determination of proteins , fats and moisture in pharmaceutical preparation in pharmaceutical analysis department.

1. e-mail: Pharmaravi.2011@gmail.com 1

2. PROTEINS
Introduction
Introduction
Proteins are large, complex, organic compounds and
are composed mostly of amino acids linked with
peptide bonds
2

3. Proteins differ from each other according to the type,
number and sequence of amino acids that make up the
polypeptide backbone
Proteins are important constituents of foods for a
number of different reasons
They are a major source of energy, as well as containing
essential amino-acids
oLeucine,
oLysine, oIsoleucine and
oTryptophan, oValine
o Methionine,
3

4. •Structures of Proteins
Primary structure
Secondary structure
 Tertiary structure
 Quaternary structure
4

5. Primary structure
•It is the linear sequence of amino acids joined
together by peptide bond.
•It is simple and unfolded structure of polypeptide
chains.
5

6. Secondary structure
 The primary structure of protein folds to forms
secondary structure.
 It is regular, rigid and tubular
Tertiary structure
 Two or more secondary structure combines to
form a tertiary structure.
 It is a three dimensional folding structure by
completes folding of the sheets and helices of a
secondary structure.
6

7. Quaternary structure
Quaternary structure refers to the way
individual polypeptides combine to form
complexes Quaternary structure
7

8. Amino acid
 An amino acid is a small organic molecule
that, as the name indicates, contains both an
amino component and an acid component
Non-polar aliphatic R-groups
8

9. Aromatic R-groups Positively charged (= basic) R-groups
Negatively charged (= acidic) R-groups
9

10. Qualitative analysis of Proteins
Precipitation reactions
Colour Reactions of Proteins
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.
Precipitation by salts
To 2 ml of protein solution add equal
volume of saturated (NH4)2SO4 solution
White precipitation is formed 10

11. Precipitation by heavy metal salts
To 2 ml of protein solution, add few drops of
Heavy Metals (lead acetate or mercuric
nitrate) solution, results in white precipitation
Precipitation by alkaloidal reagent
To a few ml of sample solution add 1-2 ml of
picric acid solution. Formation of
precipitation indicates the presence of
proteins
11

12. Precipitation by organic solvents
To a few ml of sample solution, add 1 ml of
alcohol. Mix and keep aside for 2 min.
Formation of white precipitation indicates the
presence of protein
Precipitation by heat
Take few ml of protein solution in a test tube
and heat over a flame. Cloudy white
precipitation is observed
Precipitation by acids
To 1 ml of protein solution in test tube, add few
drops of 1% acetic acid, white precipitation is
formed 12

13. Colour Reactions of Proteins
Proteins give a number of colour reactions
with different chemical reagents due to the
presence of amino acid
Biuret test
The Biuret test is a chemical test used for
detecting the presence of peptide bonds
 In the presence of peptides, a copper (II)
ion forms violet-colored coordination in an
alkaline solution
13

14. To 2 ml of protein solution in a test tube add
10% of alkaline (NaOH) solution. Mix and add 4-5
drops of 0.5% w/v copper sulphite (CuSO4)
solution
Formation of Purplish Violet Colour indicates
the presentation of proteins 14

15. Xanthoproteic Test
To 2 ml of protein solution add 1 ml conc.HNO 3
Heat the solution for about 2 minutes and cool
under tap water
A yellow colour is obtained due to the nitration of
aromatic ring
Add few drops of 40% w/v NaOH solution
 The yellow colour obtained initially changes to
orange 15

16.  Millon’s Test
When Millon’s reagent is added to a protein,
a white precipitation is formed, which turn
brick red on heating
Phenols and phenolic compounds, when
mixed with Hg(NO3)2 in nitric acid and traces
16

17. Ninhydrin Test
When protein is boiled with a dilute solution
of ninhydrin, a violet colour is produced
Proteins Hydrolysis
Amino acids
Amino Acids + Ninhydrin
Keto acid + NH3 + CO2 + Hydrindantin
NH3 + Ninhydrin
Pink colour 17

18. Hopkin- Cole’s Test
To a few ml of protein solution in a test tube
add few drops of formaldehyde solution
(1:500) and 2 drops of HgSO4 (Oxidant)
Mix thoroughly and add very gently 2-4 ml of
conc.HgSO4 along the sides of the test tube
The formation of violet coloured ring at the
junction of the two layers is Observed
18

19. Aldehyde Test
To 1 ml of protein solution in test tube add few
ml of PDAB in H2SO4.
Mix the contents and heat if necessary.
The formation of purple colour is observed
Phenol’s reagent Test
To few ml of protein solution in a test tube add 1
ml of NaOH solution (4% w/v) and 5 drops of
phenol’s reagent.
The formation of blue coloured solution
Observed
19

