a technique where the vapors in the gas above, and in ... · headspace analysis a technique where...

A technique where the vapors in the gas above, and in equilibrium with a solid or liquid is sampled - as opposed to directly sampling, the solid or the liquid.

Headspace analysis

Central questions during method development

The purpose of analysis

Analytical result



Analytical result

- which substance group? - which facilities are available? - which accuracy is required?



Sample collection

Data processing

Analytical result

direct measurement

- sensors




Sample collection

Sample preparation

Chromatography / detection

Data processing

Analytical result

direct measurement

- CLS, SPME - solvent extraction - sensors

- sample cleaning - derivatisation

- GC / LC - Mass spectrometry


10

Sensors - Chemiresistor

11

Elektrochemische Sensoren

12

AFM

13

Cantilever als Sensoren

Mehrere Si Cantilever mit unterschiedlichen Beschichtungen optisch ausgelesen

14

Cantilever als Sensoren

15

Cantilever as Sensors

z.B. Ethanol (links) durch Mustererkennung auswertbar. Auch spezifische Mischungen werden erkannt (rechts). Prozesse sind quantitativ.



Sample collection

Sample preparation

Chromatography / detection

Data processing

Analytical result

direct measurement

- CLS, SPME - solvent extraction - sensors

- sample cleaning - derivatisation

- GC / LC - Mass spectrometry


Headspace analysis

G = the gas phase (headspace): The gas phase is commonly referred to as the headspace and lies above the condensed sample phase. S = the sample phase: The sample phase contains the compound(s) of interest. It is usually in the form of a liquid or solid in combination with a dilution solvent or a matrix modifier. Once the sample phase is introduced into the vial and the vial is sealed, volatile components diffuse into the gas phase until the headspace has reached a state of equilibrium. The sample is then taken from the headspace.

A technique where the vapors in the gas above, and in equilibrium with a solid or liquid is sampled - as opposed to directly sampling, the solid or the liquid.

Headspace analysis

Simple: use a gas-tight syringe to sample the vapor above the analyte matrix and inject it in some analytic instrument.

Analytical laboratory: GC and GC/MS

GC-MS (Quad)

GC-MS (TOF)

- trace analytic - volatile analysis - product identification - screening

- isotope ratio - thermal desorption - high accurancy MS

- screening of less complex samples - Quantification of specific compound

GC-FID

EI, FID 100 pg analyte on column

CI, 5 pg analyte on column

Z-Nose

On-line monitoring of volatiles

The 7100 zNose is a gas chromatograph that fits into an attaché case and can analyze a sample in as little as 10 seconds.

Z-Nose

The 7100 zNose is a gas chromatograph that fits into an attaché case and can analyze a sample in as little as 10 seconds.

Vapor Prints can readily reveal whether a sample is tobacco or marijuana.

Gas Chromatography

Most important separation technique in headspace analysis

Chromatography (“color writing”) is a physical method of separation in which the components to be separated are distributed between two phases, one of the phases constituting a stationary bed of large surface area, the other being a fluid that percolates through or along the stationary bed. Gas Chromatography: Analyte has to be a stable gas at Temperatures < 300°C

Ettre & Zlatkis, 1967, The Practice of Gas Chromatography

Gas Chromatography

Carrier gas (nitrogen or helium)

Sample injection

Long Column (30 m) In heated oven

Detector (FID, MS…)

Gas Chromatography Velocity of a compound through the column depends upon affinity for the stationary phase

Area under curve is proportional to of compound adsorbed to stationary phase

Gas phase concentration Carrier gas

Gas Chromatography

• Requires only very small samples with little preparation • Good at separating complex mixtures into components • Results are rapidly obtained (1 to 100 minutes) • Very high precision • Only instrument with the sensitivity to detect volatile organic

mixtures of low concentrations • Equipment is not very complex (sophisticated oven)

1. Isobutane 2. n-Butane 3. Isopentane 4. n-Pentane 5. 2,3-Dimethylbutane 6. 2-Methylpentane 7. 3-Methylpentane 8. n-Hexane 9. 2,4-Dimethylpentane 10. Benzene 11. 2-Methylhexane 12. 3-Methylhexane 13. 2,2,4-Trimethylpentane 14. n-Heptane 15. 2,5-Dimethylhexane 16. 2,4-Dimethylhexane 17. 2,3,4-Trimethylpentane 18. Toluene 19. 2,3-Dimethylhexane 20. Ethylbenzene 21. m-Xylene

Temperature Control Options

Column: Petrocol DH, 100m x 0.25mm ID, 0.5µm film Oven: 35°C (2 min) to 180°C at 5°C/min, hold 5 min Carrier: helium, 20cm/sec (set at 35°C)

Example Method Isothermal 120 °C

T-Gradient

http://info.sial.com/cgi-bin/gx.cgi/Applogic+Supelco.Groups?ID=1338

split mode

2 ml/min

1 ml/min

46 ml/min

49 ml/min

Injector

splitless mode

2 ml/min

47 ml/min

0 ml/min

49 ml/min

Injector

Injector

LEAK

Most common injector problem

Analytical laboratory: HPLC

LC-UV-MS (Trap)

