Proteins

1.2 ANALYTICAL TECHNIQUE

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 1/33
 Contents
1 Introduction
(aim, basic principles, considerations, constrains,
international standardized techniques)
2 Some rapid qualitative tests
3 Kjeldahl
4 Spectroscopy UV-VIS
5 Amino acid analyzer
6 Study case

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 2/33
 1 Introduction (1/4)
To obtain:
• Protein content: nutrition labelling, pricing  melamine scandal?
• Content of a particular protein in a mixture: functional property
investigation,
• Protein content during isolation and purification of a protein
• Amino acid composition
• Nutritive value of a protein
• Nonprotein nitrogen

Sources: Nielsen 2010, p. 135.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 3/33
 1 Introduction (2/4)
Basic principles
Determinations of:
• Nitrogen
• Peptide bounds
• Aromatic amino acids
• Dye-binding capacity
• Ultraviolet absoptivity of proteins
• Light scattering properties

Sources: Nielsen 2010, p. 135.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 4/33
 1 Introduction (3/4)
Factors for consideration
• Food components: lipids and charbohidrates
• Sensitivity, accuracy, precision, speed, cost of analysis
• Purpose
• Standard (international, regional, national)

Constraints
 other components with similar physicochemical properties, e.g. :
non coded amino acids, small peptides, nucleic acids,
phospholipids, amino sugars, porphyrin, some vitamins alkaloids,
uric acid, urea, ammonium ions.

Sources: Nielsen 2010, p. 135.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 5/33
 1 Introduction (4/4)
International standardized techniques:
1. Kjeldahl method
2. Dumas method
3. Infrared spectroscopy
4. Biuret method
5. Anionic dye-binding method
6. Spectroscopy UV-VIS
7. Amino acid analyzer

Sources: Nielsen 2010, p. 136ff; AOAC International 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 6/33
 2 Some rapid qualitative tests (1/3)
1. Biuret reaction
• Reaction under alkaline conditions:
cupric ions + peptide bonds (at least 2)  violet-purplish color
• Absorbance of the color is read at 540 nm
• Constraint: urea

Sources: Nielsen 2010, p. 139; Andarwulan et al. 2011, p. 126

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 7/33
 2 Some rapid qualitative tests (2/3)
2. Lowry reaction
• More sensitif than Biuret
• Combination between reaction Cu2+ with peptide bonds and the
reduction of the Folin-Ciocalteau phenol reagent
(phosphomolybdic-phosphotungstic acid) by tyrosine and
tryptophan  bluish color (can be read at 650 nm)
• Constraint: phenolic compound

Sources: Nielsen 2010, p. 139f; Andarwulan et al. 2011, p. 129

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 8/33
 2 Some rapid qualitative tests (3/3)
3. Ninhydrin reaction
Amino acids + ninhydrin Ruhemann’s purple (blue-violet)  570 nm
Imino acids (proline & hydroxyprolin) + ninhydrin  yellow  440 nm

Sources: Fennema 1996, p. 331; Belitz et al. 2009, p. 22.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 9/33
 3 Kjeldahl (1/5)
Principle
 Quantify the N content through 3 main steps:

1. Digestion
Sample + H2SO4 + heat + catalyst  (NH4)2SO4 + CO2 + H2O
catalyst: HgO, Selenium dioxide:Copper sulfate = 3:1

2. Neutralization + destillation
(NH4)2SO4 + 2 NaOH  Na2SO4 + 2 H2O + 2 NH3
2 NH3 + 2 H3BO3  2 NH4H3BO3
indicators: methylene blue & methyl red

