Protein Analysis Methods Flashcards
(35 cards)
- Sample is digested with sulfuric acid in the presence of a catalyst
- measures total organic N (including ammonia)
- official method of analysis for crude protein
Kjeldahl Method
Advantages:
- applicable to all food types
- inexpensive ( if not using automated system)
- Accurate; official method
- modified to analyze microgram quantities
Kjeldahl method
Disadvantages:
- Measures total nitrogen; nitrogen just protein nitrogen
- time consuming
- poorer precision
- corrosive reagent used
Kjeldahl method
- samples combusted @ high temperatures (700-1000 C) with a flow of pure oxygen
- All carbon is converted to carbon dioxide
- Total nitrogen released is measured by gas chromatography using a thermal conductivity detector
Dumas (Nitrogen combustion) Method
Advantages:
- applicable to all food types
- no hazardous chemicals
- Short time (3 min)
- automated instruments can analyze up to 150 samples without attention
Dumas (Nitrogen combustion) Method
Disadvantages:
- Expensive
- measure total nitrogen; not just protein nitrogen
Dumas (Nitrogen combustion) Method
- measure the absorption of radiation by molecules in food sample
- measures the energy reflected or transmitted by the sample
- For proteins and peptides: mid-infrared and near infrared characterizes of peptide bond can be used to estimate protein content
Infrared Spectroscopy
- used to infrared milk analyzers to determine milk protein content
MID - Infrared spectroscopy
- applicable to wide range of food products (grains, cereal, meat, and dairy products)
NEAR - infrared spectroscopy
- instruments are expensive
- samples analyzed rapidly by analysts without minimal training
Both mid and near infrared spectroscopy
3 stage process
1) Digestion
2) Neutralization & distillation
3) Titration
Kjeldahl method
Reagents used in digestion step & principle
1) sulfuric acid
2) anhydrous sodium sulfate
3) copper catalyst
- nitrogen is converted into an ammonium ion
Reagents used in digestion step & principle
- Alkali containing sodium hydroxide is added to neutralize the sulfuric acid
- Ammonium ions are converted into ammonia gas
- The ammonia is distilled into a boric acid solution containing indicator methylene blue & methyl red
- ammonium borate complex formed
Note: Ammonia gas liberated during procedure can be titrated via sulphuric acid, boric acid, and HCl in excess
Neutralization & distillation step
- the amount of ammonia trapped in boric acid solution is determined by directly titrating with 0.1 HCl
- ammonium borate complex yields boric acid and chloride ions
Titration step
4 Solvent extraction methods for fats:
1) Goldfish - Continuous
2) Soxhlet - Semicontinuous
3) Mojonnier - Discontinuous
4) Chloroform-Methanol - Folch and Bligh & Dyed
Fat content measurement is based off of:
Gravimetric analysis
Ideal solvents should:
- evaporate readily
- relatively low building point
- nonflammable and nontoxic
- inexpensive
- nonhygroscopic
- penetrate sample readily
- Combustion method
- Nitrogen released measured by built in gas chromatography
Dumas method
- based on energy absorbed when a sample is subjected to a wavelength of infrared radiation
- near and mid ?
- samples can be analyzed quickly in half a minute with MINIMAL training
Infrared spectroscopy
Near: variety of foods (grains, meat, dairy, cereal)
Mid: Milk analyzers
- measures peptide bonds (at least 2)
- violet- purple color is produced
- color intensity is proportional to the protein content
- does not detect nitrogen from non protein or non peptide sources
Biuret method
- measures a combination of peptide bonds and the amino acids tryptophan and tyrosine
- protein must be isolated from sample first
- color NOT strictly proportional to protein content
Lowry Method
Measures only organic nitrogen plus ammonia
Kjeldahl method
Measures total nitrogen, including the organic fraction
Dumas method
This method gives a higher value for products that contain nitrates/nitirites
Dumas method