6. Analisa Gas Kromatografi 4

7/30/2019 6. Analisa Gas Kromatografi 4

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

M.

GuilhausUNSW1999

-Allrightsreserved.

1

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

Example Analyses

Vaq

p

KD

D

www.chem.unsw.edu.au/UGNotes/Guilhaus/

q

Vorg


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GuilhausUNSW1999

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2

Choice of Carrier Gas and Flow-rate

Speed: H2 and He arebest because smallHETP is maintained at

high flow rates (betterresolution at fastanalysis times)

Cost: H2 and N2 are

cheapest

Safety: He and N2 aresafe (H2 flammable)

He and H2 preferred forcapillary GC - fasteranalysis times

Set flow rate to obtain lowest HETP at

fastest flow rate to get best separation and

shortest analysis time.


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GuilhausUNSW1999

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3


Many different stationary phases areavailable in GC.

Stationary phases are chosen accordingto their chemical polarity.

Polarity determines the solubilityofvarious analytes.

Analytes with similar polarity to stationaryphase dissolve more readily in stationaryphase and are retained (and separated)more.

Thicker films have higher capacity(lowerresolution (larger HETPA), longerretention times).

Solubility rules apply:

Like dissolves like.

A polar stationaryphase retains and

separates polar analytes

and vice versa


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GuilhausUNSW1999

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4


Hydrocarbons, halocarbons, mercaptans,

sulphides

Ethers, ketones, aldehydes, esters, tert-amines, nitro compounds and nitrileswithout a-hydrogen

Alcohols, carboxylic acids,phenols,primary & secondary amines,nitorcompounds and nitriles with a-

hydrogen

Polyhydroxy alcohols, hydroxy acids,polyprotic acids, polyphenols

LOW

INTERMEDIATE

POLAR

VERY POLAR

Polarity Class Analytes


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5


Polysiloxanes (silicones)with various polar andnon-polar groups

Polyethylene glycols

Hydrocarbons

Esters

Polyesters

TYPICAL PHASES SE-54 CB (low polarity)

94% methyl

5% phenyl

1% vinyl

Max temp. 300C

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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These tend to elute in order of theirdiffusivity.

A good indicator is the boiling point andlower BP compounds would be expected

(usually) to elute first.

COMPOUNDS WITH SIMILAR POLARITY


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7

Chiral Stationary Phases

Chiral stationary phases may be used toseparate optical isomers (a moleculewhose mirror image is notsuperimposeable on itself is chiral).

Example

Stationary phaseincorporatesL-valine.

TheD andL enantiomers of

amino acids are separated

from each other


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8

Types of Injection

Only a small part of thesample is delivered to thecolumn

Split controlled by needle

valve

Allows high concentrationsamples with low capacitycolumn

Splitter valve can be closedfor a splitless injection

SPLIT & SPLITLESS INJECTIONseptum purge

split

carrier gas

capillary column

vaporisation

tube

septum


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9

Types of Injection

Volatiles equilibrate between

gas phase and liquid insealed vial at constant temp.

Important sampling techniquein food chemistry

Indirect analysis method -needs knowledge ofpartitioning

Not sensitive for high boilingcompounds

Easy to automate

STATIC HEADSPACE INJECTION

[X]C

[X]G

K =[

[

X]

X]

C

G


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10


Higher column temperaturesspeed up the analysis but aredetrimental to separation of

the more volatile componentsof mixtures.

A temperature programallows the separation to bespeeded up progressivelyallowing the most volatilecomponents to separate atlower temperatures first.

T1x min

T1-T2 rampy deg./min

T2z min

cooling

cycle


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Example

Separation of a wide boiling

range of hydrocarbons (a)isothermal; (b) temperatureprogram of 50-250C at

8/min

C15

C21

92 min

21 min


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12

Choice of Detector

FID

TCD

MS

ECD

FTD

FPD

Flame ionisation

Thermal conductivity

Mass spectrometer

Electron capture

Flame

thermoionisation

Flame photometric

Universal, simple operation

Mainly for inorganic compounds

Universal - gives structural information

- also can be highly selective

Compound with electronegativegroups 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.

High

Low

V.High

V.High

V.High

High

Detector Notes Sensitivity


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13

Quantification: Calibration

The purpose of calibration is to allow the size of peaks in a

chromatogram to be converted into the correspondingamount of analyte having passed through the column.

Usually the area under the peakin a chromatogram is

proportional to the amount of that compoundreaching 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 responsefactor.

Response factors must be taken into account when comparingpeak areas for different compounds.

Variation of Detector Response


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Quantification: Matrix Affects

Comparing chromatograms obtained with real samples to thosefrom artificial mixtures of pure reference compounds

(standards) may lead to errors - the sample matrix can affectthe response.

Standards should be present in an identical, or at least verysimilar, matrix as the sample.

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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uilhausUNSW1999

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Quantification with Standard Addition

Plot areas and extrapolateto -ve x-axis

0

area

amount of standard addition

Multiple Standard Addition

amount in sample

The standard is a pure sample of the analyte added in exact knownamounts to the sample (usually) at the beginning of the sample

preparation.

Response factors are not necessary but a pure reference compoundmust be available for every analyte.

The matrix is well matched


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uilhausUNSW1999

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Quantification with Internal Standards

Known amount of a reference compound (internal standard) mixedwith sample. The internal standard must...

NOT already be present in sample BE chemically inert HAVE similar retention properties NOTreact with or change the analyte.


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uilhausUNSW1999

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Quantification with Internal Standards

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

C A

A

C

Ri

i

s

s

i s

=

/

Ai=area of peak for component i

As=area of peak for standard sCs=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 relativeresponse factors


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Quantification: Response Factors

Compound ( i)(in ref. mixture)

1

23

4

Internal Standard

Conc.(nmol/mL)

5.30

4.703.50

3.40

4.90

Peak Area(arb. units)

10102

110059980

8765

12349

RArea/Conc.

1906

23412851

2578

2520

Ri/ Rs

0.76

0.931.13

1.02

1.00

A. Measurement of Relative Response Factors


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uilhausUNSW1999

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Quantification: Response Factors

Compound ( i)

1

23

4

Internal Standard

Conc.(nmol/mL)

7.14

4.990.08

3.99

4.90

Peak Area(arb. units)

12198

10422196

9152

11012

Ri/ Rs

0.76

0.931.13

1.02

1.00

B. Internal Standard AnalysisC

A

A

C

Ri

i

s

s

i s

=

/


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uilhausUNSW1999

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Quantification by Area Normalisation

May be used when all mixture components elute from columnand give responses on detector.

Assumes total area of all peaks corresponds to total amount ofsample reaching detector.

Independent of injected volume

No need to match matrix No need for internal standard

Disadvantage: first condition rarely met.


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uilhausUNSW1999-Allrightsreserved.

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Quantification by Area Normalisation

Measure each peak area

Correct each area by dividing by the response factor Add corrected areas

Compute percentage of each component from ratio ofcorrected area to total area

Method and example

Compound

Methyl acetateMethyl propionate

Methyl butyrate

Area

1640045200

30200

R

0.600.78

0.88

Area/R

2733357949

34318

119600

%

22.948.5

28.6


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Examples of Analyses

1 cocaine

2 codeine3 morphine

4 quinine

Alkaloids


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Examples of Analyses

1 17-a-estradiol

2 dihydroequiline3 testosterone

5 estrone

6 equiline

Steroids

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