Essential Oil Analysis

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 Classical Analysis
 Spesific Grafity the most frequent physicochemical
properties
Specific gravity / Relative density is the ratio of the density of a
substance to the density (mass of the same unit volume) of a
reference substance. The reference substance is nearly
always water for liquids or air for gases.
 Optical activity (by using a polarimeter) with the angle of
rotation depending oil nature, the length of the column
through which the light passes, the applied wavelength, and
the temperature.

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 Refractive index is represented by the ratio of the sine of the angle of
incidence (i) to the sine of the angle of refraction (e) of a beam of light
passing from a less dense to a denser medium, such as from air to the
essential oil

where N and n arethe indice f h ore and the less dense medium

Purity assessment of essential oils is based on water solubility; the

test, which reveals the presence of polar substances, such as alcohols,
glycols and their esters, and glycerin acetates

Oil is added to a saturated solution of sodium chloride, which after

homogenization is divided into two phases; the volume of the oil, which is
the organic phase, should remain unaltered; volume reduction indicates the
presence of water-soluble substances
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 The estimation of melting and congealing points, as well as the
boiling range of essential oils, is also of great importance for identity
and purity assessments

The use of qualitative information alone is not suficient to

correctly characterize an essential oil, and quantitative data are of
extreme importance

Essential oils are often analyzed by means of chromatographic

methods

Planar chromatography being well represented by thin-layer

chromatography (TLC) and paper chromatography (PC). In both
techniques, the stationary phase is distributed as a thin layer on a flat
support, in PC being self-supporting, while in TLC coated on a glass,
plastic, or metal surface; the mobile phase is allowed to ascend through the
4 layer by capillary forces
 TLC is a fast and inexpensive method for identifying substances
and testing the purity of compounds, being widely used as a preliminary
technique providing valuable information for subsequent analyses.
Advanced of TLC
•Low cost
•Short analysis time
•Ease of sample preparation
•All spots can be visualized
•Sample cleanup is seldom necessary
•Adaptable to most pharmaceuticals
•Uses small quantities of solvents
•Requires minimal training
•Reliable and quick
•Minimal amount of equipment is needed
•Densitometers can be used to increase accuracy of spot concentration

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Spotting the Sample
 The analyte must be in a suitable solution for spotting and
any solvent can be used; in other words the analyte must be
in solution.
Polarity
 Polarity of solutes; Polar and non-Polar
 Polar solutes: alcohols (ROH), acids (RCOOH), amines
(RNH2)
 Polar solvents: Methanol, ethanol, acetic acid
 Non-Polar solutes: hydrocarbons, ketones (compared to
methanol)
 Non-Polar solvents: hexane, toluene (compared to
methanol)
Solvent Eulotropic Series
Solvent E-value
 Toluene 0.29
 Chloroform 0.40
 Acetone 0.56
 Ethyl Acetate 0.58
 Ethanol 0.88
 Methanol 0.95
 Acetic Acid/Ammonia High
 Water High
Calculation of Solvent Polarity
 Efinal = xE1 + xE2 + ……..xEn
 x = volume fraction of solvent
 E = E value of solvent
 Example
 25 mL CHCL3 + 75 mL MeOH
 0.25 x 0.40 + 0.75 x 0.88 = 0.76
Like Dissolves Like
 Polar molecules favor polar solvents and vice versa
 Polar solutes migrate faster in polar mobile phase
 • Spots can be visualized by two basic techniques:
– Ultraviolet light at 254 nm (shortwave UV). Long wave UV (365
nm) is used less commonly.
– Staining to make spots visible
A universal visualization reagent is a iodine or sulfuric acid
solution. When sprayed on your plate, the plate is heated and your
spots are charred which can be seen by eye.
e.g. p-Anisaldehyde – sulfuric acid
For detection of phenols, sugars, steroids, and terpenes
Spray with a solution of freshly prepared 0.5ml p-anisaldehyde in 50ml
glacial acetic acid and 1ml 97% sulfuric acid. Heat to 105°C until
maximum visualization of spots.
Results: phenols, terpenes, sugars, and steroids turn violet, blue, red,
grey or green.

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 GC and GC-MS
for Volatile Organic Compounds

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 Gas Chromatography
 Function
 Components
 Common uses
 Chromatographic resolution
 Sensitivity

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 Function
 Separation of volatile organic compounds
 Volatile – when heated, VOCs undergo a phase transition into
intact gas-phase species
 Separation occurs as a result of unique equilibria established
between the solutes and the stationary phase (the GC
column)
 An inert carrier gas carries the solutes through the column

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 Components
 Carrier Gas, N 2or He, 1-2 mL/min
 Injector
 Oven
 Column
 Detector

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 Syringe

Injector
Detector

Gas tank

Column

Oven
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 Injector
 A GC syringe penetrates a septum to inject sample into the
vaporization camber
 Instant vaporization of the sample, 280 C
 Carrier gas transports the sample into the head of the
column
 Purge valve controls the fraction of sample that enters the
column

