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ACOM > part 3 analysis of lipids > Flashcards

part 3 analysis of lipids Flashcards

1
Q

What is the composition of a fatty acid?

A

A fatty acid is composed of a carboxylic group (R-COOH), and a long aliphatic hydrocarbon chain (alkyl chain).

  • medium chain C10-C14
  • long chain C16-C18
  • very long chain > C20
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2
Q

What are the main structural characteristics of FAs?

A

Naturally occuring FAs normally contain an even number of carbon atoms. FAs with odd number of carbon atoms are less common.

Saturated FAs do not contain double bonds. Unsaturated FAs contain one or more (polyunsaturated) double bonds.

The most common isomer: cis (z) (on the same side)

Some FAs are branched or hydroxylated

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3
Q

Name a few important fatty acids

A
  • Palmitic acid: saturated FA, 16:0
  • stearic acid: 18:0
  • oleic acid: 18:1(9)
  • linoleic acid: 18:2(9,12)
  • linolenic acid: 18:3(9,12,15)
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4
Q

Link between double bonds of FA and retention time

A

The presence of double bonds is resulting in a different polarity, higher interactions with the stationary phase, and therefore in different retention time

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5
Q

Which types of analysis can be applied for the identification of lipid materials (oil/fat)?

A

Types of analysis:
- Qualitative analysis: identification of each FA –> MOLECULAR MARKERS

  • Semiquantitative analysis: relative distribution of FAs (e.g. %) –> MOLECULAR DISTRIBUTION (fingerprinting) & COMPOUND RATIOS (ratio between two specific fatty acids) & ISOTOPE RATIO (13C/12C)
  • Quantitative analysis: FA absolute concentration (e.g. ng/g in the sample)
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6
Q

Some uncommon fatty acids

A

Castor oil marker: ricinoleic acid (an hydroxylated oleic acid)

Tung oil marker: elaeostearic acid (not usual because trans conformation and conjugated double bonds)

Bacteria marker: anteiso-C15:0 (branched fatty acids with an odd number of carbon atoms)

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7
Q

Marker of vegetable oil

A

Palmitic/stearic acid

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8
Q

Procedure for the preparation of a sample before analysis of fatty acids by GC-MS

A

Analysis of total fatty acids because bound and free FA cannot be distinguished

  1. Hydrolysis - chemical degradation with KOH/H2O and CH3OH to transform TAG (triacylglyceride) into soaps (R-OO- M+)
  2. solvent (liq-liq) extraction using n-hexane
  3. acidification treatment with HCl to transform soaps into free fatty acids (neutralization)
  4. methylation with BF3/MeOH –> fatty acids methyl esters
  5. extraction with chloroform
  6. GC-MS

Salts of fatty acids are formed by alkaline hydrolysis (cf ppt3)

Alternative procedure: For high temp GC-MS, silylation with BSTFA is performed. With this technique, free and bound fatty acids can be distinguished

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9
Q

Acylglycerols

A

Most important class of lipids, which consists in esters of glycerol with fatty acids. Commonly known as triglycerides

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10
Q

DEF Lipids

A

Organic substances of biological origin which are soluble in non-polar solvents (n-hexane, dichloromethane, ethyl ether, ethyl acetate). They can either be in the form of oil (liquid) or fat (solids). Those materials are more resistant to degradation from environmental conditions and ageing due to their non-polar nature (hydrophobicity).

Examples of lipid-based materials and their uses: dairy and animal fats/plant oils (cooking, lightening, ingredients of cosmetics, balms, medications), drying oils (binders, varnishes, coatings).

