Lecture 7- Lipid Analysis Flashcards
What are lipids?
Soluble in organic solvents, insoluble/sparingly soluble in water
What is the FDA definition of lipids?
Total fat is the sum of fatty acids C4-C24 calculated as TG
What are oils?
TG that are liquid at room temp
What are fats?
TG that are solid at room temp
What is the bond that hold the glycerol molecule to the TG?
Ester bond
What are the 3 classes of lipids?
- Simples
- Compound
- Derived
Simple Lipid
Ester of fatty acids with alcohol
Compound lipid
Compounds containing groups in addition to an ester of a fatty acid with an alcohol ( phospholipids, cerebosides, sphingolipids)
Derived Lipids
substances drived from neutral lipids or lipid compounds ( FA, sterols, hydrocarbons)
Why is it important for measure fat content?
- Nutriton
- Health/diet restrictions
- Food quality
- Economic considerations
What makes it possible to seperate lipids from proteins and carbohydrates?
Insolubility
What is the hydrophobicity of lipids depend on?
Molecular constituents
What is crude fat?
In non polar lipids, TG
–> using non polar organic solvent
What method extracts crude fat?
Dry extraction
What are the methods of dry extraction?
Soxhelt and goldfish
What is total lipids?
Non polar and polar lipids
How are total lipids extracted?
wet extraction
What are the methods of wet extraction?
Babcock, berger, Majonnier, Bligh&Dyer
What are the two types of fat extraction based on type of chemical?
Solvent and non solvent
What are the solvent methods?
Soxhelt, Goldfish, Majonnier, Bligh & Dyer
What are the non solvent methods?
Babcock, Gerber
What is crude fat refered to in the determination?
Residue obtained after removal of the non polar organic solvent used to extract dried sample
The extraction of food material by a non polar solvent will give an estimate of…
Crude fat, no total lipds
What is the major lipid components in food?
TG
What does Crude fat mainly measure?
TG
What is the overview of crude analysis?
Dry food samples–> (extraction w/ non polar organic solvent) –> solution of fat in the solvent–>(evaporation of organic solvent –> residue (Crude fat)
The non polar organic solvent only extracts…
non polar materials
What are the ideal solvent properties?
Should have a high solvent power for lipids and low or no solvent power for proteins, amino acids, and carbohydrates
Should evaporate readily and leave no residue
Should have a relatively low boiling point
Should not leave behind any residue
Should be nonflammableand nontoxic
Should penetrate sample particles readily
Should be nonhygroscopic
Should be cost effective
What are some examples of polar organic solvents used?
Hydrocarbon ( petroleum ether, low boiling point, volatile, non polar)
Diethyl (more polar, absorbs O2 and forms peroxides which in presence of heat could explode)
n-Butanol ( more polar than other solvents)
Petroleum Ether
- low boiling point
- mainly hexanes and pentanes
- more hydrophobic than diethyl ether and therefore selective for more hydrophobic lipids
Three ways for Dry extraction?
Discontinuous Extraction
Intermittent Extraction
Continuous Extraction
What is disconinuous extraction
high ratio of organic solvent sample
What is intermittent extraction
Solvent is in contact with sample intermittently (soxhlet)
What is continuous extraction?
solvent is always in contact with the sample for given period of time (Goldfish)
How do you avoid lipid oxidation?
Using inert atmosphere
Preparing smaple under low temp
Avoiding excessive light
Steps in sample preparation of wet analysis
Removal of water: To enhance penetration of non-polar solvents
Reduction of particle size: Grinding (of nuts) enhances surface area
Separation of the lipid from bound proteins and/or carbohydrates through the use of alkaline or acid hydrolysis
Describe the importance of acid hydrolysis in wet extraction. (2 reasons)
Enable penetration of solvent into the sample
Break both covalently and ionically bound lipids into easily extractable lipid forms
Meat sample–(hydrolysis by HCl)–> Solvent extraction for crude fat
What are the lipid in dairy, bread, flour and animal products like? Is direct extraction efficient?
Bound to proteins and carbohydrates and direct extraction with non polar solvents is inefficient
What is discontinuous extraction
Simplest version of extraction
No continuous flow of solvent
Sample is extracted with fixed volume of solvent
What are the disadvantages of discontinuous extraction
Needs large amount of solvent Low efficiency (as solvent extracts the fat, its ability to extract fat decreases)
What is intermittent extraction?
semi continuous solvent
soxhlet extraction
What is the soxhlet instrument?
Consist of a flask (2) for holding the solvent (1), a section containing the siphoning unit (Soxhelt flask) (6, 7), paper thimble for holding food (4), and a condenser (9).