20. Color Reactions of Proteins
Test Composition of Reagent + Result (Color) Group Responsible Importance
Ninhydrin Triketohydrin Hydrate Blue or Purple Free amino and free Test for amino acid, peptides
COOH in determining amino acids
Biuret NaOH + CuSO4 Violet Peptide linkages + Tripeptides up to protein
Millon’s Hg in HNO3 Red Hydroxyphenyl group + Tryptophan
Xanthoproteic Conc. HNO3 Lemon yellow Benzene ring + Tyrosine, Phenyl alanine,
Tryptophan
Hopkins-Cole Glyoxylic acid and conc. Violet ring Indole group + Tryptophan
H2SO4
Liebermann Conc. HCl , sucrose Violet Indole group + Tryptophan
Erlich’s Diazo Pb(OAc)2 Sullfanilic acid in Red orange – + Histidine and Tyrosine
HCl + NH4OH lighter orange
Sakaguchi 10% NaOH, ά naphtol, Intense red color Guanidine + Arginine
alkaline hypobromite
Acree-Rosenheim HCHO conc. H2SO4 Violet ring Indole group +Tryptophan
Reduced Sulfur KOH, Pb(OAc)2 Black ppt Sulfur + Cystine, Cystein and
methionine
Br water Br.H2O, amyl alcohol Pink Indole group + Tryptophan
Molisch ά naphtol in alcoholic Violet ring Carbohydrates Glycoprotein
H2SO4
Adamskiewez Glacial Acetic acid and Reddish violet Indole group + Tryptophan
conc. H2SO4 ring at the
junction 20

21. Quantitative Analysis of Proteins
21

22. Kjeldahl method
The Kjeldahl method was developed in 1883
by a brewer called Johann Kjeldahl
 A food is digested with a strong acid so
that it releases nitrogen which can be
determined by a suitable titration technique.
The amount of protein present is then
calculated from the nitrogen concentration of
the food
22

23. Kjeldahl method
Principles
Digestion Neutralization Titration
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
23

24. Enhanced Dumas method
A sample of known mass
Combustion (900 oC)
CO2, H2O and N2
Nitrogen
Thermal conductivity detector
The nitrogen content is then measured
24

25. Methods using UV-visible spectroscopy
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
Principles
Direct measurement at 280nm
Biuret Method
Lowry Method
Dye binding methods
Turbimetric method 25

26. Direct measurement at 280nm
Tryptophan and tyrosine absorb ultraviolet
light strongly at 280 nm
The tryptophan and tyrosine content of
many proteins remains fairly constant, and so
the absorbance of protein solutions at 280nm
can be used to determine their concentration
Biuret Method
A violet-purplish color is produced when
cupric ions (Cu2+) interact with peptide
bonds under alkaline conditions
The absorbance is read at 540 nm
26

27. Lowry Method
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
somewhere between 500 - 750 nm depending on
the sensitivity required
27

28. Other Instrumental Techniques
Measurement of Bulk Physical Properties
Measurement of Adsorption of Radiation
Measurement of Scattering of Radiation
Methods Based on Different Solubility Characteristics
Salting out
Isoelectric Precipitation
Solvent Fractionation
Ion Exchange Chromatography
Affinity Chromatography
Separation Due to Size Differences
Dialysis
Ultra-filtration
Size Exclusion Chromatography
Two Dimensional Electrophoresis
28

29. 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.
29

30. Fats
Lipids can be defined as Esters of Fatty
acids and are naturally occurring
 Lipids consist of numerous fatlike 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
30

31. Types of fats
CnH (2n+1) CO2H
CnH(2n-1)CO2H
31

32. Classification
Classification
I. "Simple" Carboxylic esters
A. Fats or glycerides (esters of fatty acids with glycerol
e.g. acylglycerols)
Monoglycerides
Diglycerides
Triglycerides
B. Waxes
II. Complex carboxylic esters
•Glycerophospholipids
•Glycoglycerolipids
•Glycoglycerolipid sulfates
32

33. III. Complex lipids (containing amides)
•Sphingolipids
•Glycosphingolipids
IV. Precursor and derived lipids
•Acids (including phosphatidic acid and bile acids)
•Alcohols (including sterols)
•Bases (Sphinganines, etc.)
V. Hydrocarbons
•Straight-chain
•Simple branched
•Polyisoprenoid
VI. Lipid vitamins and hormones
33

34. Qualitative Analysis of Fats
1.Solubility test
2.Microscopic Properties
3.Physical test
4.Emultion formation
5.Sackowski’s test
6.Libermann-Burchrd’s test
7.Zak’s reaction 34