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 Splitless (100:90) vs. Split (100:1)
Syringe Syringe

Injector Injector

He
He

Purge valve
closed Purge valve
18 GC column GC column open
 Split or splitless
 Usually operated in split mode unless sample limited
 Chromatographic resolution depends upon the width of
the sample plug
 In splitless mode the purge valve is close for 30-60 s,
which means the sample plug is 30-60 seconds
 As we will see, refocusing to a more narrow sample plug
is possible with temperature programming

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 Splitless & Spit Injection

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 Open Tubular Capillary Column

0.32 mm ID
Mobile phase
(Helium)
flowing at 1 Liquid
mL/min Stationary 0.1-5 mm
phase

15-60 m in length

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 FSOT ( Fused Silica Open Tubular)
columns
 Coated with polymer, crosslinked
 Polydimethyl siloxane (non-polar) ex. HP-1, DB-1
 Poly(phenylmethyldimethyl) siloxane (10% phenyl)
 Poly(phenylmethyl) siloxane (50% phenyl)
 Polyethylene glycol (polar) ex. HPWAX, DBWAX
 Poly(dicyanoallyldimethyl) siloxane
 Ploy(trifluoropropyldimethyl) siloxane

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 Polar vs. nonpolar
 Separation is based on the vapor pressure and polarity of the
components.
 Within a homologous series (alkanes, alcohol, olefins, fatty
acids) retention time increases with chain length (or
molecular weight)
 Polar columns retain polar compounds to a greater extent
than non-polar
 C18 saturated vs. C18 saturated methyl ester

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 C16:0 C18:2
C18:1

C16:1 C18:0

Polar RT (min)
column

C18:2

C18:1
C16:0

C16:1 C18:0

RT (min)
Non-polar column

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 Oven
 Programmable
 Isothermal- run at one constant temperature
 Temperature programming - Start at low temperature and
gradually ramp to higher temperature
 More constant peak width
 Better sensitivity for components that are retained longer
 Much better chromatographic resolution
 Peak refocusing at head of column

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 Typical Temperature Program
220C

160C

50C

26 0 60
Time (min)
 Detectors
 Flame Ionization Detectors (FID)
 Electron Capture Detectors (ECD)
 Electron impact/chemical ionization (EI/CI) Mass
spectrometry

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 FIDs
 Effluent exits column and enters an air/hydrogen flame
 The gas-phase solute is pyrolized to form electrons and ions
 All carbon species are reduced to CH2+ ions
 These ions collected at an electrode held above the flame
 The current reaching the electrode is amplified to give the signal

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 FID
 A general detector for organic compounds
 Very sensitive (10-13 g/s)
 Linear response (107)
 Rugged
 Disadvantage: specificity

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 ECD
 Ultra-sensitive detection of halogen-containing species
 Pesticide analysis
 Other detectors besides MS
 IR
 AE

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 Mass Spectrometry

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 What kind of info can mass spec give
you?

 Molecular weight
 Elemental composition (low MW with high resolution
instrument)
 Structural info (hard ionization or CID)

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 How does it work?

 Gas-phase ions are separated according to mass/charge ratio and

sequentially detected

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 Par ts of a Mass Spec

 Sample introduction
 Source (ion formation)
 Mass analyzer (ion sep.) - high vac
 Detector (electron multiplier tube)

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 Sample Introduction/Sources
Volatiles
 Probe/electron impact (EI)
 GC/EI
Involatiles
 Direct infusion/electrospray (ESI)
 HPLC/ESI
 Matrix Assisted Laser Adsorption (MALDI)
Elemental mass spec
 Inductively coupled plasma (ICP)
 Secondary Ion Mass Spectrometry (SIMS)
 surfaces

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 EI, CI
 EI (hard ionization)
 Gas-phase molecules enter source through heated probe or GC
column
 70 eV electrons bombard molecules forming M+* ions that
fragment in unique reproducible way to form a collection of
fragment ions
 EI spectra can be matched to library stds
 CI (soft ionization)
 Higher pressure of methane leaked into the source (mtorr)
 Reagent ions transfer proton to analyte

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 EI Source
Under high vacuum
filament

70 eV e-

To mass
analyzer

GC column

anode
repeller Acceleration
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slits
 EI process
 M + e-
M+*

f f
1
2 f f4
3

This is a remarkably reproducible process. M

will fragment in the same pattern every time
using a 70 eV electron beam
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 Ion Chromatogram of Safflower Oil

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 EI spectrum of phenyl acetate

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 CI/ ion-molecule reaction
 2CH 4+ e-  CH5 + and C
H+
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 CH5+ + M  MH+ + CH
4

 The excess energy in MH+ is the difference in proton

affinities between methane and M, usually not enough to give
extensive fragmentation

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 Links
http://www.shsu.edu/~chm_tgc/sounds/flashfiles/GC.swf

http://www.restek.com/Chromatography-Columns/GC-Columns/GC-
Column-Cross-Reference-Columns-by-Phase

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