Solid-phase Microextraction Analyses of

Flavor Compounds in Foods

 Continuous Solvent Extraction

Condenser

:::
::: Beverage sample

Solvent (Ether) Holes at the bottom

Water Bath of the tube
Steam distillation and continuous solvent extraction
Kuderna-Danish Assembly for Concentration of
Isolated Compounds by Solvent Evaporation

Water Bath
Gas chromatogram of Orange Juice Flavor

1
10 11
17

3
13

16 Total GC peak area

15
6 in 0 kGy: 2.99 × 107
8
9

5 12
7
14
 Objectives of Solid Phase Microextraction

Conventional Sample Preparation

• Time and Labor Intensive
• Multiple Steps
• Loss of Sample
• Errors in each steps
• Contamination
To produce sample with highest compound concentration,
lowest level contamination and shortest sample preparation time
Instrumental Analysis of Volatile Compounds

• Static headspace analysis

• Dynamic headspace analysis

• Solid phase microextraction

 III. Headspace Analysis

Static headspace analysis

Syringe
Silicone rubber
stopper

Can
 VI. Dynamic Headspace Analyzer

Diagram of dynamic headspace sampler and gas chromatographer

Detection Limits and Precision of Organic
Volatiles in Water a

Technique Detection Limit Precision (% rsd)

with FID (ppb)

SPME 0.05-0.3 1-3

Static Headspace 1-2 1-3

Dynamic Headspace 0.003-0.005 1-8

Direct Injection 17-240 2-13

a; Methyl chloride, chloroform, dioxane, TCE, benzene, toluene, xylene, and 1,2,4-trimethylbenzene
 Solid Phase Microextraction

Solid Phase Microextraction has been commercially

available for 10 years and new applications are being
developed for flavor and food analyses rapidly
 Solid phase microextraction

A technique that uses a short, thin, solid rod of fused

silica, coated with absorbent polymer for extraction
of volatile compounds

Principle : Equilibrium partitioning of the analyte

between the coating fiber and headspace.
SPME Fiber with Holder
Diagram of SPME Extraction

Fiber

Headspace SPME
SPME Analysis of Volatile Compounds

Plunger

Adjustable depth
gauge

Barrel

Coated SPME
fiber

Water bath
 Principles of Headspace SPME

nf : The # of moles of flavor compounds on

fiber coating

nf= Kfh VfVsCo

Kfh Vf+Khs Vh+Vs
Khs : Partition coefficient of flavor
compounds between headspace and solution

Concentration of compounds on fiber coating

Kfh =
Concentration of compounds in headspace

Vf, Vs, Vh: Volume of fiber coating, solution,

and headspace, respectively
Co: Initial concentration of flavor compounds
in the solution
Effects of Different Fibers on the Volatile
Compound Extraction of Soybean Oil

• CB/PDMS:Carboxen/Polydimethylsiloxane
• PDMS: Polydimethylsiloxane
• CW/DVB: Carbowax/Divinylbenzene
• PA: Polyacrylate.
Effects of Different Fibers on the
Hexanal Analysis in Oil
2,000

1,600
Relative Hexanal Peak Size

1,200

800

400

0
CB/PDMS PA PDMS CW/DVB
 Effects of Different Fibers on the
Hexanal Analysis

Hexanal Peak in Electronic Count

Mean CV (%)

CB/PDMS 499 4.2

PA 739 7.2
PDMS 966 3.2
CW/DVB 1,520 10.7

CV: Coefficient Variation (%) for n =5

Significant difference (P<0.05)
Carboxen, Polydimethylsiloxane
Poyacrylate
Carbowax and Divinylbenzene
Reproducibility for the Determination of Major Flavor
Compounds in a Single Strength Orange Juice

Ethyl butyrate α -Pinene Octanal Limonene Decanal

Replicates (ppm) (ppm) (ppm) (ppm) (ppm)

1 0.432 1.378 1.089 251.05 1.005

2 0.400 1.391 1.050 254.28 0.925
3 0.391 1.343 1.054 248.26 0.987
4 0.380 1.389 1.059 256.25 0.995
5 0.403 1.402 1.020 255.71 1.015
6 0.397 1.470 1.010 260.01 1.007

SD 0.017 0.042 0.029 4.130 0.033

Average 0.400 1.395 1.047 254.26 1.989
CV(%) 4.36 3.00 2.71 1.63 3.32
Effect of G.C.Injection Temperature on Soybean
Oil Volatile Compound Analysis

230 °C

250 °C
Effect of Fiber Coating Thickness on the Absorption
for the Extraction of 0.1 ppm Benzene

