Solidphase Microextraction Analyses of Flavor Compounds in Foods

1
Solid-phase Microextraction Analyses of Flavor
Compounds in Foods
2
Continuous Solvent Extraction
Condenser

Beverage sample
Solvent (Ether)
Holes at the bottom of the tube
Water Bath
3
Steam distillation and continuous solvent
extraction
4
Kuderna-Danish Assembly for Concentration of
Isolated Compounds by Solvent Evaporation
Water Bath
5
Gas chromatogram of Orange Juice Flavor
6
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

7
Instrumental Analysis of Volatile Compounds

• Static headspace analysis
• Dynamic headspace analysis
• Solid phase microextraction

8
III. Headspace Analysis
Static headspace analysis
Syringe

Silicone rubber
stopper
Can
9
VI. Dynamic Headspace Analyzer
Diagram of dynamic headspace sampler and gas
chromatographer
10
Detection Limits and Precision of Organic
Volatilesa in Water
Technique
Detection Limit with FID (ppb)
Precision ( rsd)
1-3
SPME
0.05-0.3
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-trimethylbenze
ne
11
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
12
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.
13
SPME Fiber with Holder
14
Diagram of SPME Extraction
Fiber
Headspace SPME
15
SPME Analysis of Volatile Compounds
Plunger
Adjustable depth
gauge
Barrel
Coated SPME
fiber
Water bath
16
Principles of Headspace SPME
nf The of moles of flavor compounds on fiber
coating Khs Partition coefficient of flavor
compounds between headspace and
solution Kfh Vf, Vs, Vh Volume of fiber
coating, solution, and headspace,
respectively Co Initial concentration of flavor
compounds in the solution
nf
KfhVfVsCo
KfhVfKhsVhVs
Concentration of compounds on fiber coating
Concentration of compounds in headspace
17
Effects of Different Fibers on the Volatile
Compound Extraction of Soybean Oil

• CB/PDMSCarboxen/Polydimethylsiloxane
• PDMS Polydimethylsiloxane
• CW/DVB Carbowax/Divinylbenzene
• PA Polyacrylate.

18
Effects of Different Fibers on the Hexanal
Analysis in Oil
2,000

1,600
1,200
Relative Hexanal Peak Size
800
400
1
0
CB/PDMS
PA
PDMS
CW/DVB
19
Effects of Different Fibers on the Hexanal
Analysis
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
Hexanal Peak in Electronic Count
Significant difference (Plt0.05)
Carboxen, Polydimethylsiloxane Poyacrylate Carbowa
x and Divinylbenzene
20
Reproducibility for the Determination of Major
Flavor Compounds in a Single Strength Orange Juice
?-Pinene (ppm)
Octanal (ppm)
Limonene (ppm)
Decanal (ppm)
Ethyl butyrate (ppm)
Replicates
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
21
Effect of G.C.Injection Temperature on Soybean
Oil Volatile Compound Analysis
22
Effect of Fiber Coating Thickness on the
Absorption for the Extraction of 0.1 ppm Benzene
100
100 ?m
80
60
Mass (ng)
56 ?m
40
20
15 ?m
0
0
200
400
600
Time (Seconds)
23
Effect of Distribution Constants on the
Absorption Profile of 0.1 ppm Analyte
30
Kfs 831 (p-Xylene)
25
20
Mass (ng)
15
Kfs 294 ( Toluene)
10
Kfs 125 ( Benzene)
5
0
0
1000
2000
3000
Isolation Time (Seconds)
Kfs Partition coefficient of flavor compounds
between fiber coating and solution
24
Effect on Isolation Temperature on the GC
Chromatogram of Headspace Polyaromatic Compounds
Extracted at 25 C
Extracted at 130 C
Extracted at 200 C
25
Effect of Water and Microwave Heating on the
Chromatograms of Headspace Polyaromatic Compounds
100
Water Heating
80
Microwave Heating
60
Mass Extracted (ng)
40
20
0
1
2
3
4
5
Compound Number
1, Naphthalene 2, Acenaphthylene 3, Benzene
4, Fluorene 5,Anthracene
26
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)
27
Effect of Benzene Concentration in Water on
Absorption by Fiber
1000
Cs 10 ppm
100
Cs 1 ppm
10
Mass (ng)
Cs 0.1 ppm
1
0.1
0
100
200
300
400
500
600
Time (Seconds)
28
Effect of Various Salts on the Concentration of
Volatile Compounds in the Headspace
No Salt
Sodium Chloride
Sodium Sulfate
Normalized (FID response)
Potassium
Carbonate
Benzene
Dioxane
29
Matrix Effect on SPME Recovery of Terpene Alcohols
Detector Response
Cltronellol
Geranol
30
SPME Analysis of Volatile Compounds
Plunger
Adjustable depth
gauge
Barrel
Coated SPME
fiber
Water bath
31
Effects of Temperature and Time on the
Equilibrium of Flavor Compounds Between the SPME
Coating and the Headspace of Orange Juice
30
25C
25
40C
20
50C
FID response
15
60C
10
80C
5
0
60
0
10
20
30
40
50
Adsorption Time (minutes)
32
SPME Analysis of Volatile Compounds
Plunger
Adjustable depth
gauge
Barrel
Coated SPME
fiber
Water bath
33
Gas chromatogram of Orange Juice Flavor
1
11
10
17
3
13
Total GC peak area
in 0 kGy 2.99 ? 107
16
15
6
8
9
12
5
7
14
34
Regression Equations between Flavor Compounds
(ppm) and GC Peak Areas (Electronic Counts)
Concentration range (ppm)
Compounds
Regression Eq
R2
Ethyl butyrate
Y0.2891X0.015
0.99
0.1-1.2
n-Octanal
Y0.4913X0.003
1.00
0.1-1.3
Decanal
Y0.2010X0.066
0.99
0.1-1.1
?-Pinene
Y0.3428X0.092
0.99
0.2-2.0
Limonene
Y17.922X9.462
0.99
20-50
Y Compound part per million, X Electronic
counts of GC peak area
35
Isolation Time Effect on Soybean Oil Volatile
Compounds by SPME
40
36
Isolation Temperature Effect on Soybean Oil
Volatile Compounds by SPME
30
PV 10
25
PV 50
20
Relative Peak Size
15
10
5
0
35
45
60
Isolation Temperature (C)
37
Isolation Temperature Effect on Soybean Oil
Volatile Compounds by SPME for 60 min.
Soybean Oil 1 (PV1)
Soybean Oil 2 (PV50)
Isolation Temperature
Total
Area
Total
Area
CV()
CV()
Mean
Mean

