
Physical Methods
Polarimetry
Refractive Index
Density
Infra Red
Immuno Assay

12. Test Name Procedure Observation
Molisch Test 1ml test soln + 2drops of alpha
napthol, mix and add Conc
H2SO4
along sides of test tube
Purple ring appears at the
junction of two layers
Fehling’s Test 1ml of test soln + 1ml of
Fehling’s reagent, heat on
BWB for 5 mins.
Reddish Brown ppt
Benedict’s Test 1ml of test soln + 1ml of
Benedict’s reagent, heat on
BWB for 5 mins.
Reddish Brown ppt
Barfoed’s Test 1ml of test soln + 2ml of
Barfoed’s reagent, heat on
BWB for 2 mins.
Green, red or yellow ppt
COLOR REACTIONS OF CARBOHYDRATES

13. Test Name Procedure Observation
Seliwanoff Test 0.5ml of sample + 2ml of
Seliwanoff’s reagent, heat on
BWB for 2 mins
Red product
Hydrolysis Test 6ml of 1% Sucrose + 2 drops
of Conc HCl, heat on BWB
for 5 mins
Then test for Fehling &
Benedict and all other
previous test
Iodine Test 0.5ml of fresh starch soln + 1
drop of I2 soln
0.5ml dextrin + 1 drop I2
soln
Dark bule color
Yellow-Negative
Violet color
Osazone Test 0.5g Phenyl hydrazine + 1
spoon Sodium acetate + 2ml
glucose, heat on BWB for
45min
Broomed shaped crystals
(Glucose)

14. LANE EYNONMETHOD:
Determines the concentration of Reducing Sugars in a sample
The reducing sugars in the carbohydrate solution react with
the copper sulfate present in the flask. Once all the copper
sulfate in solution has reacted, any further addition of
reducing sugars causes the indicator(Methylene Blue) to
change from blue to white.
PRINCIPLE:

15. a
12.5g sample + 25 ml of 10% neutral lead soln + some qty
of alumina cream. Make upto 250ml with lead acetate
Filter and collect filterate
100ml filterate + 10ml 10% soln of Potassium Oxalate,
make upto 500 ml. shake well
Filter and collect filterate
Subject for analysis
SAMPLE PREPARATION

16. In 250ml of Erlenmeyer flask add 10ml of Felhing’s reagent
Add 15ml sugar soln, boil for 15sec, till faint blue color appears
Add 2-5 drops of 1% aqueous soln of methylene blue, continue
heating
Add sugar soln, until the reduction of dye.
Amount of Sugar = Factor X 100
Titer Value
Factor can be obtained from literature
METHOD:

17. GRAVIMETRIC METHOD
Determines the concentration of reducing sugars
PRINCIPLE:
Carbohydrates are oxidized in the presence of heat and an excess of copper
sulfate and alkaline tartrate under carefully controlled conditions which
leads to the formation of a copper oxide precipitate.
reducing sugar + Cu2+ + base oxidized sugar + CuO2
The amount of precipitate formed is directly related to the concentration of
reducing sugars in the initial sample.
The concentration of precipitate present can be determined gravimetrically
or titrimetrically.

18. ANTHRONE METHOD
Colorimetric method of determining the concentration of the total sugars in a
sample.
Sugars react with the anthrone reagent under acidic conditions to yield a blue-
green color.
The sample is mixed with sulfuric acid and the anthrone reagent and then
boiled until the reaction is completed. The solution is then allowed to cool and
its absorbance is measured at 620 nm.
This method determines both reducing and non-reducing sugars because of the
presence of the strongly oxidizing sulfuric acid

19. PROCEDURE:
Take 3 test tubes
Blank Standard Unknown
Glucose (ml) 0.0 0.5 0.5
Distilled water (ml) 1.0 0.5 0.5
Anthrone (ml) 4.0 4.0 4.0
Mix well, place in BWB for 10 mins, cool to RT, Read Optical density
Conc of Unknown = Abs of Unknown X Conc of Std
Abs of Std

20. ANALYSIS OF
POLYSACCHARIDES
Starch is the most common digestible polysaccharide found in foods
Processed food samples are
dried, ground.
Dispersed in 80% ethanol
Mono & Oligosaccharides are
soluble
Starch is insoluble and can be
separated by filtering
Sample Preparation

21. Gravimetrically
Titrimetrically
Physical
Methods
• By collecting, drying
and weighing the
precipitate
• By determining the
amount of iodine
required to precipitate
the starch
• Density, Refractive
Index, Polarimetry
ANALYSIS METHOD FOR STARCH
To starch
add Enzyme
Breakdown
to Glucose
Subject to
analysis