3. Titration
2 NH4H3BO3 + 2 HCl   2 NH4Cl + 2 H3BO3

Sources: Nielsen 2010, p. 136ff; AOAC 1999, Ch. 33, p. 10ff; Andarwulan et al. 2011, p. 120
AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 10/33
 3 Kjeldahl (2/5)
Calculation
% N = (corrected HCl vol.) x N HCl x 14.007 x 100%
mg of sample
= ml x (mol/1000 ml) x (g N/mol) x (1/mg sampel) x 100%
= (g N/1000 mg sampel) x 100%
= (g N/g sampel) x 100%
= % w/w (w.b.)
%P =%NxF

w: weight
w.b.: wet basis
F: conversion factor (see Nielsen 2010, p. 137)

Sources: Nielsen 2010, p. 137; AOAC 1999, Ch. 33, p. 10ff; Andarwulan et al. 2011, p. 120
AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 11/33
 3 Kjeldahl (3/5)
Advantages
1. Applicable to all types of foods
2. Relative inexpensive (manual system)
3. Official method for crude protein content

Limitation
1. Calculate total N  crude protein
How to recognize protein nitrogen and nonprotein nitrogen of the
sample? precipitation by tricloroacetic acid (TCA)
2. Time consuming (2 hours)  Dumas method (3 minutes)
3. Poorer precision  Spectroscopy method
4. Corrosive reagent  Dumas method

Sources: Nielsen 2010, p. 138; AOAC 1999, Ch. 33, p. 12ff.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 12/33
 3 Kjeldahl (4/5)
Alternative: Dumas method
Principles

samples combustion reduction detection

Advantages GC
• No hazardous chemical
• Quick

Disadvantages
• Expensive
• Crude protein
• Sample with high content of fat  explosion
Sources: Nielsen 2010, p. 138; AOAC 1999
AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 13/33
 3 Kjeldahl (5/5)
Precipitation of protein by TCA
2,2,2-trichloroacetic acid
• The most efficient precipitating agent for native protein
• Reversible association reaction
• Protect the native conformation
of protein
• Without cleaving the protein
• A stable “molten globule-like“
 parcially structured inter-
mediate state
• Stabilizing the β-strands at the
N- and C- terminal ends

Sources: Rajalingam et al. 2009, p. 980ff

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 14/33
 4 Spectroscopy UV-VIS (1/7)
Basic principles of spectroscopy
 Interaction of electromagnetic radiation with matter
 UV, Vis, IR, NMR

Sources: Nielsen 2010, p. 380

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 15/33
 4 Spectroscopy UV-VIS (2/7)

Sources: Nielsen 2010, p. 389

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 16/33
 4 Spectroscopy UV-VIS (3/7)
Assay method Principle Wave lenght Interferences
Biuret Cu + peptide  purple 540 nm Amines, ammonium sulfat

Lowry Biuret reaction + reduction by 650 nm Phenol, aromatic amino

tyrosine and tryptophan acids, reducing sugar,
ammonium sulfat
Bradford Protein + Coomassie Brilliant 595 nm Detergents
Blue (red  blue)
Bicinchoninic Under alkaline condition: 562 nm High concentrations of
acid (BCA) protein and peptides reduce metals, strong reducing
Cu2+ to Cu+, then react with agents, chelating agents
BCA  purple
UV 280 Determination of tyrosine and 280 nm Nucleic acids, phenols,
tryptophan in protein aromatics
Dye-Binding Reaction with excess amount Amino black Starch, Ca, P
of anionic dye 615 nm;
Orange G
Sources: Nielsen 2010, p. 138ff; Upstone 2000, p. 13 485 nm
AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 17/33
 4 Spectroscopy UV-VIS (4/7)
Calculation
Sampel : 50 ml whole goat milk with total solid 13.57%
Specific gravity at 20oC is 1.033 kg/L
Assay method: BCA (562 nm), dilution: 1 ml  100 ml
Average absorbance of 1 ml diluted sample was 0.4576
Calculate the protein content both wet basis and dry basis!
The absorbance analysis for BSA standard (Nielsen 2010, p. 145):
BSA (mg/ml) Mean absorbance
0.2 0.25
0.4 0.53
0.6 0.75 y = 1.11 x + 0.058
0.8 0.95
1.0 1.15

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 18/33
 4 Spectroscopy UV-VIS (5/7)
Result
Protein content of the sample:
3.48% w.b.
25.64% d. b.