What are the 2 main components of Soxhelt apparatus that contain the sample and the solvent?
Soxhelt flask and paper thimble
Soxhlet extraction steps?
The sample is placed in filter paper thimble.
2. The solvent builds up in the extraction chamber.
3. The solvent accumulates in the extraction chamber
(soxhelt flask) for 5-10 mins that facilitate the extraction
of crude fat from the sample.
4. The solvent+crude fat siphons back to the boiling flask.
5. Dismantle the unit, place the bottom flask containing solvent and fat on a heating mantle and evaporate the solvent to obtain crude fat
6. Measure the crude fat (weight or volume) left in the flask.
What are some variables that need to be considered with crude fat determination?
Sample preparation
Choice of solvent (petroleum and diethyl ether)
Extraction ( temp, time and particle size)
What is continuous extracton method?
Goldfish method
What is the goldfish method?
Based on closed loop of solvent evaporating being condensed
- Procedure onvolves placing the sample between boiling solvent and cold surface
- Solvent from boiling flask (Baker) continuously flows over the sample place in a ceramic thimble
- Boilin continues, teh solvent vaporizes, condenses on the cold surfaces and washed back through the sample to the boilding falsk
What are the limitations of goldfish?
Channeling-formation of solvent channel in the sample and fire hazard
What happens to the solvent in goldfish extraction?
Solvent condenses and flows over the sample
The solvent also evaporates
The sample tube collects the sample in the sample tube
What are the most important wet extraction methods? (3)
Babcock and Gerber
Majonner
Bligh and Dyer
What is the Babcock Method
Non solvent wet extraction
- measure total lipids
- Sample contain water
- It is still recognized as a reference method for milk fat
What is the principle?
Wet sample + H2SO4 + heat -> Centrifugation -> Fat coalesces/ (blob) of fat measured Volumetrically by the graduated portion of the Babcock bottle.
H2SO4 digests protein to liberate the fat, generates heat and releases the fat.
Total fat is measured volumetrically
It is not applicable to products containing chocolate or added sugar without modification because of charring of chocolate and sugar by sulfuric acid.
What is the Gerber MEthod?
Non solvent wet extraction
Similar to babcock
Besides H2SO4, isoamyl alcohol added to prevent the charring of sugar
Help releasing lipid from organic material
Majonnier Method (Base/alkaline method)
Can be used for liquid and solid milk samples
What is the principle of the Majonnier method
NH4OH Ethanol \+ Petroleum Ether Diethyl ether --> Centrifugation - Decant ether solution into previously weighed dish --> Evaporation --> Majonnier Fat Dish
What are the 4 regaents of Majonnier method?
1-NH4OH dissolves protein of casein micelle membrane and fat globule membrane to separate any bond lipids from proteins; if the sample is sour, it neutralizes the acidic sample.
2-Ethanol prevents milk from becoming viscous (prevents possible gel formation).
3-Diethyl ether and Petroleum ether solubilize and extract fat;
What prevents absorption of water by diethyl ether and dissolves non plar lipids?
Mix of petroleum ether with diethyl ether
What is the Bligh and Dyer MEthod mainly used for?
Initially used for fish and seafood–> now for meat and meat products
What is the principle of the Bligh and dyer method?
Extraction of lipids with a mixture of Chloroform: Methanol: Water
Monophasic to biphasic state
What is the method of bligh and dyer?
Food samples are mixed/homogenized in a chloroform-methanol solution (CHCl3) and the homogenized mixture is filtered into a collection tube.
•A 0.88 % potassium chloride (KCl) aqueous solution is added to the chloroform-methanol mixture containing the extracted fats to form two phases: the aqueous phase (top) and the chloroform phase containing the lipid (bottom).
•The phases are further separated in a separatory funnel or by centrifugation.
•Evaporation of the chloroform, to quantify total fat by weight.
What are other instrumental methods?
Infrared Method: fat can absorb IR energy at a wavelength 3.73μm;
2-Specific Gravity (Foss-Let Method):
Fat is determined as a function of the specific gravity of a sample solvent extract.
3-Nuclear Magnetic Resonance (NMR).
Flow path of bligh-dyer method
sample–> extraction fat by monophasic (extractio solvent)–> Add CHCl3 or water to change to biphsic state–> Aq phase containes non fat materials and CHCl3 contains FAT –> Seperation by separatory funnel–> remove CHCl3
Which sample would have a higher fat content: once with higher or lower E absorption? (IR method)
Higher
What is the standard method of milk analysis?
Gerber method
What is added to prevent charring of sugar in wet extractions of fat in samples containing high CHO?
isoamyl alcohol
What 2 methods are discontinuous solvent extractions?
Majonnier
Bligh and Dyer
