Outline

Separation Techniques Gas Chromatographic Analyses

Choice of Carrier Gas and Flow Rate Stationary Phase and Film Thickness Types of Injection Column Temperature and Program Choice of Detector Calibration Methods
Standard Addition Response Factor Internal Standard Area Normalisation

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Vorg

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Example Analyses

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -4

Choice of Carrier Gas and Flow-rate

Speed: H2 and He are best because small HETP is maintained at high flow rates (better resolution at fast analysis times)
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Cost: H2 and N2 are cheapest Safety: He and N2 are safe (H2 flammable) He and H2 preferred for capillary GC - faster analysis times

Set flow rate to obtain lowest HETP at fastest flow rate to get best separation and shortest analysis time.

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Stationary Phase and Film Thickness

Many different stationary phases are available in GC. Stationary phases are chosen according to their chemical polarity . Polarity determines the solubility of various analytes. Analytes with similar polarity to stationary phase dissolve more readily in stationary phase and are retained (and separated) more. Thicker films have higher capacity (lower resolution (larger HETPA), longer retention times).
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Solubility rules apply: Like dissolves like. A polar stationary phase retains and separates polar analytes and vice versa

CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -4

Stationary Phase and Film Thickness

Polarity Class
LOW

Analytes
Hydrocarbons, halocarbons, mercaptans, sulphides Ethers, ketones, aldehydes, esters, tertamines, nitro compounds and nitriles without a-hydrogen Alcohols, carboxylic acids,phenols, primary & secondary amines, nitorcompounds and nitriles with ahydrogen Polyhydroxy alcohols, hydroxy acids, polyprotic acids, polyphenols

INTERMEDIATE

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POLAR

VERY POLAR

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Stationary Phase and Film Thickness

TYPICAL PHASES Polysiloxanes (silicones) with various polar and non-polar groups Polyethylene glycols Hydrocarbons Esters Polyesters
SE-54 CB (low polarity) 94% methyl 5% phenyl 1% vinyl Max temp. 300C
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OV-1701 CB (medium polarity) 88% methyl 7% phenyl 5% cyanopropyl Max temp. 280C Carbowax (polar) Max temp. 220C
- O - CH2 - CH2 - O - n

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Stationary Phase and Film Thickness

COMPOUNDS WITH SIMILAR POLARITY These tend to elute in order of their diffusivity. A good indicator is the boiling point and lower BP compounds would be expected (usually) to elute first.
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Chiral Stationary Phases

Chiral stationary phases may be used to separate optical isomers (a molecule whose mirror image is not superimposeable on itself is chiral).
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Example Stationary phase incorporates L-valine. The D and L enantiomers of amino acids are separated from each other

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Types of Injection
SPLIT & SPLITLESS INJECTION
septum septum purge

Only a small part of the sample is delivered to the column Split controlled by needle valve Allows high concentration samples with low capacity column Splitter valve can be closed for a splitless injection

carrier gas
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vaporisation tube split

capillary column

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Types of Injection
STATIC HEADSPACE INJECTION Volatiles equilibrate between gas phase and liquid in sealed vial at constant temp. Important sampling technique in food chemistry Indirect analysis method needs knowledge of partitioning Not sensitive for high boiling compounds Easy to automate
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[X]G [X]C

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K=

[ X]C [ X]G

Column Temperature and Program

Higher column temperatures speed up the analysis but are detrimental to separation of the more volatile components of mixtures. A temperature program allows the separation to be speeded up progressively allowing the most volatile components to separate at lower temperatures first. T2 z min T1-T2 ramp y deg./min T1 x min cooling cycle

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Column Temperature and Program

Example
Separation of a wide boiling range of hydrocarbons (a) isothermal; (b) temperature program of 50-250C at 8/min

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92 min

C15
21 min

C21

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Choice of Detector
Detector FID Flame ionisation Notes Universal, simple operation Sensitivity High