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 19/33
 4 Spectroscopy UV-VIS (6/7)
Advantages
1. Simple and rapid
2. No corrosive agent
3. Does not measure nonprotein N
4. More precise than Kjeldahl method
5. Some methods are official
• Biuret: cereal, meat, soybean
• Dye-binding: wheat flour, soy products, meat, milk
• IR: milk, grains, cereal, meat, dairy product

Sources: Nielsen 2010, p. 138ff; AOAC International 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 20/33
 4 Spectroscopy UV-VIS (7/7)
Limitation
1. Instruments are quite expensive
2. Some methods are not common for food mixture (need
precipitation and extraction)
3. Protein standard and calibration are needed
4. Some coumpounds may interfere the analysis
5. The basic principles and limitations of each essay should be taking
into consideration in selecting the method

Sources: Nielsen 2010, p. 138ff; AOAC International 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 21/33
 5 Amino acid analyzer (1/9)
To obtain:
Compositions of the amino acids of a protein
Quantify each amino acids of a protein
 Quantitative and qualitative

Basic principle
1. Release amino acids by hydrolysis
2. Separation using chromatographic techniques
3. Quantification using amino acids standard

 instrument: ion-exchange chromatography (often), reversed-phase

liquid chromatography, gas-liquid chromatography

Sources: Nielsen 2010, p. 271; AOAC International 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 22/33
 5 Amino acid analyzer (2/9)

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 23/33
 5 Amino acid analyzer (3/9)
5.1 Hydrolysis
 boil in 6N HCl for 24h

Consideration
• Tryptophan is destroyed by acid hydrolysis
• Methionine, cysteine, threonine, serine are progressively
destroyed during hydrolysis
• Asparagine and glutamine are converted to aspartic and glutamic
acid
• Isoleucine and valine are hydrolyzed more slowly than other aa
• Tyrosine may be oxidized

Sources: Nielsen 2010, p. 271; AOAC International 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 24/33
 5 Amino acid analyzer (4/9)
Solution:
• Threonine and serine  3 periods of hydrolysis (24, 48, 72 h) 
estimated from zero time assuming first-order kinetics
• Valine and isoleucine estimated from 72 h hydrolysate
• Cysteine and cystine are converted into more stable compound,
i.e. cysteic acid by hydrolysis in performic acid and then hydrolized
in 6N HCl
• Tryptophan is hydrolyzed in basic solution prior to chromatography
or analyse using other methods

Sources: Nielsen 2010, p. 271; AOAC International 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 25/33
 5 Amino acid analyzer (5/9)
5.2 Partitioning by chromatographic technique
• Separation is based on the relative solubility of the sample
between two phases, mobile and stationary
• Normal phase: polar stationary phase and non-polar solvent
• Reverse phase: non-polar stationary phase and a polar
solvent

Mobile Phase Stationary Phase

Solvent Bonded Phase

Sources: Nielsen 2010, p. 271; AOAC International 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 26/33
 5 Amino acid analyzer (6/9)
Ion exchange chromatography
Anion exchange  the inert surface is interacting with anions

“stuck”  lower the pH or increase the salt concentration of the buffer

Sources: Nielsen 2010, p. 483ff
AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 27/33
 5 Amino acid analyzer (7/9)
5.3 Detector
 Amino acids  ninhidrin methods  Spectofotometer:
• UV
• Single wavelength (filter)
• Variable wavelength (monochromatic)
• Multiple wavelengths (PDA)
• Fluorescence
• Electrochemical
• Mass spectrometric

Sources: Nielsen 2010, p. 271; AOAC International 1999

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 28/33
 5 Amino acid analyzer (8/9)
5.4 Calculation
The chromatograph (caution exchange chromatography)

