PeakList:
1.vanillin
Column: UltraC8
Catalog #:
9103565
Dimensions: 150 x 4.6mm
Particle Size: 5µm
Pore Size:
100Å
Sample:
Inj.:
10µL
Conc.:
5% solution of vanilla extract
Solvent:
40% ethanol
Conditions:
Mobile phase:A:1.2% acetic acid
B:methanol
Time (min.) %B
0.0
20.0
5.0
20.0
15.0
40.0
20.0
40.0
25.0
20.0
Flow:
1.0mL/min.
Temp.:
28°C
Det.:
UV@ 254nm
0 2 4 6 8 10 12 14 16 18min.
Introduction
Vanilla extracts and flavorings are used in awide range of food
products, includingdairyproducts, beverages, bakedgoods, and
confections. They also are used as a backgroundnote or flavor
enhancer inproducts such as sauces, soups, andvegetables. In
fact, vanilla is the only flavorwith aUSFederalDepartment of
Agriculture (FDA) standardof identity (21CFR169), which
states that vanilla extractmust contain the extractivematerial
from13.35oz. of vanilla beans per gallon and at least 35%
alcohol byvolume. If the alcohol content is less than35%, the
solution is “vanilla flavor.” Imitationvanilla extract contains
natural and artificial flavorings, includingvanillin, andhas
alcohol as the solvent. Imitationvanilla flavors containvanillin,
ethyl vanillin, or other acceptable flavoringmaterials, withor
without “real” vanilla.
The flavor profile of vanilla contains over 250 components,
withvanillinpresent at levels between0.5-2%.Acuringprocess
creates the characteristic odor and flavor bypromoting an enzy-
matic reaction that transforms glucovanillin andother sub-
stances intovanillin andother aromatic compounds. Ingeneral,
vanillin is the component of vanilla extract that is imitated in
artificial flavorings. UnitedStates Pharmacopoeia (USP)
definedvanillin canbe synthesized from sources such as lignin,
a byproduct of the paper industry. Ethyl vanillin also is synthe-
sized for use invanilla flavorings, andhas a taste very similar to
vanillin. Figure1 shows the chemical structures for vanillin and
ethyl vanillin. The aromatic rings on these compounds allow
sensitive detectionbasedon theirUVabsorbance at 254nm.
Analysis
In theAssociationofOfficialAnalytical Chemists (AOAC)
Method990.25, flavor compounds invanilla extract and artifi-
cial vanilla flavor are analyzedusingHPLC. The analytes are
separatedon aC8 column andquantitatedby comparing their
UVabsorbance at 254nm to an external standard. The analysis
is performed isocratically, with amobile phase of acidified
water:methanol (90:10) and a flow rate of 2.5mL/min. Even at
this high flow rate, the run time is long (approximately40min-
utes) and there is significant broadeningof the late-eluting ethyl
vanillinpeak.
An efficient separation canbe performedusing theRestekUltra
C8 reverse phase column (150x4.6mm, with5µmparticles)
HPLC
Applications
note
Restek Corporation • (800) 356-1688 • (814) 353-1300 •
#59186
HPLC
1
2
AnalysisofVanillin andEthylVanillin inVanillaFlavorsUsingUltraC8Column
Figure 2
TheUltraC8 column achieves excellent resolution
of vanillin and ethyl vanillin.
LC_0148
PeakList:
Conc.:
(mg/mL)
1. vanillin
0.12
2. ethyl vanillin
0.04
Column:
UltraC8
Catalog #:
9103565
Dimensions: 150 x 4.6mm
Particle Size: 5µm
Pore Size:
100Å
Sample:
Inj.:
10µL
Solvent:
40% ethanol
Conditions:
Mobile phase:A:1.2% acetic acid
B:methanol
Time (min.) %B
0.0
20.0
5.0
20.0
15.0
40.0
20.0
40.0
25.0
20.0
Flow:
1.0mL/min.
Temp.:
28°C
Det.:
UV@ 254nm
and a gradient pumpprogram, such as acidifiedwater:methanol
(80:20 to60:40). Byusing a gradient program, the run time canbe
reduced to25minutes at a flow rate of 1mL/min (Figures 2 and3).
Figure 2 shows the analysis of a standard solutionof vanillin and
ethyl vanillin. Using these parameters, the k' values for vanillin and
ethyl vanillin are 4.8 and6.8, respectively. The resolutionof these
compounds is excellent under these run conditions. Figure 3 shows
the injectionof a solutionof vanilla extract. In addition tovanillin,
there are other aromatic compounds present in the extract.Vanillin is
well resolved from these components, and the vanillin canbe easily
quantitated in this sample. Other flavor components present invanil-
la extracts and flavorings also canbe analyzedusing this procedure.
Vanillin
EthylVanillin
CHO
CHO
OCH 3
OH
OH
OC 2 H 5
Figure 1
Ethylvanillin isasyntheticmolecule thathasasimilar taste to
vanillin,but isapproximately2-3 timesstronger.
6
8 10 12 14 16 min.
1
Figure 3
Theanalysis of a5% solutionof a vanilla extract
allows easyquantitationof vanillin.
LC_0149