Examples of plant oils: almond, balanos, castor, linseed, moringa, coconut, palm, poppy, sesame…

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11
Q

Classification of lipids

A

SAPONIFIABLE vs. NON-SAPONIFIABLE

  • saponifiable: fatty acids bound with glycerol (acylglycerols), with glycerolo-3-phosphate (phosphoglycerides), or with superior alcohols (waxes)
  • non-saponifiable: they do not produce soaps (do not contain fatty acids) such as terpenes (abietic acid) or steroids (cholesterol)

SIMPLE vs. POLAR/COMPLEX

  • simple = fatty acids + glycerol (ex: lipid deposits, neutral lipids, triacylglycerols)
  • polar = fatty acids + glycerol + sugar/phosphates (ex: membrane lipids, phospholipids, chloroplasts, glycosylglicerides, plasmatic membranes, phosphoglycerides)
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12
Q

What are sterols?

A

Sterols are good molecular markers because they are already specific. Sterols are non-saponificable lipids. They are alcoholic steroids (generally contain an OH group in position C-3
and an alkyl chain in C-17). Steroids have a characteristic hydrocarbon
skeleton (perhydro-1,2-cyclopentenophenanthrene ring system). Differences
in alkyl substituents, double bond positions, etc. can be species-specific and
useful in source apportionment studies.

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13
Q

Lipidic markers of food of animal origin, of plants, of diagenesis, of yeast and fungi, and of animal faecal matter

A
  • food of animal origin: cholesterol
  • plants: sitosterol
  • diagenesis: cholestanol
  • yeast/fungi: ergosterol
  • animal faecal matter: coprostanol
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14
Q

Another type of non-saponificable lipids: natural resins

A

Plant resins are principally composed of terpenoids. Compounds based on the C5 isoprene
repeating unit. According to the number of carbon atoms, terpens are divided into:
- 10 MONO
- 15 SESQUI
- 20 DI
- 30 TRI terpens
Mono and sesquiterpenes are rather volatile and rarely found in ancient manufacts.

Examples of diterpenoid resins: pine resins, strasbourg resins, venice turpentine, sandarac
Typical component: tricyclic diterpenoid acids

Like sterols, trcylic diterpenoid acids can be source specific. The hydrocarbon skeleton is preserved during ageing/diagenesis but chemical transformation can occur changing the chemical pattern (functional groups, side chains, saturation degree).
The product analyzed is giving info on its precursor.

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15
Q

DEF Derivatisation

A

Derivatisation is a chemical reaction that transforms the compound of interest into a derivative with improved characteristics for its analysis.
In GC with non-polar stationary phases, it is common practice to reduce the polarity of the
analytes by replacing the hydrogen of –O-H (or =N-H) groups with a proper substituent R. The reaction is accomplished with a derivatizing reagent.

The most common reaction are:

  • methylation: -R is a methyl group -CH3 (used commonly to transform an acid into a methyl ester)
  • trimethylsilylation: -R is a trimethylsilyl group, -Si(CH3)3 (also increases the ionization efficiency so intensity of the peak is also higher)

Derivatization is particularly used on acid and alcohol.
Acid –> Methyl ester
Alcohol –> Trimethylsil ether

Examples of methylating reagents: boro trifluoride/methanol (BF3/CH3OH), tetramethylammonium hydroxide (TMAH)

Examples of silylating reagents: HMDS (hexamethyldisilazane), BSTFA (N,O-bis( trimethylsilyl)trifluoroacetamide)

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16
Q

Main class of acylglycerols

A
  • triacylglycerols = triglycerides TAG
  • diacylglycerols = diglycerides DAG
  • monoacylglycerols = monoglycerides MAG

SIMPLE = only one type of fatty acid
vs. MIXED

17
Q

Analysis of acylglycerols with GC

A

DAG & MAG with a low molecular weight could be analyzed by GC, but hydroxy groups are making them non-volatile. They need to be derivatize through sylilation (trimethylsylation for GC-MS analysis: replace -COR in -OCOR by -SiMe3)

Normal GC can’t deal with triacylglycerol = transformation into fatty acids

For HPLC (liq chromato), analysis of acylglycerols can be directly performed.