22. ANALYSIS OF FIBERSDietaryFiber
Cell Wall
Polysaccharides
Non Cell Wall
Polysaccharides
Lignin
Cellulose – Principal structural component of
all cell wall associated with Hemicellulose &
Lignin
These are Indigestable Carbohydrates but not
derived from Cell wall of plants, Guar &
Locust gum, agar, alginates etc
Non carbohydrate polymer containing 40
aromatic sub units

23. METHOD OF ANALYSIS FOR FIBERS
Two methods are available:
Gravimetrically: by weighing the mass of insoluble fiber fraction isolated from
sample( Crude Fiber Method & Total, Insoluble and soluble Fiber Method)
Chemically: by breaking down the fiber into its constituents monosaccharides
Remove
Lipid
Add 1.25%H2SO4
& NaOH
Insoluble
Residue
Remove
Lipid
Add Enzyme & 95%
alcohol
Insoluble
Residue
Crude Fiber Method Total, Insoluble and Soluble Fiber Method

24. PROTEINS
Proteins are large, complex, organic compounds
composed mostly of amino acids linked with peptide bonds.
Qualitative Analysis Quantitative Analysis
a) Precipitation Reactions a) Kjeldhal Method
b)Color Reactions b) Enhanced Dumas Method
• Biuret Test c)UV Spectroscpic Method
• Xanthoprotein Test d) Lowry Method
• Millon Test
• Ninhydrin Test
• Hopkin’s Test
• Aldehyde Test
• Phenol Reagent Test
ANALYSIS OF PROTEINS

25. PRECIPITATION REACTIONS
Protein exist in colloidal solution due to hydration of polar
groups (-COO, NH3
+, -OH)
They can be precipitated by dehydration or neutralization
of polar groups.
Test Observation Inference
By Salts 2ml Protein soln + equal vol of
ammonium Sulphate
White ppt
Heavy Metal Salts 2ml of Protein soln + few drops of Lead
acetate
White ppt
Alkaloidal Reagent Sample + 1-2ml of Picric acid soln Formation of ppt
Organic Solvents Sample + 1ml alcohol, keep for 2mins Formation of ppt
Heat Sample is heated over flame Cloudy White ppt
Acids Sample + few drops 1% Acetic acid White ppt

26. Test Observation Inference
Biuret 2ml sample + 10% NaOH, Mix, + 4-5
drops of 0.5% W/V Copper Sulphite
Soln
Purplish Violet color
Xanthoprotein 2ml sample + 1ml Conc HNO3
heat for 2mins, cool + few drops of
40% W/V NaOH
Yellow color changes to
orange
Millon’s Test 2ml Sample + Millon’s Reagent White ppt, turns to brick red
on heating
Ninhydrin 2ml Sample + dil. Soln of ninhydrin Violet color
Hopkin Cole’s Test 2ml Sample + few drops of HCHO + 2
drops HgSO4 ,mix + 2-4ml of HgSO4
along sides of test tube
Violet color ring at the
junction of two layers
Aldehyde Test 1ml Sample + Few ml PDAB in H2SO4
Mix and heat
Purple Color

27. QUANTITATIVE ANALYSIS
Kjeldhal Method:
This is a more convenient and simpler method for the estimation of nitrogen and is
largely used for the estimation of nitrogen in fertilizers, food stuffs, drugs, etc.
PRINCIPLE:
A known mass of the organic compound is digested (heated) with conc.H2SO4 in
presence of potassium sulphate and a little copper sulphate or mercury in a long -
necked flask called Kjeldahl’s flasks. Potassium sulphate raises the boiling point of and
thus ensures complete reaction while copper sulphate or mercury catalyses the reaction.

28. The food sample to be analyzed is weighed into a digestion flask
(NH4)2SO4 + 2 NaOH
2NH3 + 2H2O + Na2SO4
H3BO3 (boric acid)
NH4
+ + H2BO3
- (borate ion)
H+
H3BO3
Digestion Neutralization Titration
PROCEDURE:

29. This method is applicable to all organic compounds containing nitrogen
Principle: A known mass of the organic substances is heated with excess of
copper oxide in an atmosphere of CO2 .Carbon, hydrogen and sulphur (if
present) are oxidised to CO2, H2O, SO2 while nitrogen gas is set free. Any oxide
of nitrogen that may be formed is reduced back to free nitrogen by passing over a
hot reduced copper gauze.
ENHANCED DUMAS METHOD

30. A sample of known mass
CO2, H2O and N2
Combustion (900 oC)
Nitrogen
Thermal conductivity detector
The nitrogen content is then measured
PROCEDURE:

31. These methods use either the natural ability of proteins to absorb (or
scatter) light in the UV-visible region of the electromagnetic spectrum, or
they chemically or physically modify proteins to make them absorb (or
scatter) light in this region
METHODS USING UV-VISIBLE SPECTROSCOPY
PRINCIPLES
Direct measurement at 280nm
Biuret Method
Lowry Method

32. a)Direct Measurement At 280nm:
Tryptophan and tyrosine absorb ultraviolet light strongly at 280 nm.
The Protein content of tryptophan and tyrosine remains fairly
constant, and so the absorbance of protein solutions at 280nm can be
used to determine their concentration
b)Biuret Method:
When cupric ions (Cu2+) interact with peptide bonds under alkaline
conditions a violet-purplish color is produced .And absorbance is read at
540 nm

33. The Lowry method combines the Biuret reagent with another
reagent (the Folin-Ciocalteu phenol reagent) which reacts
with tyrosine and tryptophan residues in proteins.
 This gives a bluish color which can be read between 500 - 750 nm
depending on the sensitivity required
500nm- for determination of high conc Proteins
750nm-for determination of low conc Proteins
C) Lowry Method

34. AMINO ACID ANALYSIS
 Amino acid analysis is used to determine the amino acid
composition of proteins.
 A protein sample is first hydrolyzed (e.g. using a strong acid) to
release the amino acids, which are then separated using
chromatography,
e.g., ion exchange, affinity or absorption chromatography.

35. FATS
Lipids can be defined as “Esters Of Fatty Acids” and are naturally occurring.
Lipids consist of numerous fat like chemical compounds that are insoluble in
water but soluble in organic solvents.
Lipid compounds include Monoglycerides, Diglycerides, triglycerides,
phosphatides, cerebrosides, sterols, terpenes, fatty alcohols, and fatty acids

36. CLASSIFICATION
I. Simple Carboxylic Esters
a) Fats or Glycerides- Acylglycerols
b)Waxes
II Complex Carboxylic Esters
Glycerolipids
Glycoglycerolipids
Glycoglycerolipid Sulphates
III. Complex Lipids (Amides)
Sphingolipids & Glycosphingolipids
IV.Precursor & Derived Lipids
Acids (Phosphatidic & Bile acid)
Alcohols (Sterols)
Bases ( Sphinganines)
V.Hydrocarbons (Straight & Branched)
VI.Lipid Vitamins & Hormones

37. ANALYSIS OF FATS
Qualitative Analysis Quantitative Analysis
Solubility Test Saponification Value
Microscopic Properties Iodine Value
Physical Test Hydroxyl Value
Emulsion Formation Acid Value
Sackowski’s Test
Libermann Burchard Test
Zak’s Reaction

38. QUALITATIVE ANALYSIS OF FATS
Test Observation Inference
Solubility Test a) Few drops of oil + 1-2ml Carotene.
b)Sample + Chloroform/ Benzene
a) Formation of 2 layers
(Insoluble)
b) Soluble
Microscopic Properties Observed through Microscope Rhombic shaped crystals
Physical Test Spot Sample on filter paper & Observe Greasy spot penetrates filter paper
Emulsion Formation Spot drop of oil on watch glass & 2-3 drops of
water
Oil droplet is broken into fine
droplets
Sackowwski’s Test 2ml organic soln + 2ml of Conc H2SO4
& boil for 3mins
Upper layer(Chloroform)-Red
Lower layer(H2SO4)-Yellow
Libermann Burchard 2ml of organic soln in chlororform + 5-6 drops
acetic anhydride + 2 drops conc H2SO4
Rose to Bluish green color soln
Zak’s Reaction 2ml of organic soln in chlororform + Ferric
chloride + Conc H2SO4
Red colored soln

39. QUANTITATIVE ANALYSIS OF FATS
Number of moles =
Saponification value:
The number of milligrams of Potassium hydroxide required to saponify 1gm
of fat under the conditions specified.
No. of Moles = Mass of oil
Relative atomic mass
Iodine Value:
The mass of Iodine in gms that is consumed by 100gms o chemical
substance.
Used to determine “the amount of Unsaturation of fatty acids”.
Higher the Iodine number, the more C=C bonds are present in the fat.

