1. Headspace Analysis – A Summary of Possibilities
Ray Perkins, Anatune Ltd.
Andreas Hoffmann, GERSTEL GmbH.
Dan Carrier, Anatune Ltd.

3. Competitive product analysis
Product development
Quality control
Product safety
Competitive product analysis
Who is this talk aimed at – consumer products
Product development, QC, competitive product analysis
In all cases the way a product smells and tastes is really important.
Why is liquid injection a problem? All of these products contain large amounts of involatile material and many contain water. So if we use dilute and shoot we clag up the inlet column or mass spectrometer
In contrast headspace techniques are good because we only transfer gas phase material into the instrument – what goes in comes out the other end without causing trouble on the way!

4. Static Headspace Sampling SHS Solid Phase Micro Extraction SPME
Dynamic Headspace Sampling
DHS
Fully Evaporative Dynamic Headspace
FE-DHS
Headspace Analysis
With headspace analysis there are lots of variations on the theme. In our applications lab we do a lot of work matching different HS techniques to individual customers problems.
Today I want to do two things:
Give some insight into the relative performance of several important headspace techniques in the cotext of consumer product analysis.
Introduce a new and potentially very useful headspace technique called fully evaporative dynamic headspace sampling..

5. GERSTEL serves the analytical the needs of the consumer product industry World-wide and Anatune serves this market in the UK and Ireland.
Analysts working in this field, need to employ a wide variety of HS techniques along with GC-MS and this has driven the evolution of the GERSTEL MPS sampler

6. Instrumentation
The MPS is a single robotic platform that is designed to support pretty much any HS technique, without the need for significant reconfiguration of the instrument, so it is easy for analysts to chose the HS technique that best meets their immediate needs.
We ahe a system configured like this in our lab – which is in constant use as do the other apps labs in the GERSTEL family World-wide, an all of the techiques I am about to discuss were carried out on a system like this.

7. Volatile & Semi-Volatile Organics
SDE
Simultaneous Distillation Extraction
GERSTEKL in Germany approached by customer wanting an alternative to SDE (Simultaneous Distillation Extraction)
Thiis in a standard procedure in flavour & aroma research
It enables volatile organics to be extracted from an aqueous product by distilation and for them to be concentrated in an organic solvent, leaving water and involatile matrix behind.
Provides good results except very polar and very water-soluble compounds, may cause in some cases building of artefacts.
But...very labour and time intensive, only 1-2 sample per day and apparatus possible (plus cleaning, high solvent usage, …)

8. Perfume oil (45 compounds), spiked into these matrices:
Test Samples
Perfume oil (45 compounds), spiked into these matrices:
1 % in shampoo
1 % in dishwasher detergent
1 % in fabric softener
1 % in washing powder
0.5 % in vanishing cream
Compare various HS techniques and see how the compare with SDE
First made a synthetic perfume mixture with 45 compounds – including know problem compounds
Then add this to various matrices for test purposes

9. Ethyl-2-methyl butanoate
Test Standard
5.00
10.00
15.00
20.00
25.00
30.00
35.00
500000
Dipropylene glycol (matrix compound)
ß-Ionone
Musk T
Ethyl-2-methyl butanoate
b.p.130oC
Benzyl cinnamate
b.p. 229oC
Ethylene brassylate
b.p. 330oC
Chromatogram of perfume oil (liquid injection), 45 compounds by straight liquid injection split conditions
Matrixcompound (Dipropylenglycol) is also important to be able to reproduce e.g. a competitor‘s product
BP wise, ranges from 130 C to 390 C
MP wise -68 C 53 C room temp
a-Pinene

10. Test Standard Peak Areas Normalised
The determined peak areas of each analyte from the liquid injection are normalised to 100% through applying an individual conversion factor they are brought to the same scale (e.g. 100%). The obtained pattern serves to define what a perfect recovery would look like.
When analyzing an unknown sample containing the same analytes and applying the conversion factors to the determined peak areas, a comparison between the normalised results is easy. If e.g. the same pattern is visible, the constitution of the sample as seen by the GC-MS, is identical to the standard.

11. Static Headspace of Shampoo (2g spiked with 1% perfume oil)
5.00
10.00
15.00
20.00
25.00
30.00
35.00
1e+07
1.1e+07
1.2e+07
1.3e+07
1.4e+07
Describe how SHS works – simple 2 phase system
2 g Shampoo, spiked with 1% perfume oil, static headspace at 80°C
Resulting chromatogram far away from liquid injection, no sign of the matrix visible

12. Static Headspace of Shampoo
Normalised
That‘s also visible using the normalization procedure, This pattern is completely different from that obtained by liquid injection, so definatley not a good alternative to SDE It looks totally different….