LC-UV

- LC-fluorescence - screening - product identification

- preparative sample cleaning

Problem: Analytes in headspace analytics are often more volatile than solvent => high background, poor MS BUT: Derivatisation makes metabolites acessible

NO

F

FF

F

F

NH2O

F

FF

F

FO

3/32

Ionenmobilitätsspektrometrie

Einfache Einheiten zur Bestimmung der Beweglichkeit von Ionen in der Gasphase

3/33


3/34


Nach Injektion (t = 0) ist Ion mit spezifischer Driftzeit nachweisbar

Miniaturisierung für Sensortechnik

Kopp

lung

mit

LC

Analytical laboratory: GC and GC/MS

GC-MS (Quad)

GC-MS (TOF)

- trace analytic - volatile analysis - product identification - screening

- isotope ratio - thermal desorption - high accurancy MS

- screening of less complex samples - Quantification of specific compound

GC-FID

EI, FID 100 pg analyte on column

CI, 5 pg analyte on column

36

Mass Spectrometry

http://masspec.scripps.edu/ Georg Pohnert

37

Mass Spectrometry

40 60 80 100 120 140 160 180

100

rel.

Int.

(%)

55 70

41

83

148 106 112 150

m/z

ClD

D

O+ HOOC

OH

O

0

100 140 180 220 260 m/z

0

50

100 209 [M+H]+

rel.

Int.

(%)

Electron impact ionisation

Elektrospray ionisation

Mass spectrum: 2D-Representation of relative signal intensity vs mass / charge ratio

2/38

Instrumentation

2/39

Mass analysators

•Sektorfield

•Quadrupol

•Ion trap

•Tof

•Orbitrap

R>70.000 (Standard 1000), Massenbereich 2000-5000 amu, langsam (5 scans s-1) sperrige Instrumente

Nominalmassenauflösung, Massenbereich 600-2000 amu, schnell, gute Quantifizierung, billige Instrumente

Nominalmassenauflösung, Massenbereich 600-2000 amu, schnell, schlechte Quantifizierung, eingeschränkte Bibliothekssuche, flexible Experimente, billige Instrumente

Hochauflösung, Massenbereich 107 amu..., schnell, eingeschränkte Quantifizierung, mittlere Preiskategorie

R 200.000, Massenbereich bis 50.000, sehr Empfindlich, schnell, teuer

3/40

GC MS

Vorgeschaltete Chromatographie erlaubt Auftrennung von Signalen

3/41

GC MS Zweidimensionales Chromatogramm wenn Totalionenstrom gemessen wird

3/42

GC MS Zweidimensionales Chromatogramm wenn Totalionenstrom gemessen wird

3/43

GC MS Massenspektren nach einfachem Abzug des Untergrundsignals

3/44

GC MS Massenspektren erlauben oft Bibliothekszuordnungen

3/45

GC MS Verfolgen der Ionenspur erlaubt selektive Detektion von Signalen

TIC

m/z = 121

Sampling techniques: Direct injection

• Heated needle • No enrichment • Low sensitivity

Direct injection with automated devices

Sampling techniques: Direct injection

Consequence of low sensitivity: No broad application BUT: If feasible a perfect solution since cheap and quick!

Sampling techniques: Purge and Trap

• Way to measure dilute samples by concentration of constituents

• Trap constituents under low temperature • Heat trap to release constituents and send to GC column

N2

Trap . . . . . .

. . . . .

. . .

. . .

. . .

.


• Way to measure dilute samples by concentration of constituents

• Trap constituents under low temperature • Heat trap to release constituents and send to GC column

N2

Trap . . . . . . . . . . . .

Prinzip: (Purge-Phase)

He, N2

Probe

Umschalten des Ventils in der Desorptionsphase

Analyt gelangt zum GC

Eingang in der Desorptionsphase


SPME

Enrichment of volatiles

SPME on fiber

derivatisation

CLS activated carbon

CLS Carboxen

Tenax

Closed loop stripping Solid phase microextraction

SPME Solid Phase Micro Injection

SPME Solid Phase Micro Injection – Method development


Extraction time

On fiber derivatisation Step 1: impregnation of the fiber

Step 2: fiber-incubation into the headspace of the sample

headspace

F

F

F

F

FO

NH2

bouquet of aldehydes

Volatile release in chemical defense

Diplodus holbrooki

Lytechinus variegata

Dictyota dichotoma

Dictyota dichotoma exhibits an activated defense against marine herbivores

Solid phase micro-extraction (SPME)

0 1 2 3 4 5 6 7 8 Time (min)

30 40 50 60 70 m/z

27.8

31.8 43.7

39.7 27.1 44.8 54.8 58.7

Intact algae

30 40 50 60 70 m/z

27.8

46.7 61.7 31.8

44.7

60.8

34.8 26.9 43.8

39.7 48.7 57.7 36.8 55.7 64.8

Wounded algae

Rela

tive

Abun

danc

e

After wounding there is an up regulation of volatile compounds with a complex pattern.