TCD MS

Thermal conductivity Mass spectrometer

Mainly for inorganic compounds

Low
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Universal - gives structural information V.High - also can be highly selective Compound with electronegative groups or highly conjugated pesticides etc. P and N containing compounds. Important in food industry. Selective but complex to operate P, S, Sn compounds. Food industry. V.High

ECD

Electron capture

FTD

Flame thermoionisation

V.High

FPD

Flame photometric

High
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CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -4

Quantification: Calibration
Variation of Detector Response
The purpose of calibration is to allow the size of peaks in a chromatogram to be converted into the corresponding amount of analyte having passed through the column. Usually the area under the peak in a chromatogram is proportional to the amount of that compound reaching the detector (within the linear response range of the detector). For a particular detector, each compound will give a different response per mole sensed by the detector - molar response factor. Response factors must be taken into account when comparing peak areas for different compounds.
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Quantification: Matrix Affects

Comparing chromatograms obtained with real samples to those from artificial mixtures of pure reference compounds (standards) may lead to errors - the sample matrix can affect the response. Standards should be present in an identical, or at least very similar, matrix as the sample.
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Taking into account response factor and matrix considerations, three calibration methods emerge as most important: Standard Addition Internal Standard Area Normalisation

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Quantification with Standard Addition

The standard is a pure sample of the analyte added in exact known amounts to the sample (usually) at the beginning of the sample preparation. Response factors are not necessary but a pure reference compound must be available for every analyte. The matrix is well matched
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area

Multiple Standard Addition

Plot areas and extrapolate to -ve x-axis

amount of standard addition amount in sample

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Quantification with Internal Standards

Known amount of a reference compound (internal standard) mixed with sample. The internal standard must... NOT already be present in sample BE chemically inert HAVE similar retention properties NOT react with or change the analyte.

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Quantification with Internal Standards

Relative response factors must be applied in the analysis for the number of moles of component i

Ci =

C Ai s As Ri / s
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Ai=area of peak for component i As=area of peak for standard s Cs=concentration of internal standard Ri/st = relative response factor of component i to standard s Analysis is independent of the volume injected (excellent precision) Internal standard present in matrix of sample but care must be taken that matrix is matched in determination of relative response factors
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Quantification: Response Factors

A. Measurement of Relative Response Factors
Compound ( i ) (in ref. mixture) 1 2 3 4 Internal Standard Conc. (nmol/mL) 5.30 4.70 3.50 3.40 4.90 Peak Area (arb. units) 10102 11005 9980 8765 12349

R Area/Conc.
1906 2341 2851 2578 2520

R i / Rs

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0.76 0.93 1.13 1.02 1.00

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Quantification: Response Factors

B. Internal Standard Analysis
Compound ( i )

Cs Ai Ci = As Ri / s
Conc. (nmol/mL)
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R i / Rs

Peak Area (arb. units) 12198 10422 196 9152 11012

1 2 3 4 Internal Standard

0.76 0.93 1.13 1.02 1.00

7.14 4.99 0.08 3.99 4.90

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Quantification by Area Normalisation

May be used when all mixture components elute from column and give responses on detector. Assumes total area of all peaks corresponds to total amount of sample reaching detector.
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Independent of injected volume No need to match matrix No need for internal standard Disadvantage: first condition rarely met.

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Quantification by Area Normalisation

Method and example
Measure each peak area Correct each area by dividing by the response factor Add corrected areas Compute percentage of each component from ratio of corrected area to total area Area 16400 45200 30200
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Compound Methyl acetate Methyl propionate Methyl butyrate

R
0.60 0.78 0.88

Area/R 27333 57949 34318 119600

% 22.9 48.5 28.6

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Examples of Analyses
Alkaloids
1 cocaine 2 codeine 3 morphine 4 quinine
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Examples of Analyses
Steroids
1 17-a-estradiol 2 dihydroequiline 3 testosterone 5 estrone 6 equiline
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