40. Hydroxyl Value:
It is expressed as the mass of Potassium hydroxide in milligrams
equivalent to the hydroxyl content of one gram of the chemical
substance
Acid Value:
The mass of Potassium hydroxide in milligrams that is required to
neutralize one gram of chemical substance

41. DETERMINATIONOF
ASHVALUE
Remnant of crude drug after incineration
contains mostly inorganic salts and non-volatile
inorganic components known as “ASH”
Ash value can be determined by:
 Total ash
 Acid insoluble ash
 Water soluble ash

42. DETERMINATION OF TOTAL ASH
Incinerate 2-3gm of air dried sample in
silica crucible
At about 450oC, until free from carbon.
Cool and weigh.
Collect the residue &
incinerate it .
If carbon free ash content
cannot be obtained
Then pass on
to ashless
filter paper
Later on filter paper
Calculate the %ash with reference to air
dried drug

43. DETERMINATION OF ACIDINSOLUBLE
ASH
Boil the ash
obtained in
Total ash with
25ml 2M HCl
for 5mins
Collect the
residue on
ashless filter
paper
Wash with hot
water & ignite
Cool in
dessicator and
weigh.
Calculate the % acid insoluble ash with
reference to the air dried drug

44. DETERMINATION OF
WATER SOLUBLE ASH
Boil the ash obtained in total ash with
25ml of water for 5mins
Collect insoluble matter on ashless
filter paper
Wash with hot water, ignite for
15min at a temp 450oC
Substract the wt of the insoluble
matter from the wt of the ash taken
Calculate the % of water soluble ash with reference to the air dried drug

45. MOISTURE CONTENT
Moisture is expected component of crude drug which must
be eliminated as far as possible
Methods Available:
Loss on drying
Azeotropic Volumetric Method
Karl Fischer Titration
Colorimetric Method

46. AZEOTROPIC VOLUMETRIC METHOD
The Azeotropic Method gives a direct measurement of water (or) other volatile
constituents present in crude drugs being examined when sample is distilled
together within an immiscible solvent such as toluene to xylene, the water
present in the sample is absorbed by the solvent.
Dean Stark Apparatus

47. Procedure:
Crude drug was
weighed in flask
Add toulene.
Connect apparatus
Receiving flask is
filled with toulene
Heated until no more
water is distilled over
Both toulene and
water is distilled over
As water is heavier
than toulene, it sinks
to bottom of receiving
tube
When the apparatus
is cooled. Toulene
and water is
separated completely
The volume of water
distilled is given by
% = 100 X N
W
W= wt in gm of material
examined
N= No of ml of water

48. KARL FISCHER TITRATION
Most extensively used chemical method
for the determination of Total Moisture.
It can be applicable to very small
quantities of mositure
The reagent contains solution of Iodine, Sulphurdioxide and Pyridine in dry
Methanol constitutes KFR (Karl Fischer Reagent)
I2 + SO2 + 3C5H5N + H2O 2C5H5N + 2HI + C5H5N+SO3
C5H5N+SO3 + CH3OH C5H5N
OSO2CH3
H

49. Primary standardization of reagentPlace36mlofdrymethanol
intitrationvessel
Add KFR & 150-
350gnm of sodium
tartarate
TitratewithKFR
Determine endpoint
using Pt electrode
Determineaccuratevolume
ofKFRrequired
Calculate Water
equivalent factor
F = wt in mg of Sodium tartarate X 0.1566
ml of KFR

50. Test for sample
% Water Content = Vol of KFR X Water Eq factor
Wt of Sample

51. Determination of nitrogen
The two most commonly used methods for the estimation of
nitrogen are:
• Duma’s Method,
• Kjeldhal Method

52. conclusion
Finally it can be concluded that, though the Proximate
analysis and Dietary fiber analysis produce satisfactory
results, still there is a need for development of few other
advanced versatile methods,
Eg: Where in case of non-ruminants amino acid value is
more important than total protein.

53. References:
1.http://www.aquaculture.ugent.be/Education/coursematerial/online%20courses/
ATA/analysis/carb-mon.html
2. Hedge, J E and Hofreiter, B T (1962) In: Carbohydrate Chemistry 17 (Eds
Whistler R L and Be Miller, J N) Academic Press New York.
3.http://www.britannica.com/EBchecked/topic/479680/protein/72530/The-
isolation-and-determination-of-proteins#toc72531
4.http://csb.stanford.edu/class/public/readings/Molecular_Architecture_I_Lecture
2/Voet_and_Voet_BOOK_00_Chapter6_Protein_Structure.pdf
5.http://www.biology.arizona.edu/biochemistry/problem_sets/aa/aa.html; 2003.
6.http://quizlet.com/8801657/recreate_set/
7.http://quizlet.com/8801729/color-reactions-of-proteins-flash-cards/
8.http://people.umass.edu/~mcclemen/581Proteins.html
9.http://www.sigmaaldrich.com/analytical-chromatography/analytical-
reagents/amino-acid-analysis.html.
10.O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall: Protein Measurement
with the Folin Phenol Reagent, J. Biol. Chem. 193 (1951) 265 - 275.