100

80
100
µ m
Mass (ng)

60

56 µ m
40

20
15 µ m
0
0 200 400 600
Time (Seconds)
 Effect of Distribution Constants on the
Absorption Profile of 0.1 ppm Analyte

30

25 Kfs = 831 (p-Xylene)

Mass (ng)

20

15

10 Kfs = 294 ( Toluene)

5 Kfs = 125 ( Benzene)
0
0 1000 2000 3000

Isolation Time (Seconds)

Kfs: Partition coefficient of flavor compounds between fiber coating and solution
 Effect on Isolation Temperature on the GC
Chromatogram of Headspace Polyaromatic Compounds

Extracted at 25 °C

Extracted at 130 °C

Extracted at 200 °C
 Effect of Water and Microwave Heating on the
Chromatograms of Headspace Polyaromatic Compounds

100
Water Heating
Mass Extracted (ng)

80 Microwave Heating

60

40

20

0
1 2 3 4 5
Compound Number

1, Naphthalene: 2, Acenaphthylene: 3, Benzene: 4, Fluorene: 5,Anthracene

 Effect of Stirring on the Absorption for the
Extraction of 1 ppm Benzene in Water

40 2,500 rpm

30 400 rpm
Mass (ng)

20
0 rpm
10

0
0 200 400 600
Time (Seconds)
 Effect of Benzene Concentration in Water on
Absorption by Fiber

1000
Cs = 10 ppm
100
Mass (ng)

Cs = 1 ppm
10

1 Cs = 0.1 ppm

0.1
0 100 200 300 400 500 600

Time (Seconds)
 Effect of Various Salts on the Concentration
of Volatile Compounds in the Headspace
(FID response)

No Salt

Sodium Chloride

Sodium Sulfate

Potassium
Carbonate
Normalized

Benzene Dioxane
Matrix Effect on SPME Recovery of
Terpene Alcohols
Water
water-salt
12% Ethanol
Detector Response

12% Ethanol-salt

Cltronellol Geranol
SPME Analysis of Volatile Compounds

Plunger

Adjustable depth
gauge

Barrel

Coated SPME
fiber

Water bath
Effects of Temperature and Time on the Equilibrium of
Flavor Compounds Between the SPME Coating and the
Headspace of Orange Juice
30

25 25°C

40°C
FID response

20

50°C
15

60°C
10

5 80°C

0
0 10 60
20 30 40 50

Adsorption Time (minutes)

SPME Analysis of Volatile Compounds

Plunger

Adjustable depth
gauge

Barrel

Coated SPME
fiber

Water bath
Gas chromatogram of Orange Juice Flavor

1
10 11
17

3
13

16 Total GC peak area

15
6 in 0 kGy: 2.99 × 107
8
9

5 12
7
14
 Regression Equations between Flavor Compounds
(ppm) and GC Peak Areas (Electronic Counts)

Concentration
Compounds Regression Eq R2 range (ppm)

Ethyl butyrate Y=0.2891X+0.015 0.99 0.1-1.2

n-Octanal Y=0.4913X+0.003 1.00 0.1-1.3
Decanal Y=0.2010X+0.066 0.99 0.1-1.1
α -Pinene Y=0.3428X+0.092 0.99 0.2-2.0
Limonene Y=17.922X+9.462 0.99 20-50

Y: Compound part per million, X: Electronic counts of GC peak area

Isolation Time Effect on Soybean Oil
Volatile Compounds by SPME
40

60 °C

45 °C
30
35 °C
Relative Peak Size

20

10

0
0 30 60 90 120 150
Isolation Time (min)
Isolation Temperature Effect on Soybean Oil
Volatile Compounds by SPME
30
PV 10
25
PV 50
Relative Peak Size

20

15

10

0
35 45 60

Isolation Temperature (C)

 Isolation Temperature Effect on Soybean Oil
Volatile Compounds by SPME for 60 min.