35. 1.Solubility test
Few drops of oil in an test tube
Few ml of oil sample
1-2 ml of carotene
Chloroform Benzene
The formation of two layers
(Insoluble)
Results in the soluble solution
35

36. 1. Microscopic Properties
Lipids appear in white shining chombic shape
crystals
2. Physical test
•A little quantity of oil on a filter paper
Few minutes
The greasy spot penetrating the filter paper
36

37. 4. Emultion formation
A drop of oil on a watch glass
Place carefully 2-3 drops of water over it
Oil droplet is broken into fine droplets,
indicates the process of emulsification
37

38. 5. Sackowski’s test
2 ml of Organic solution (oil) in Chloroform
3 minutes 2 ml of conc.H2SO4
Upper chloroform layer shows red colour and
lower H2SO4 layer shows yellow colour
6. Libermann-Burchrd’s test
2 ml of Organic solution (oil) in Chloroform
(CHCL3)
5-6 drops of Acetic anhydride & 2
drops of conc.H2SO4
Rose colour to Bluish Green coloured solution38

39. 7. Zak’s reaction
2 ml of Organic solution in Chloroform (CHCL 3)
FeCL3 in Acetic Acid conc. H2SO4
Red coloured solution
Quantitative analysis of fats
•Saponification value
•Iodine value
•Hydroxyl value
•Acid value
39

40. Saponification value
The number of milligrams of potassium
hydroxide required to saponify 1gm of fat
under the conditions specified
Mass of oil
Number of moles =
Relative atomic mass
40

41. Iodine value
The mass of iodine in grams that is
consumed by 100 grams of a
chemical substance
Used to determine the amount of
unsaturation in fatty acids
The higher the iodine number, the more C=C
bonds are present in the fat
41

42. Hydroxyl value
It is expressed as the mass of
potassium hydroxide (KOH) in
milligrams equivalent to the hydroxyl
content of one gram of the
chemical substance
Acid value
The mass of potassium hydroxide (KOH)
in milligrams that is required to
neutralize one gram of
chemical substance 42

43. Water / Moisture Determination
Karl Fischer Method
The Water Determination Test (Karl Fischer
Method) is designed to determine water
content in substances, utilizing the
quantitative reaction of water with iodine and
sulfur dioxide in the presence of a lower
alcohol such as methanol and an organic
base such as pyridine, as shown in the
following formulae
H2O+I2+SO2 + 3 C5H5N 2(C5H5N +H) I- + C5H5N + SO3
C5H5N + SO3 + CH3OH (C5H5N +H) O- SO2 +OCH3
43

44. 1.Volumetric titration
Iodine required for reaction with water is
previously dissolved in water determination
TS, and water content is determined by
measuring the amount of iodine consumed as
a result of reaction with water in a sample
Volume(ml) of TS for Water
determination consumed X f (mg/ml)
Water = X 100
Weight of sample (mg)
44

45. 2. Coulometric Titration
2. Coulometric Titration
First, iodine is produced by electrolysis of
the reagent containing iodide ion, and then,
the water content in a sample is determined
by measuring the quantity of electricity which
is required for the electrolysis (i.e., for the
production of iodine), based on the
quantitative reaction of the generated iodine
with water. 45

46. References:
1)http://www.britannica.com/EBchecked/topic/479680/protein/72530/The-isolation-an
2)http://csb.stanford.edu/class/public/readings/Molecular_Architecture_I_Lecture2/V
3)http://www.biology.arizona.edu/biochemistry/problem_sets/aa/aa.html; 2003.
4)http://quizlet.com/8801657/recreate_set/
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6)http://people.umass.edu/~mcclemen/581Proteins.html
7)http://www.sigmaaldrich.com/analytical-chromatography/analytical-
reagents/amino-acid-analysis.html
8)Chemical Reagents
9)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.
10) Sargent, M.G.: Fiftyfold amplification of the Lowry protein assay. Anal.
Biochem. 163 (1987) 476-481.
11) Smith, P.K. et al.: Measurement of protein using bicinchoninic acid. Anal.
Biochem. 150 (1985) 76-85.
12) Katan MB, Mensink RP, Zock PL. Trans fatty acids and their effect on
lipoproteins in humans. Annu Rev Nutr 1995; 15:473-493.
13) Firestone D (May-Jun 1994). "Determination of the iodine value of oils and fats:
summary of collaborative study". J AOAC Int. 77 (3): 674–6. PMID 8012219
14) http://www.ffcr.or.jp/zaidan/FFCRHOME.nsf//$FILE/B43.pdf
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48. THANKYOU