13. Static Headspace all Matrices
Normalised
Same result for the other samples
Note that the overall pattern of recovery is characteristic of the HS technique used but that there are also differences dus to the effects of different matrices. You will see that pattern thoughout...
Does that mean static headspace has no use – far from it – it gives a very good representation of the smell of the product to a consumer.

14. And that initial aroma can be very evocative and very important!

15. SPME (DVB/Car/PDMS) Shampoo
5.00
10.00
15.00
20.00
25.00
30.00
35.00
1e+07
1.1e+07
1.2e+07
1.3e+07
1.4e+07
1.5e+07
1.6e+07
1.7e+07
1.8e+07
1.9e+07
2e+07
2.1e+07
2.2e+07
Next lets consider Solid Phase Micro Extraction
Describe how it works – 3 phase system
2 g Shampoo, spiked with 1% perfume oil, SPME (headspace) at 80°C
Looks better – more late running compounds than static headspacee.
But still no sign of the dipropylene glycol so an important charateristic of the perfume is missing from this chromatogram.

16. SPME (DVB/Car/PDMS) Shampoo
Normalised
The fingerprint confirms this..
Why the difference – less volatile compounds partition weakly into the gas phase, but strongly into the SPME fibre, so the fibre removes them from the headspace and this drives the mass transfer from the matrix into the headspace...

17. SPME (DVB/Car/PDMS) all Matrices
Normalised
Critical are the following compounds….
Except Hydroxycitronellal all solids don‘t migrate into the SPME fibre.
Hydroxycitronellal
Ethylene Brassilate
Ethyl Maltol
Ethyl Vanillin
Maltol
Vanillin
Frambinon
Benzyl Cinnamate

18. Dynamic Headspace Sampling
Purge Gas Out
Adsorbent Tube
MFC
Purge Gas In
If removal of high mw compounds from the headspace works – how an we improve on SPME?
Next tried dynamic HS
How DHS works
Sample Vial

19. Instrumentation GERSTEL Multi-purpose sampler (MPS)
Dynamic Headspace System (DHS)
The various elements of a DHS system...
Thermal Desorption Unit (TDU)
Cooled Injection System

20. Dynamic Headspace System
Loan vial into temperature controlled chamber
Move cooled trap zone over vial
Push tube and needles down so that the needlles pierce the septum on the vial

21. Finally once the volatiles have been trapped move the tube to the TDU and desorb it onto a cryogenically cooled injection system for focusing and injection onto the column.

22. Dynamic Headspace of Shampoo
5.00
10.00
15.00
20.00
25.00
30.00
35.00
1e+07
1.2e+07
1.4e+07
1.6e+07
1.8e+07
2e+07
2.2e+07
2.4e+07
2.6e+07
2.8e+07
3e+07
3.2e+07
3.4e+07
3.6e+07
3.8e+07
4e+07
4.2e+07
4.4e+07
4.6e+07
4.8e+07
Chromatogram classical DHS
Only 10 mL purge volume possible, split 1:50 (instead of 1:10)
Good recovery of early running peaks only. Why?
Still no matrixpeaks!!!

23. Dynamic Headspace of Shampoo
Why is that?
An improvement can only be reached if the liq. sample/vapor phase equlibrium can be displaced, only then less volatile compounds would enter the vapor phase.
Due to the high amount of analytes the sampling volume had to be choosen so small (10 mL) that de facto only a large volume static headspace analysis has taken place.

24. But…if the analyte concentration is too high…
What about reducing the sample size dramatically?

25. Matrix independent headspace gas chromatographic
analysis. The Full Evaporation Technique
M. Markelov, J. P. Guzowski, Analytica Chimica Acta, 276 (1993) 235.
1μL～
One could reduce the sample size to e.g. 1 µL…
Thermostate the vial and analyse the gas phase….
That is the original idea of Michael Markelov
Matrix effects are eliminated, since the equlibrium has totally moved to the gas phase (at least in theory)
80℃ GC

26. Sample Preparation: Dilute Sample with Methanol (1:9), Shake
Place 20 µL of Methanolic Phase in Empty Vial
Analyze
Problem: how to place such small quantities of shampoo, washing powder, etc.?
Solution: dilute with Methanol
FET can be combined with any HS technique!

27. Key Insight
The Full Evaporative Technique can be combined with ANY means of headspace sampling!
This is simple static headspace – FET suddenly we are seeing compounds of all volatilities!
This isn’t a pretty chromatogram – but it is jam packed with useful information!