35
C
9,847
15,304
0.85
2.89

45
C
10,985
3.52
18,060
1.08

4.51
5.43
60
C
12,978
24,869
CV Coefficient Variation () for n
5 Significant difference (Plt0.05)
38
Chromatograms of Soybean Oil Volatile Compounds
by SPME
39
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 Hexan
al 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-Oc
tenal 11.53 3.37 Nonanal 14.00 2.86 t-2-Non
enal 14.29 0.55 2-Decenal 18.69 34.3
40
Effect of Isolation Temperature on Corn Oil
Volatile Compounds by SPME
25C
45C
60C
35C
41
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 Hexa
nal 3.62 5.39 Heptanal 5.36 1.83 2-Heptenal
6.21 29.52 2-Pentylfuran 8.59 2.53 2,4-Hept
adienal 10.88 7.69 t-2-Octenal 11.51 18.07 N
onanal 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
42
Chromatograms of Soybean Oil and Corn Oil
Soybean Oil
Corn Oil
43
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

44
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

45
Conclusion
The SPME-GC is a

• Reproducible
• Economic
• Simple
• Sensitive

for the analysis of volatile compounds in foods.
46
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47
Effects of Microwave Processing on the Flavor of
Orange Juice by SPME Analysis
Control
48
Control
1 min. microwave processing
49
2 min. microwave processing
4 min. microwave processing
50
Natural Cereal Quaker Oats Company Products
StoryWhat are the ideal analytical method for
flavor isolation from foods?How to improve the
sensitivity of flavor isolation by SPME?
51
(No Transcript)
52
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.

53
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 60C. 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 60C,
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. .
54
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-decadienalwer
e major compounds of oxidized soybean oil. For
statistical analysis of data, an analysis of
variance (ANOVA) test were conducted
55
Effect of Liquid and Headspace Sampling Methods
on the Absorption of Compounds
56
Lipid Oxidation of Oil

• Loss of nutritional value
• Polymer formation
• Formation of volatile compounds

57
Effect of Isolation Temperature on Volatile
Compounds of Corn Oil
25
20
Pentane
Hexanal
15
2-Heptenal
Relative Peak Area
10
2-Decenal
t,c,-2,4-
Decadienal
5
0
25
35
45
55
65
Isolation Temperature (C)
58
Effect of Isolation Temperature on Volatile
Compounds of Corn Oil
25
20
Pentane
Hexanal
15
2-Heptenal
Relative Peak Area
t,c,-2,4-
10
Decadienal
5
0
25
35
45
55
65
Isolation Temperature (C)
59
Conclusion
The SPME-GC is a

