extraction and analysis of lipids

7/28/2019 Extraction and Analysis of Lipids

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Extraction andAnalysis of LipidsPauline Bianca R. AlfonsoEdessa Joy R. Sumagaysay

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Lipids

Lipids are compounds of biological origin

that dissolve in nonpolar solvents, such as

chloroform and diethyl ether.

Unlike carbohydrates and proteins, which

are defined in terms of their structures,

lipids are defined by the physical operation

that we use to isolate them.

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Structural Types

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EXTRACTION OF LIPIDS FROM FOODS AND

BIOLOGICAL MATERIALS

Lipids in nature are associated with other molecules via

(a) van der Waals interaction, e.g., interaction of several lipidmolecules with proteins;

(b) electrostatic and hydrogen bonding, mainly between lipids andproteins; and

(c) covalent bonding among lipids, carbohydrates, and proteins.

Therefore, to separate and isolate lipids from a complexcellular matrix, different chemical and physical treatmentsmust be administered.

Water insolubility is the general property used for theseparation of lipids from other cellular components. Completeextraction may require longer extraction time or a series orcombination of solvents so that lipids can be solubilized fromthe matrix. 4


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Properties of Solvents and Their

Mode of Extraction Lipids containing no distinguishable polar groups

(e.g., TAGs or cholesterol esters) are highly soluble in

hydrocarbon solvents such as hexane, benzene, or

cyclohexane and in more polar solvents such as

chloroform or diethyl ether, but remain insoluble in

polar solvents such as methanol.

Polar lipids are only sparingly soluble in hydrocarbon

solvents unless solubilized by association with otherlipids; however, they dissolve readily in more polar

solvents, such as methanol, ethanol, or chloroform5


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Extraction Methods with Single

Organic Solvent In gravimetric methods, lipids of the

sample are extracted with a suitable

solvent continuously, semi continuously, or

discontinuously.

The fat content is quantified as weight loss

of the sample or by weight of the fat

removed.

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Goldfisch method

Gives a continuous flow of boiling solvent to flow

over the sample (held in a ceramic thimble) for a

long period.

This gives a faster and more efficient extractionthan semicontinuous methods but may result in

incomplete extraction due to channeling.

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Soxhlet Method

The solvent accumulates in the extraction

chamber (sample is held in a filter paper

thimble) for 510 minutes and then

siphons back to the boiling flasks.

This method requires a longer time than

the continuous method, provides a

soaking effect for the sample, and doesnot result in channeling.

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Process

Sample is dried, ground into small particles and placed in a

porous thimble.

The flask is heated and the solvent evaporates and moves up

into the condenser where it is converted into a liquid that

trickles into the extraction chamber containing the sample.

As the solvent passes through the sample it extracts the lipids

and carries them into the flask. The lipids then remain in the

flask because of their low volatility.

At the end of the extraction process the flask containing thesolvent and lipid is removed, the solvent is evaporated and the

mass of lipid remaining is measured .

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SchmidBoudzynskiRatzlaff (SBR) methods

There is no continuous flow of solvent and

the sample is extracted with a fixed

volume of solvent.

After a certain period of time the solvent

layer is recovered, and the dissolved fat is

isolated by evaporating the organic

solvent.

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Methods Using Nonorganic Solvents

Due to environmental concerns and potential health hazards

of organic solvents, nonorganic solvents have become popular.

The use of microwave digestion for isolating lipids has recently

been reported. It is suggested that microwave energy, by

increasing the rotational force on bonds connecting dipolarmoieties to adjacent molecules, reduces the energy required

to disrupt hydrophobic associations. Hydrogen bonding, and

electrostatic forces, thus helping to dissolve all kinds of lipids.

Microwave technology has allowed the development of rapid,

safe, and cost-effective methods for extracting lipids and does

not require that samples be devoid of water.

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Supercritical fluid extraction (SFE)

When carbon dioxide is compressed at a temperature(31.1C) and pressure (72.9 atm) above its critical point, itdoesnt liquify but attains a dense gaseous state thatbehaves like a solvent. Thus, it is called supercritical CO2

(SC-CO2). Use of SC-CO2 for lipid extraction significantly reduces

the use of organic solvents, avoids waste disposalproblems, eliminates the use of potentially toxic andflammable solvents, and reduces the extraction time.