0

100

Rela

tive

Abun

danc

e

0

100

N TMA 59 m/z

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

100

Time (min)

S DMS 62 m/z

Accurate mass confirms identity: Trimethylamine: calculated 59.0732 m/z, measured 59.0735 m/z; Dimethylsulfide: calculated 62.0189 m/z, measured 62.0190 m/z

Solid phase micro-extraction (SPME)

Bioassay

• choice assay with amphipods

• two pellets of artificial food: control and treated • amphipod food preference was observed every five minutes

control food

treated food

Artificial seaweeds containing volatiles

Enteromorpha spp. with agar

3.

4.

2.

TMA, DMS and/or acrylic acid 1.

SPME verification of recovery: 1 hour 50 % 6 hours 10 %

45 °C

Amphipods fed on trimethylamine diet

0

50

100

% a

mph

ipod

ass

ocia

tion

with

eac

h pe

llet

300 µg/ml TMA 3 mg/ml TMA p = 0.008

N

40 µg/ml DMS

S

400 µg/ml DMS

Amphipods fed on dimethylsulfide diet

0

50

100

% a

mph

ipod

ass

ocia

tion

with

eac

h pe

llet

Possibly a stimulating effect on amphipods?

40 µg/ml acrylic acid

p = 0.046

H H 2 C

C C O O H

400 µg/ml acrylic acid

Amphipods fed on acrylic acid diet

0

50

100

% a

mph

ipod

ass

ocia

tion

with

eac

h pe

llet

Blend recognition of released volatiles lead to avoidance reaction

p = 0.016 p = 0.003

S

N H H 2 C C

C O O H

300 µg/ml TMA, 40 µg/ml DMS & acrylic acid

3 mg/ml TMA, 400 µg/ml DMS & acrylic acid

Amphipods fed on mixture diet

0

50

100

% a

mph

ipod

ass

ocia

tion

with

eac

h pe

llet

Emilania huxleyi as dominant bloom forming DMS producer

Dimethylsulfide / Dimethylsulfoniopropionate

H3CS

CH3ca. 1 pg cell-1

Emilania huxleyi as dominant bloom forming DMS producer


H3CS

CH3

ca. 1 pg cell-1

Multiplied by all DMS producing phytoplankton cells 17–34 Tg S year-1 (17-34 million tonnes). 90 % of the biogenic sulphur emissions from oceans and roughly half of the global biogenic sulphur emission.


DMS is involved in primary metabolism (osmoregulation, antioxidant, cryoprotection) of micro- and macroalgae.

H3CS

CH3

OH

O

O-

O

S+

CH3

H3C+

Effect of zooplankton grazing on DMS release by phytoplankton (black dots)

Dacey and Wakeham 1986

DMS release during grazing

Gymnodium sp.

Labidocera sp.

Steinke M. LIMNOLOGY AND OCEANOGRAPHY 51 (4): 1925-1930 JUL 2006

Herbivore stimulation through DMS

Herbivore stimulation through DMS

Steinke M. LIMNOLOGY AND OCEANOGRAPHY 51 (4): 1925-1930 JUL 2006

Multiple roles of Metabolites

Birds are attracted to DMS that presumably indicates food sources

Multiple roles of Metabolites

Pohnert et al. Trends in Ecol. Evol. 2007

RI SPME GC/MS of Oxylipins

O

pent-1-en-3-one

OH

(Z)-hex-3-en-1-ol

O

(Z)-pent-2-enal

O

(E)-hex-2-enal

O

(2E,4E)-hexa-2,4-dienal

OH

oct-1-en-3-ol

Oacrylaldehyde

O

(E)-but-2-enal

OH

(Z)-pent-2-en-1-ol

O(Z)

-hex-3-enal

OH

pent-1-en-3-ol

t/min

100

Chemical interaction in Plankton

Allelopathy

Chemical communication

Prey location

Defense

O

O

Chemical defense of Thalassiosira rotula?

Aldehydes from T. rotula inhibit copepod egg cleavage

bioassays with sea urchin eggs control t = 4 h with 1.5 mg l-1

decadienal control t = 10 min

Miralto et al. (1999) Nature 402, 173

Activated chemical defense of Thalassiosira rotula

Angewandte Chemie Int. Ed. (2000) 112, 4506

1

2

3

4

5

deca

trie

nal

[fmol

/cel

l]

decatrienal quantification from diatom cultures using SPME/GC/MS

intact cells

100 200 300 400 500 t [sec]

O



1

2

3

4

5

deca

trie

nal

[fmol

/cel

l]


O

100 200 300 400 500 t [sec]

mechanical wounding

intact cells




O

wound-activated defense to overcome dilution effects not beneficial for the single diatom but might function as community-based defense

1

2

3

4

5

deca

trie

nal

[fmol

/cel

l]

100 200 300 400 500 t [sec]

mechanical wounding

intact cells

Diverse oxylipins form the wound reaction of diatoms

MEPS (2002) 245, 33; Angewandte Chemie Int. Ed. (2000) 112, 4506

O

O

O

O

O

O4

O12

O10

5 6 7 8 9 10 11 12

t [min]

50

Irel.

O

HOOC

O COOH

a technique where the vapors in the gas above, and in ... · headspace analysis a technique where...

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