Soybean Oil 1 (PV:1) Soybean Oil 2 (PV:50)

Isolation Temperature
Total Area Total Area
CV(%) CV(%)
Mean Mean

35°C 9,847 0.85 15,304 2.89

45°C 10,985 3.52 18,060 1.08

60°C 12,978 4.51 24,869 5.43

CV: Coefficient Variation (%) for n =5

Significant difference (P<0.05)
Chromatograms of Soybean Oil Volatile Compounds by SPME
Volatile Compounds in the Headspace of Soybean
Oil by SPME-GC-MS

Compounds Retention Time (min) Relative (%)

Pentane 1.38 3.65
Pentanal 2.06 5.31
Hexanal 3.84 23.5
2-Butanone 3.97 9.09
Heptanal 5.90 2.70
2-Heptenal 6.45 4.76
2-Pentylfuran 8.40 4.77
2,4-Heptadienal 10.99 5.04
t-2-Octenal 11.53 3.37
Nonanal 14.00 2.86
t-2-Nonenal 14.29 0.55
2-Decenal 18.69 34.3
Effect of Isolation Temperature on Corn Oil Volatile
Compounds by SPME
25°C 45°C

35°C 60°C
Volatile Compounds in the Headspace of
Corn Oil by SPME-GC-MS
Compounds Retention Time (min) Relative (%)

Pentane 1.29 13.03

Pentanal 1.88 5.52
Hexanal 3.62 5.39
Heptanal 5.36 1.83
2-Heptenal 6.21 29.52
2-Pentylfuran 8.59 2.53
2,4-Heptadienal 10.88 7.69
t-2-Octenal 11.51 18.07
Nonanal 13.88 6.27
t-2-Nonenal 14.23 1.33
2-Decenal 18.61 4.93
t,t-2,4-Decadienal 20.20 1.17
t,c-2,4-Decadienal 20.70 2.71
Chromatograms of Soybean Oil and Corn Oil

Soybean Oil

Corn Oil
Improving Sensitivity of Solid Phase
Microextraction

• Fiber Thickness
• Extraction Temperature and Time
• Sample Agitation and Concentration
• Direct sampling versus Headspace Sampling
• Selection of Proper Fiber
• Saturation of Sample with Proper Salts
• Maximum Ratio of Sample to Headspace Volume
• Large Sampling Vial
Theory of Solid Phase Microextraction

It is essential to understand the theory to develop and

optimize SPME method for maximizing sensitivity and
minimizing isolation and desorption times
• Compound partition between fiber and sample for
absorption of compound to the fiber
• Like “dissolves” like
• The isolated and concentrated compound desorbs
from the fiber into an analytical instrument
 Conclusion
The SPME-GC is a
• Reproducible
• Economic
• Simple
• Sensitive
for the analysis of volatile compounds in
foods.
Effects of Microwave Processing on the
Flavor of Orange Juice by SPME Analysis

Control
Control

1 min. microwave
processing
2 min. microwave
processing

4 min. microwave
processing
 Natural Cereal Quaker Oats
Company Products Story

What are the ideal analytical method

for flavor isolation from foods?

How to improve the sensitivity of

flavor isolation by SPME?
 Discussion
• SPME has preference to some volatile compounds of vegetable oil
according to the types of fiber and isolation conditions,
2-heptenal showed twice increase and 2-decenal decreased 85% as
isolation temperature increased from 25 °C to 60 °C in corn oil sample,
Pentanal, Hexanal, and t,t-2,4-decadienal showed increasing trend.

• One possible explanation for this phenomenon was further oxidation of

corn oil during isolation at higher temperature. However, the decrease of
2-decenal could not be explained.

• The second explanation could be the equilibrium achievement between

fiber and volatile compounds. In case of pentane, the equilibrium state
was achieved at every isolation temperature.
Contrary to soybean oil, t,c-2,4-decadienal, a typical volatile
oxidation compound of vegetable oil from linoleic acid, could be
isolated at the condition of 60 min at 25,35, 45, and 60°C. Corn oil
has more linoleic acid content (more than 60 %) and higher oxidation
stage PV90.

If the soybean oil sample was exposed 12 hours at 60°C, the amount
of t,c-2,4-decadienal increased to 20% and high molecular weight
compounds such as 2-undecenal, cyclododecane,and docecanal could
be isolated (Data not shown).

This result indicated that the degree of oxidation of vegetable could

be judged by SPME by detecting marker volatile compounds like
hexanal or t,c-2,4-decadienal.
.
By increasing the injection temperature from 230 °C to 250 °C,
more volatile compounds could be detected. If fiber was
contaminated with liquid oil, different chromatogram was
detected. In this case, t,c-2,4-decadienal and t,t-2,4-
decadienalwere major compounds of oxidized soybean oil.
For statistical analysis of data, an analysis of variance
(ANOVA) test were conducted
 Effect of Liquid and Headspace Sampling
Methods on the Absorption of Compounds