Lipids so extracted are not subjected to hightemperatures during the extraction process.

The main drawback of SC-CO2 is equipment cost and theextraction of nonfat materials, such as water. 14


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Process

Pressurized CO2 is heated above a certain criticaltemperature to become supercritical fluid

This fluid behaves like a gas to easily penetrate into asample and extract lipid while it also behaves like a liquid

to dissolve a large quantity of lipids. The CO2 extracts the lipid, and forms a separate solvent

layer, which is separated from the aqueous components.

The pressure and temperature of the solvent are then

reduced which causes the CO2 to turn to a gas, leavingthe lipid fraction remaining.

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INDIRECT METHODS OF TOTAL LIPID

DETERMINATION

These methods are really not lipid extraction

methods, but they are gaining popularity

because they are rapid and largely

nondestructive. Most of these methods rely on a standard

reference procedure and must be calibrated

against a methodology to be validated.

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Low-Resolution Nuclear Magnetic

Resonance Spectroscopy

Time domain low-resolution nuclear magnetic resonance

(NMR) (referred to as wideline NMR) and frequency domain

NMR could be used to determine the total lipid content of

foods.

This method can be used to determine the contents of water,oil, and solidfat and solid-to-liquid ratio of the sample. Time

domain NMR has been used to analyze the fat content of

foods, including butter, margarine, shortening, chocolate,

oilseed, meat, milk and milk powder, and cheese.

Frequency domain NMR distinguishes food components by

resonance frequency (chemical shift) of the peaks in the

spectrum. The pattern of oil resonances reflects the degree of

unsaturation and other chemical properties.17


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Principle

In time domain NMR, signals from the hydrogen nuclei

(1H or protons) of different food components are

distinguished by their different rates of decay or nuclear

relaxation.

Protons of solid phases relax (signal disappear) quickly,

while protons in the liquid phase relax very slowly.

Protons of water in the sample relax faster than protons

of the lipid.

The intensity of the signal is proportional to the numberof protons and, therefore, to the hydrogen content. Thus,

the intensity of the NMR signal can be converted to oil

content of the sample using calibration curves or tables.18


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Chromatographic Procedures for

Lipid Characterization

Lipid extracts are complex mixtures of individual

classes of compounds and require further separation

to pure components if needed.

Analysis of chemical components of lipid (e.g., lipidclasses, fatty acids, trans fatty acids, sterols,

tocopherols, pigments, etc.) primarily involves

chromatographic and spectroscopic methods.

Usually a combination of separation techniques isused to achieve a high degree of purity of respective

lipid components and this could be analytical (for

quantitation) or preparative.19


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Gas Chromatography

This method involves partitioning of the components ofthe lipid mixture in the vapor state between a mobile gasphase and a stationary nonvolatile liquid phase dispersedon an inert support.

Analysis of fatty acid composition by GC usually requiresderivatization of fatty acids to increase their volatility.

Fatty acid methyl esters (FAME) may be prepared bydifferent transmethylation techniques and thenseparated on GC columns and detected by flameionization detection (FID).

The gas phase for GC is usually nitrogen or helium forpacked columns and helium or hydrogen for capillarycolumns.

The identification of chromatographic peaks is based oncomparison of their retention times with those ofauthentic samples.

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Analysis of Triacylglycerol (TAG)

GC analysis of TAG of food lipids may also provide

information about positional distribution of fatty acids in

the molecules.

Naturally occurring TAGs that are purified by TLC can

then be resolved without derivatization on the basis of

their carbon number or molecular weight using capillary

GC equipped with 8- to 15-m-long columns coated with

methylphenyl-, methyl-, or dimethylsilicone (nonpolar

capillary). Use of helium or hydrogen as a carrier gas for separation

of TAG on such columns requires higher temperatures

than those employed for separation of methyl esters.21


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Mono- and diacylglycerols have to be convertedto trimethylsilyl (TMS) or tert-butyldimethylsilyl

ethers (TBDMS) for complete resolution.

A combined GC and mass spectrometric

technique has been applied for determining

molecular species in the glycerol esters.

TMS or t-BDMS derivatives of glycerol esters

separated on GC may be subjected to massspectrometric analysis in order to obtain

information on their molecular structure22

extraction and analysis of lipids

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