Steam Distillation Gas Chromatography

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Steam Distillation Gas Chromatography CHMBD
447 Penn State Erie, The Behrend College
October 9-19, 2006
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• Distillations
• Steam Distillation
• A. Definition
• Steam distillation arises from an interesting
  curiosity of immiscible systems
• The distillation of liquids that are fully
  miscible is governed by Raoults Law -
  Clausius-Clapeyron
• The mixture will have its own unique boiling
  point
• The contribution of each component to the vapor
  phase is related to its partial vapor pressure
  and mole fraction
• In the distillation of immiscible liquids, the
  two act as two separate liquids

A
B
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• Distillations
• Steam Distillation
• A. Definition
• The total vapor pressure above an immiscible
  system is equal to the sum of the vapor pressures
  independent of their relative amounts
• The mixture will boil at a temperature typically
  lower than either liquid
• Consider a mixture of iodobenzene and water
• At 98 oC the value of each vapor pressure is

46 torr
714 torr
760 torr

Mixture boils!
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• Distillations
• Steam Distillation
• A. Definition
• Within the vapor phase, iodobenzene would only
  have a mole fraction of 0.06 (46 torr/760 torr)
• But because it has a larger molecular weight (204
  vs. 18 grams per mole) about 0.7 grams of
  iodobenzene are collected for every gram of water
• In the gas phase the two are fully miscible, but
  once the vapor condenses the two are no longer
  miscible and can be physically separated

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• Distillations
• Steam Distillation
• B. Uses/Apparatus
• This method is typically used to extract the
  volatile components of plants for use in
  perfumery, flavors or aromatherapy products.
• Steam distillation is used in the industries that
  produce these products as well as amateur (or
  questionable) set-ups like this one

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• Isolation of Natural Products
• This isolation of a natural product from its
  native matrix is one of the oldest examples of
  applied organic chemistry medicines and herbal
  remedies prepared by early human civilizations
  are good examples of this
• This field undergoing explosive growth as we
  attempt to find interesting molecules in nature
  that can be used for medicinal purposes as well
  as for flavorings, dyes and cosmetics from a
  natural rather than synthetic source
• The American Chemical Society (ACS) publishes
• a Journal that covers recent developments in this
  
• highly interesting and important field
• Papers start with the isolation of the
• animal or plant from its native environment
• followed by the various separation and
  identification
• techniques used to identify each component
• Recently, the initial assays of the
  anti-microbial,
• anti-carcinogenic and toxicity behavior of each
• component are also reported

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When these isolations are for medicinal purposes,
the field is known as Pharmacognosy "Pharmacognos
y" derives from two Greek words, "pharmakon" or
drug, and "gnosis" or knowledge. Like many
contemporary fields of science, pharma-cognosy
has undergone significant change in recent years
and today represents a highly interdisciplinary
science which is one of five major areas of
pharmaceutical education. Its scope includes
the study of the physical, chemical, biochemical
and biological properties of drugs, drug
substances, or potential drugs or drug substances
of natural origin as well as the search for new
drugs from natural sources. Research problems
in pharmacognosy include studies in the areas of
phytochemistry, microbial chemistry,
biosynthesis, biotransformation, chemotaxonomy,
and other biological and chemical sciences.
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Experiment 3 Steam Distillation of an Essential
Oil
Each student will be assigned a spice A steam
distillation will be performed to extract any
organic compounds from within the plant material
that have a vapor pressure leaving the heavier
materials behind We will be using a modification
of the distillation apparatus in the text
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Experiment 3 Steam Distillation of an Essential
Oil
This way, excess water that builds up can be
drained
You will continue to distill until you collect
100-300 mL of distillate
Steam from the lines in the lab is pumped into a
separatory funnel water separator
Some spices give a lot of organic extract which
you can see physically floating on the water
surface others dont
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Experiment 3 Steam Distillation of an Essential
Oil
The liquid will be transferred to a large
separatory funnel and extracted with methlyene
chloride CH2Cl2
The essential oil extract is soluble in methlyene
chloride, not water So draining the lower
methylene chloride layer, and evaporating the
methylene chloride, will afford the extracted oil

H2O
CH2Cl2
you will obtain a weight of the material (it
wont be much) and calculate a recovery based
on the original mass of plant material
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Experiment 3 Steam Distillation of an Essential
Oil
qualitatively to determine which components are
in the oil
The following week we will analyze the essential
oil by gas chromatography
and quantitatively to determine the relative
amounts of each component
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GAS CHROMATOGRAPHY HP 5890 Series II

• I. Introduction
• A. What is chromatography?
• Chromatography is the separation of the
  components of a mixture based on the physical
  process of each components adsorption or
  partition between a stationary and mobile phase
• The stationary phase is usually a solid or
  immobile liquid through or over which the mobile
  phase and the component mixture pass and is
  usually contained to produce a long path known as
  a column
• The mobile phase is usually a liquid or gas which
  passes through or over the stationary phase. The
  mobile phase initially contains the mixture to be
  separated as it is introduced onto the stationary
  phase
• The process of a component mixture moving through
  the column is known as elution
• Chromatography in its various applications
  is the most efficient and powerful means for the
  separation of organic compounds