3.5
Relative response

3
2.5
2
1.5
1
0.5
0

Dimethylphenol

Trichlorophenol
Toluene
Methyl chloride

Xylene

2,4,6-
2,6-
 Lipid Oxidation of Oil

• Loss of nutritional value

• Polymer formation

• Formation of volatile compounds

Effect of Isolation Temperature on Volatile
Compounds of Corn Oil

25

20
Relative Peak Area

Pentane

15 Hexanal

2-Heptenal

10 2-Decenal

t,c,-2,4-
Decadienal
5

0
25 35 45 55 65

Isolation Temperature (C)

Effect of Isolation Temperature on Volatile
Compounds of Corn Oil

25

20
Relative Peak Area

Pentane

15 Hexanal

2-Heptenal

10 t,c,-2,4-
Decadienal

0
25 35 45 55 65
Isolation Temperature (C)
 Conclusion
The SPME-GC is a
• Reproducible
• Economic
• Simple
• Sensitive
for the analysis of volatile compounds in
vegetable oils.
Depleted Area Around the Fiber During Static
Sampling of Compounds
Theory of Solid Phase Microextraction

It is essential to understand the theory to develop and

optimize SPME method for maximizing sensitivity and
minimizing isolation and desorption times
• Compound partition between fiber and sample for
absorption of compound to the fiber
• Like “dissolves” like
• The isolated and concentrated compound desorbs
from the fiber into an analytical instrument
Geometry of Headspace Sampling by SPME
High Velocity Carrier Gas Stripping Effect on
the Desorption of SPME
1.0

0.8

0.6
n/no

0.4

0.2

0
0 0.2 0.4 0.6 0.8 1.0

Dft/(b-a)2
Diagram for the Isolation of Headspace Flavor
Compounds of Orange Juice by SPME
Desorption Conditions of Solid Phase
Microextraction
Desorption process involves inserting the fiber into the hot
GC injection port. As the temperature increase, the
distribution coefficient between fiber and injection port
decreases and the fiber’s ability to retain compounds
quickly diminishes. Additionally, the constant flow of
carrier gas within the GC injection port helps discharge the
compounds from the fiber and transfer them to a cool
column for refocusing. Typical desorption of two minutes
is adequate to release all compounds form the fiber.
Injection Port of Gas Chromatography
The injector liner is a significant factor in assuring good
results when a SPME fiber is desorbed. The inner diameter
of the insert should be between 0.7 to 0.8 mm. An insert of
smaller diameter will not allow the fiber sheath to penetrate
the injector. The large inserts of 2 -4 mm I.d. will result in
the broadening of early eluting peaks.
SPME fibers generally desorb under hot isothermal
condition. Rapid desorption from the fiber is necessary for
sharp peaks without sample carry over. Injection
temperature is normally 10-20°C below the temperature
limit of the fiber and/or the maximum GC column
temperature
ANALYSIS OF VOLATILE COMPOUNDS
OF SOYBEAN OIL BY SPME-GC-MS

D. F. Steenson, J.H. Lee and D. B. Min

Department of Food Science and Technology

The Ohio State University

SPME Fiber with Holder
Graphic Diagrams of SPME/sample System
Configuration
 Boundary Layer Model Showing the
Different Regions of SPME
Fiber
Core

Fiber
Coating
Concentration

Boundary layer Sample

Distance
Effects of Exposure Time on the Absorption
of Compounds
100

80

60
n/n (%)

40

20

0
0 1 2 3 4 5

Ds/δ Kfs (b-a)t

Singlet Oxygen Lipid Oxidation in Foods

David B. Min

Department of Food Science and

Technology

The Ohio State University

Columbus, OH USA
 Major Discovery of Oxygen Properties

• Oxygen was discovered by Priestly in 1772.

• Diatomic molecule by Avogadro in 1811.
• Even number electron by Faraday in 1848.
• Paramagneitic property by Mulliken in 1928.
• Singlet oxygen by Childe in 1931.
• Singlet oxygen for photooxidation by Schonberg in 1935.
• Singlet oxygen was rediscovered by Foote in 1964.
4-Keto-6-nonenal Formation From Linolenic Acid
CH3 CH2 CH CH CH2 CH CH CH2 CH CH CH2 (CH2)6 COOH
1
O2
CH3 CH2 CH CH CH2 CH CH CH2 CH CH CH2 (CH2)6 COOH
O O

CH3 CH2 CH CH CH2 CH CH CH2 CH CH CH2 (CH2)6 COOH

O• O•
CH3 CH2 CH CH CH2 CH CH CH2 CH
1
O2 O

CH3 CH2 CH CH CH2 CH CH CH2 CH

O O O

CH3 CH2 CH CH CH2 CH CH CH2 CH

•
O• O

CH3 CH2 CH CH CH2 C CH2 CH2 CH

O O
4-Keto-5-nonenal Formation From Linolenic Acid

C H 3 C H 2 C H C H C H 2 C H C H C H 2 C H C H C H 2 (C H 2 ) 6 C O O H
1
O2
C H 3 C H 2 C H C H C H 2 C H C H C H 2 C H C H C H 2 (C H 2 ) 6 C O O H
O O

