Fatty Acids Analysis Using GC - Theory and Practice

Gas Chromatography

Gas-liquid chromatographyAdsorbed inner surface of capillary column

Gas-solid chromatographyPorous polymer beads or zeolites

Separates vaporized sample as a consequence of partition between a stationary phase and a mobile phase

Application Areas for GCComposition Analyses

Fatty Acid AnalysisCholesterol, Tocopherols, and PhytosterolsAmino Acid AnalysisSpices

Quality AnalysesLipid Oxidation: hexanal and hydrocarbonsVolatiles - Odor and Flavor Compounds

GC UnitGC OvenColumnDetectorSample InjectorControl System

Inlet ConditionsSplit/Splitless Inlet

Sample ConcentrationInlet Temperature

High Enough to Volatilize SamplePeak Tailing

Autosampler

Oven Temperature Conditions

Initial and Final TemperatureColumnSample

Constant TemperatureProgrammed Temperature

Uniform IncreaseMultiple Step Increase

Important Parameters to be Considered in GC Analysis

Column Selection Column Flow Rate Oven Temperature ConditionsInlet ConditionsInjector: Auto/ManualDetector Selection Gas Selection: Depending on Detector

Selection of ColumnSampleStationary Phase TypeStationary Phase Film ThicknessColumn IDColumn Length

Stationary PhaseBonded Phase

Stationary phases are immobilized/crosslinked within the tubing

Nonbonded PhaseStationary phases are coated on the wall of tubing

The stationary phase dictates the min/max temperatures at which column can be used.

Selection of Phase TypeDifferences in the chemical and physical properties of the injected organic compounds and their interactions with the stationary phase are the basis for the separation process

Phase Polarity: “Like Likes Like”Nonpolar Molecules: n-AlkanesPolar Molecules: Compounds with one or more atoms of Br, F, Cl, O, N, P, SPolarizable Molecules: with one or more of double or triple carbon-carbon bonds

Stationary Phase Film Thickness

Increasing film thickness Increases the maximum sample capacity Increases analyte retention time Reduces column efficiencyReduces the upper temperature limit

Thin films are good for analytes with high boiling pointsThick films are good for analytes with low boiling points (volatile organics and gases)

Phase Ratio (Beta value)

Expresses the ratio of the gas volume and the stationary phase volume in a column

Column radius (um)Beta value = ------------------------------------------------ = r/2df

2 x phase film thickness (um)

Selection of a Column using a Phase Ratio

_________________________________________Beta value Sample Components < 100 Highly volatile, Low MW

compounds100-400 General purpose analyses, wide

range of compounds> 400 High MW compounds__________________________________________

Column Length

Shorter column (< 15-m) is good for screening or simple samples A longer column (> 60-m) is good for complex or volatile analyses

provides greater resolution than a shorter columnincreases analyses timeincreases the pressure to move the sample through the column

Theoretical Plate

A series of discrete but continuous, narrow, horizontal plate in a column at which equilibrium of solute between the mobile and stationary phase is assumed to take place.N = L/H

(N = number of theoretical plate, L = column length, H = the height equivalent to a theoretical plate)

Column efficiency = N1/2

Effect of Column ID on the Characteristics of GC Column

_____________________________________________ __Column Sample Efficiency Optimum flow ID Capacity (ng) TPN/m rate (mL/min)0.2 mm 5-30 5000 0.40.25 mm 50-100 4170 0.60.32 mm 400-500 3330 1.00.53 mm 1,000-2,000 1670 2.80.75 mm 10,000-15,000 1170 5.62 mm 20,000 2000 20(packed column)________________________________________60-m capillary column, 2-m packed column. 0.25 um film for 0.25and 0.32 mm columns and 1.0 um for megabore columns

Column Flow Rate

Column DiameterColumn LengthOven Temperature

Detectors

Flame Ionization DetectorThe number of ions produced is proportional to the number of reduced carbon atoms in the flame

Thermal Conductivity DetectorThe thermal conductivity of hydrogen and helium are about 6-10 times greater than those of most organic compounds.

Electron Capture DetectorSensitive to halogens, peroxides, quinones, and nitro groups. Important tool for chlorinated insectidides

Flame ionization Detector

Chromatogram from Flame Ionization Detector

Retention Times of Compounds

Mass Selective DetectorElectron Impact: Bombarding the sample with high energy electrons, which produces a family of positive particles whose mass distribution is characteristic of the parent species. The complex mass patterns that result are used for identificationChemical Ionization: Use reagent gas. More gentle than EI and less fragmentation. Methane is the most common reagent. Isobutane and ammonia are also used.

Mass Selective Detector

Ion Source

EI Fragmentation

3-Phenyl-2-propenal (C9 H8 O), MW = 132.16

3-Pentanol (C5 H12 O), MW = 88.15

Compounds Identification

Sample Preparation and Injection

Solvent ExtractionFolch SolutionSaponification

DerivatizationSample Injection

Auto-injectionManual injectionOn-column injection

Derivatization

Improves thermal stability of compoundsIncreases volatility of compoundsIncreases sensitivityImproves separation

Derivatizing Reagents

AcylationConverts compounds that contain active hydrogens (-OH, -SH, and -NH) into esters, thioesters, and amide. Fluorination for ECD.

AlkylationAddition of alkyl group (aliphatic or aliphatic-aromatic) to an active functional (H) group. (e.g., esterification of fatty acids)

SilylationReplacement of active H group by a silyl group.

Fatty Acid Methyl Esters (FAME)

Methanolic hydrogen chloride5% anhydrous hydrogen chloride in methanol30 minutes at 50°C

Methanolic sulfuric acid1 to 2% concentrated sulfuric acid in methanol. Destruction of PUFANot recommended for polyunsaturated fatty acids

Boron trifluoride-methanol12 to 14% boron trifluoride in methanol 45 minutes at 100°C

Advantages and Limitations of GC

AdvantagesVery powerful tool for quantification and qualification of various compoundsSample preparation is easyEasy to operate

LimitationsNon-volatiles cannot be analyzed For solvent-soluble compounds onlyLimited for small compounds