18
Q

3 types of chemical degradation of TAG

A
  • hydrolysis: water with catalyst H+ or HCl –> glycerol + fatty acid
  • methanolysis: methanol and a catalyst HCl or BF3 –> glycerol + methyl ester of fatty acid
    Most efficient way because both degradation into FA and derivatization into methyl ester
  • alkaline hydrolysis:
    1. alkaline hydrolysis (aqueous KOH) –> saponification –> soap (R-COO- + glycerol)
    2. soaps (R-COO-) –> acidification (HCl) –> free fatty acids (R-COOH)
    3. extraction in hexane and methylation –> product: fatty acid methyl ester
    4. GC analysis
19
Q

Strategies for identification of organic materials

A
  • fingerprinting (fatty acid distribution) –> comparing the distribution
  • molecular marker (specific compounds)
  • compound ratio (ratio between peak area) ex: palmitic/stearic or azelaic/palmitic ratios in the identification of siccative oils in painting layers
  • carbon isotopic ratio with GC-IRMS: measurement of the difference between the isotopic ratio of the sampled material and a reference material (marine carbonate). This difference in content of 13C (delta) is caused by mass discrimination in chemical-physical processes (phase compartment enriched at different levels of one isotope). The composition in isotopes is measured with mass spectrometer. Organic materials containing C atoms are forming CO2 through combustion.
    -> isotopic ratio of CO2 in atm: -7‰
    -> carbonate: 0‰
    -> coal: -20 to -30‰
    -> terrestrial biomass, products of photosynthesis: -26‰
    -> algal biomass: -20‰
    Even plants of the same compartment can have different delta since their way of fixing atm CO2 through photosynthesis may be different.
20
Q

Markers of:

  • olive oil
  • linseed oil
  • cow milk
  • siccative oils
A
  • Olive oil: oleic acid (18:1)
  • Linseed oil: polyunsaturated fatty acids
  • Cow milk: palmatic acid (16:0) & oleic (18:1)
  • Siccative oil: azelaic acid –> dicarboxylic acid with 9 atoms (formed by degradation of polyunsaturated fatty acids such as linoleic, linolenic…)
21
Q

What can make the interpretation of fatty acids difficult?

A
  • Similarity in fatty acid composition
  • Original fatty acid pattern modified by degradation
  • Mixtures of different oil/fat materials
  • Analytical factors: matrix interferents, contamination…
22
Q

What are the main processes of degradation of fatty acids?

A

Physical degradation (phase distribution)

  • volatilisation (most short-chain fatty acid volatilize –> distribution shifting toward heavier FA)
  • dissolution
  • precipitation

Bio/chemical degradation:

  • photodegradation (light, oxygen)
  • hydrolysis (water)
  • oxidation –> formation of more oxygenated compounds
  • thermal –> formation of double bonds, isomerization
  • biodegradation (microorganisms)

TAG –> DAG + FA –> MAG + FA
Ester bonds broken and FA formed
Catalyzed by lipase, produced by microorganisms

23
Q

What are waxes?

A

Waxes are another class of organic materials belonging to the class of lipids. They are heterogeneous materials, solid at room temperature, hydrophobic. They are used as sealants, waterproofing agents, surface coatings, protective varnishes, balms, cosmetics, fuel, casting agents… They are obtained from animal origin (beeswax of bees, chinese wax (secretion of scale insect), lanolin (wool of sheep)), vegetal origin (carnauba wax (leaves of palm trees), roots), or mineral origin (paraffin from petroleum distillation)

Their structure is generally composed of long chain aliphatic hydrocarbons. The common structure is: esters between long chain carboxylic acids and long chain alcohols.

Chromatograms of beeswax: very complex, composed of homologous series of linear aliphatic hydrocarbons with odd number of C + free fatty acids + long-chain monoesters of alkanoic acids

24
Q

Procedure for measurement of isotopic ratio of single fatty acids by GC-IRMS

A

one possible application: identification of food and diet in ancient population

  1. extraction of lipids
  2. hydrolysis
  3. hexane extraction (removal of non-saponificables)
  4. methylation with BF3-CH3OH
  5. FAME analysis by GC-IRMS

ACOM (5 decks)

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