• Reproducible
• Economic
• Simple
• Sensitive

for the analysis of volatile compounds in
vegetable oils.
60
Depleted Area Around the Fiber During Static
Sampling of Compounds
61
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

62
Geometry of Headspace Sampling by SPME
63
High Velocity Carrier Gas Stripping Effect on the
Desorption of SPME
n/no
Dft/(b-a)2
64
Diagram for the Isolation of Headspace Flavor
Compounds of Orange Juice by SPME
65
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 fibers 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.
66
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 10-20C below the temperature limit of
the fiber and/or the maximum GC column temperature
67
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

68
SPME Fiber with Holder
69
Graphic Diagrams of SPME/sample System
Configuration
70
Boundary Layer Model Showing the Different
Regions of SPME
Fiber Core
Fiber Coating
Boundary layer
Sample
Concentration
Distance
71
Effects of Exposure Time on the Absorption of
Compounds
100
80
60
40
20
0
0
1
2
3
4
5
Ds/?Kfs(b-a)t
72
Singlet Oxygen Lipid Oxidation in Foods

• David B. Min
• Department of Food Science and Technology
• The Ohio State University
• Columbus, OH USA

73
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.

74
4-Keto-6-nonenal Formation From Linolenic Acid
75
4-Keto-5-nonenal Formation From Linolenic Acid
76
4-Keto-5-nonenal Formation From 4-keto-6-nonenal
77
4-Keto-nonanal Formation From Linoleic Acid
C
H
C
H
C
H
(
C
H
)
C
O
O
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
2
6
3
2
2
2
2
2
1O2
C
H
C
H
C
H
C
H
C
H
(
C
H
)
C
O
O
H
C
H
C
H
C
H
C
H
C
H
C
H
2
2
6
2
3
2
2
2
2
O
O
C
H
C
H
(
C
H
)
C
O
O
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
2
6
2
3
2
2
2
2
O
O


C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2
2
2
1O2
O
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2
2
2
O
O
O
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2
2
2

O
O

C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
C
H
2
3
2
2
2
2
2
O
O
78
2- Pentyl furan Formation From 4-keto-nonanal
C
H
C
H
C
H
C
H
C
H
C
H
C
C
H
C
H
2
3
2
2
2
2
2
O
O
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
C
H
2
3
2
2
2
O
O
H
H
H
O
2
C
H
C
H
C
H
C
H
C
C
H
C
H
2
3
2
2
2
C
C
H
H
O
79
2- (2-Pentenyl) furan Formation From
4-keto-6-nonenal
C
H
C
H
C
H
C
H
C
H
C
H
C
C
H
C
H
2
3
2
2
2
O
O
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
C
H
2
3
2
O
O
H
H
H
O
2
C
H
C
H
C
H
C
H
C
H
C
C
H
2
3
2
C
C
H
H
O
80
2- (1-Pentenyl) furan Formation From
4-keto-5-nonenal
C
H
C
H
C
H
C
H
C
H
C
H
C
C
H
C
H
3
2
2
2
2
O
O
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
C
H
3
2
2
O
O
H
H
H
O
2
C
H
C
H
C
H
C
H
C
H
C
C
H
3
2
2
C
C
H
H
O
81
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
82
Effect of Static and Magnetic Stirring Methods on
the Absorption of Pesticides
200
Static
150
Parathion
Mass (ng)
100
Terbutryn
50
Simetryn
0
0
20
40
60
80
100
120
Absorption Time (Minutes)
83
Effect of Static and Agitation Method on the
Absorption of Benzene and Xylene
60
? Xylene-Agitation
50
? Xylene-Static
40
30
Mass Absorbed (ng)
20
? Benzene-Agitation
10
? Benzene-Static
0
0
2
4
6
8
10
Extraction Time (minutes)
84
Effect of Magnetic Stirring and Sonication on the
Absorption of Polyaromatic Hydrocarbons
1, acenaphthylene 2, acenaphthalene 3,
fluorene 4, phenaphthalene 5,anthracene 6,
fluoranthene
85
Volatile Compounds of Headspace and Liquid
Samples by SPME
86
Diagram of SPME Extraction
Direct sampling SPME
Headspace SPME