28. Static Headspace (FET)
Standard
5.00
10.00
15.00
20.00
25.00
30.00
35.00
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
360000
Static headspace (80°C), FET
1 µL perfume oil in MeOH, diluted down to the concentration of compounds in the samples
Very small peaks, but…..

29. Static Headspace (FET)
Standard
…nevertheless a significant improvement compared to classical static headspace.
Disadvantage: Too close to instruments detection limit. And an increase of sample amount would undo the FET-effect.

30. SPME (FET) Standard Dipropylene glycol Let‘s apply FET to SPME…..
5.00
10.00
15.00
20.00
25.00
30.00
35.00
200000
400000
600000
800000
Dipropylene glycol
Let‘s apply FET to SPME…..
…. and suddenly matrix information is there

31. SPME (FET) Standard Ethylene Brassilate Frambinon Ethyl Vanillin
Surprise:
Volatile compounds are discriminated!
The reason is:
The equilibrium has completely moved in favour of the vapor phase, but there still is another equilibrium, namely between the vapor phase and the SPME fiber.
And this fiber is affected by the temperature of 80°C, that drives the compounds back from the fiber into the vapor phase
Despite that the critical analytes have better recoveries
Ethylene Brassilate
Frambinon
Ethyl Vanillin
Vanillin
Maltol
Ethyl Maltol
Benzyl Cinnamate

32. SPME (FET) All Matrices Ethyl Maltol Maltol Ethyl Vanillin
The pattern repeats for all matrices...
Ethyl Maltol
Maltol
Ethyl Vanillin
Ethylene Brassilate
Vanillin
Benzyl Cinnamate
Frambinon

33. Dynamic Headspace (FET)
Standard
5.00
10.00
15.00
20.00
25.00
30.00
35.00
1e+07
1.1e+07
1.2e+07
1.3e+07
1.4e+07
1.5e+07
1.6e+07
1 µL perfume oil in MeOH, diluted down to the concentration of compounds in the samples

34. Dynamic Headspace (FET)
Shampoo
5.00
10.00
15.00
20.00
25.00
30.00
35.00
500000
1e+07
1.05e+07
1.1e+07
1.15e+07
1.2e+07
1.25e+07
Shampoo
Matrix compounds are also visible (information of matrix composition is important)
Not that much, that it disturbes chromatography

35. Dynamic Headspace (FET)
Standard vs. Shampoo
Octanal
Decanal
Lilial
Hydroxycitronellal
In dynamic headspace FET there is only a single phase system left, this can be seen in the quality of the fingerprint….
Nevertheless there are some critical compounds. Presumabely they got caught at some glass surfaces (physical effects).
In the shampoo these compounds can be clearly seen, possibly the shampoo matrix deactivates the glass surfaces.
Some other compounds show worse recovery from the shampoo…the chemical composition (e.g. pH-value,…) might be the reason for that (chemical effects)
Ethylene Brassilate
Benzyl Cinnamate
Frambinon
Ethyl Vanillin
Maltol
Vanillin
Ethyl Maltol

36. Dynamic Headspace (FET)
All Matrices

37. Dynamic Headspace (FET)
Shampoo – carryover?
An imortant criterium for analysis is the completeness of vaporization. To check this, the shampoo sample has been analyzed (blue trace) and the same vial has been run again (red trace). The results show, that the sample has been almost completely vaporized already in the first run.
That means: Since the sample has been completely vaprized, classical quantification approaches (incl. internal standard) can be applied!!!

38. SDE vs. DHS (FET)
A comparison study (performed with a shampoo with slightly different composition) between SDE with hexane, SDE with frigene and with DHS FET proves the comparibility of DHS results with the much more labour and time intensive SDE procedures.

39. Practical application of FET-DHS
Measuring specific flavour compounds in a herbal liqueur
Slight differences in the composition of these beverages can drastically alter change the taste
Some of the critical compounds are present at very low concentration and can’t be seen using static headspace.

40. Anethole was the only compound detected using static headspace
See the black trace
When run using FET DHS – blue trace you can immediately see the sensitivity gain.

41. Here is the FET DHS trace – showing much more detail
Here is the FET DHS trace – showing much more detail. The increase in signal to noise was over 100 fold

42. Estragole
Concentration: 0.16 µg/ml

43. Anethole
Concentration: 13 µg/ml

44. No one HS technique suits all purposes
Conclusions
No one HS technique suits all purposes
Full Evaporative technique can be used with any HS technique.
FET+DHS provides a more practical alternative to SDE
Conclusion