Sources: Kahn and Faiz 2008, p. 11

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 29/33
 5 Amino acid analyzer (9/9)
Advantages
• Analysis amino acids both qualitative (composition) and quantitative
• May recognize the odd amino acids
• Peptides and noncoded amino acids might be recognized
• International technique
• Rapid (about 30 minutes)

Limitation
• Expensive
• Expert analyst, basic knowledge of chromatography is needed

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 30/33
 6 Study case
Which method is the most appropriate for:
1. Protein analysis for nutritional labelling?
2. Qualitative protein test for a novel food?
3. Protein adulteration test of imported food? (melamine scandal)

4. Rapid analysis for protein content of milk and cereal?

5. Analysing the content and the composition of essential amino
acids of milk?

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 31/33
 References (1/2)
Figures
Spectroscopy UV-VIS
http://faculty.sdmiramar.edu/fgarces/LabMatters/Instruments/UV_Vis/Cary50_Pic
/conventional-spectrophotometer.png
Chemical structure of melamine http://www.sciencebase.com/images/melamine-
structure.jpg
Reaction of peptide bonds with cupric ions
http://www.demochem.de/Grafik/biuret1.gif
HPLC image
http://www.chromatographytoday.com/assets/file_store/pr_files/17842/images/t
humbnails/800w-arc_pinnacle_frontal_open_12x18cm_300dpi.jpg
Structure of TCA http://0.tqn.com/d/chemistry/1/0/d/Q/1/Trichloroacetic_acid.jpg
Anion exchange of chromatography
http://www.mikeblaber.org/oldwine/BCH4053/Lecture07/ion_exch.jpg

AZG team – Department of Community Nutrition – Faculty of Human Ecology – IPB 32/33
 References (2/2)
Nielsen SS. 2010. Food analysis. 4th Ed. Springer Science + Business Media, LLC: New York,
Dordrecht, Heidelberg, London.
Nielsen SS. 2010. Food analysis: Laboratory manual. 2nd Ed. Springer Science + Business
Media, LLC: New York, Dordrecht, Heidelberg, London.
Kusnandar F. 2010. Kimia pangan: Komponen makro. Dian Rakyat: Jakarta.
Andarwulan N, Kusnandar F, Herawati D. 2011. Analisis pangan. Dian Rakyat: Jakarta.
Winarno FG. 1997. Kimia pangan dan gizi. PT Gramedia Pustaka Utama: Jakarta.
Belitz H-D, Grosch W, Schieberle P. 2009. Food Chemistry. 4th revised and extended Edition.
Springer-Verlag: Berlin, Heidelberg.
Fennema OR. 1996. Food Chemistry. 3rd Ed. Marcel Dekker, Inc. : New York, Basel, Hongkong.
Huang T, Jander G, and de Vos M. 2011. Non-protein amino acids in plant defense against
insect herbivores: Representative cases and opportunities for further functional analysis.
Phytochemistry, Vol. 72 Issue 13, p1531-1537.
AOAC International. 1999. Official methods of analysis of AOAC international. 16th Ed. AOAC
international suite 500: North Frederick Avenue, Gaithersburg, Maryland, USA.
http://www.aoac.org/
Rajalingam D, Loftis C, Xu JJ, Kumar TKS. 2009. Trichloroacetic acid-induced protein
precipitation involves the reversible association of a stable partially structured
intermediate. Protein Science 18: 980-993.
Upstone SL. 2000. Ultraviolet/Visible Light Absorption Spectrophotometry in Clinical
Chemistry. In: Encyclopedia of Analytical Chemistry, R.A. Meyers (Ed.) pp. 1699–1714, John
Wiley & Sons Ltd, Chichester.
Kahn AS and Faiz F. 2008. Amino acid analysis using ion exchange resins. In: CODEN JNSMAC 48
(1&2): 1-17.

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