C H 3 C H 2 C H C H C H 2 C H C H C H 2 C H C H C H 2 (C H 2 ) 6 C O O H
O • O •
CH3 CH2 CH CH CH2 CH CH CH2 CH
1
O2 O
CH3 CH2 C H CH CH2 CH CH CH2 CH
O O O
CH3 CH2 CH CH CH2 CH CH CH2 CH
•
O • O

CH3 CH2 CH CH CH2 C CH2 CH2 CH

O O
4-Keto-5-nonenal Formation From 4-keto-6-nonenal

CH3 CH2 CH CH CH2 C CH2 CH2 CH

O O
-H•

CH3 CH2 CH CH CH C CH2 CH2 CH

•
O O

CH3 CH2 CH CH CH C CH2 CH2 CH

O O

CH3 CH2 CH CH CH C CH2 CH2 CH

•
O O
+H•

CH3 CH2 CH2 CH CH C CH2 CH2 CH

O O
4-Keto-nonanal Formation From Linoleic Acid
CH3 CH2 CH2 CH2 CH2 CH CH CH2 CH CH CH2 (CH2)6 COOH
1
O2
CH3 CH2 CH2 CH2 CH2 CH CH CH2 CH CH CH2 (CH2)6 COOH
O O

CH3 CH2 CH2 CH2 CH2 CH CH CH2 CH CH CH2 (CH2)6 COOH

O O
• •

CH3 CH2 CH2 CH2 CH2 CH CH CH2 CH

1
O2 O

CH3 CH2 CH2 CH2 CH2 CH CH CH2 CH

O O O

CH3 CH2 CH2 CH2 CH2 CH CH

•
CH2 CH
O
• O

CH3 CH2 CH2 CH2 CH2 C CH2 CH2 CH

O O
2- Pentyl furan Formation From 4-keto-nonanal

CH3 CH2 CH2 CH2 CH2 C CH2 CH2 CH

O O

CH3 CH2 CH2 CH2 CH2 C CH CH CH

O O
H H

H2 O

CH3 CH2 CH2 CH2 CH2 C CH

HC CH
O
2- (2-Pentenyl) furan Formation From 4-keto-6-nonenal

CH3 CH2 CH CH CH2 C CH2 CH2 CH

O O

CH3 CH2 CH CH CH2 C CH CH CH

O O
H H

H2 O

CH3 CH2 CH CH CH2 C CH

HC CH
O
2- (1-Pentenyl) furan Formation From 4-keto-5-nonenal

CH3 CH2 CH2 CH CH C CH2 CH2 CH

O O

CH3 CH2 CH2 CH CH C CH CH CH

O O
H H

H2 O

CH3 CH2 CH2 CH CH C CH

HC CH
O
 Effect of 2- and 16 ml Vial Sizes on the
Volatile Compounds in Headspace

1.8

1.2

0.6

0
Ethanol CHCl 3 TCE m-Xylene
 Effect of Static and Magnetic Stirring Methods on
the Absorption of Pesticides

200
Static Magnetic Parathion
150
Mass (ng)

100 Parathion

Terbutryn
Terbutryn
50

Simetryn Simetryn
0

0 20 40 60 80 100 120 0 20 40 60 80 100 120

Absorption Time (Minutes) Absorption Time (Minutes)
Effect of Static and Agitation Method on the
Absorption of Benzene and Xylene

60
✲ Xylene-Agitation
50
Mass Absorbed (ng)

▲ Xylene-Static
40

30

20
 Benzene-Agitation
10
▼ Benzene-Static
0

0 2 4 6 8 10
Extraction Time (minutes)
 Effect of Magnetic Stirring and Sonication on
the Absorption of Polyaromatic Hydrocarbons
Mass Extracted (ng)

30
25 Stirring
20 Sonication
15
10
5
0
1 2 3 4 5 6
Compound Number

1, acenaphthylene: 2, acenaphthalene: 3, fluorene: 4, phenaphthalene: 5,anthracene: 6, fluoranthene

Volatile Compounds of Headspace and
Liquid Samples by SPME

Headspace of Oil Analysis

Liquid Oil Analysis

Diagram of SPME Extraction

Direct sampling SPME Headspace SPME