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GAS CHROMATOGRAPHY HP 5890 Series II
I. Introduction B. History The first
reported chromatographic separation was performed
by the Russian botanist, Tswett, in 1906 as he
attempted to separate the extracts from plant
leaves by percolation of the extract through a
bed of CaCO3 He observed that different colored
pigments moved through the finely divided calcium
carbonate at different rates to give rise to
different colored bands. He thought color (and
not chemical structure) was the basis for
separation and formulated the name for the
method, chromatography, which derives from the
Greek chroma (meaning color) From 1906 to the
1940s the only applications that were derived
from this discovery were the separation of plant
carotenes (work of Kuhn and Lederer From about
1950, the number of different applications of
chromatography and the development of
instrumentational chromatographic methods has
increased rapidly with rapid acceleration. Now
thousands of papers are published annually that
concentrate on chromatographic techniques
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GAS CHROMATOGRAPHY HP 5890 Series II
I. Introduction B. History The
chromatographic methods available today

Preparative Methods
Instrumentational Methods
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GAS CHROMATOGRAPHY HP 5890 Series II
I. Introduction C. Basis for separation As
components move through the column an equilibrium
is established between the component being
adsorbed on the stationary phase of the column
and being dissolved in the mobile
phase Kcomponent Component dissolved
in mobile phase
Component adsorbed onto stationary
phase The amount of time a component requires
to move through the column Is known as the
retention time (tR) for instrumentational
chromatography or the retention factor Rf for
preparative chromatography High K
compound moves rapidly low tR (high Rf ) Low
K compound moves slowly high tR (low Rf)

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GAS CHROMATOGRAPHY HP 5890 Series II
I. Introduction C. Basis for
separation The extent of this equilibrium for a
component to prefer one phase or another is
dictated by intermolecular forces - Ionic
interactions (Ion exchange chromatography) -
Hydrogen bonding -
Dipole-dipole interactions - Van Der Wäals
forces (London Forces) Equilibrium processes are
also affected by pressure and temperature and are
constrained by the domain of time For Gas
Chromatography, these processes are more
important than for any other type of
chromatography as the component mixture is in the
gas phase

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GAS CHROMATOGRAPHY HP 5890 Series II
Example 1 Suppose you have a mixture of three
compounds and they are eluted through the
following column
bp 212 C
bp 207 C
bp 177 C
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
Which one will have the lowest retention time
(move the fastest)? Which one will have the
slowest retention time (move the slowest)?
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GAS CHROMATOGRAPHY HP 5890 Series II
Answer
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
Moves fastest, lowest retention time because it
is entirely hydrocarbon, it does not form strong
intermolecular interactions with the stationary
phase
Moves slower, medium retention time It can form
partial hydrogen bonds (electron pair donor only)
with the stationary phase which allows some
intermolecular interaction
Moves slowest, highest retention time It can
form effective hydrogen bonds (electron pair
donor and hydrogen donor) with the stationary
phase which allows considerable interaction with
the stationary phase
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography A. History In 1941,
A. Martin published a theoretical description of
the application of gas chromatography It
wasnt until 1951 when the first successful GC
separation was reported by Synge For this work
Martin and Synge were awarded the 1952 Nobel
Prize for chemistry Since that time the method
has grown, such that in conjunction with HPLC
(high performance liquid), they form the basis of
modern analytical chemistry
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography B.
Instrument General Although many models and types
of GCs exist, they all follow the same basic
layout and function The principle limitation to
the method is that all components of the mixture
to be analyzed must be capable of going into the
gas phase (and survive at that temperature for a
brief time)
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography B. Instrument
Gas Supply This provides the mobile phase
for GC The most common types of carrier gases
(mobile phase) are hydrogen, helium and
nitrogen These gases are inert and do not
chemically react with most organic mixtures The
typical pressures range from 10-100 psi
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography B. Instrument
Injector Port The sample is introduced into
the injector port via a microliter (mL) syringe
through the septum inlet (silicone rubber) The
injected volume depends on the type of column
used and the resolution desired
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography B. Instrument
Oven The oven is used to maintain the
temperature of the column, usually below the
temperature of the injector detector
ports Temperature may be varied to use
volatility as another variable in the separation

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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography B.
Instrument Column (stationary phase) There are
thousands of columns commercially available for
just about any application you can think of, but
there are two major types. The column is
housed in the oven for temperature control.
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GAS CHROMATOGRAPHY HP 5890 Series II

• II. Gas Chromatography
• B. Instrument
• Columns (stationary phase)
• Packed column
• 4-8 mm in diameter and 1-4 meters in length
  metal tubing is completely filled with the finely
  divided solid packing material (support) that is
  coated with an immobile non-volatile liquid
• can be used for the analytical separation of
  2-25 components or for the preparative
  purification of a particular component
• sample size is typically 0.1- 2 mL (analytical)
  to 5-100 mL (preparative)
• hundreds of different packings are available
  with different chemical (adsorbent) properties
  

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GAS CHROMATOGRAPHY HP 5890 Series II

• II. Gas Chromatography
• B. Instrument
• Columns (stationary phase)
• Capillary column
• 0.05-0.1 mm in diameter and 10-100 meters long
  
• glass tubing is lined with the adsorbent or
  coated with a non-volatile liquid
• used only for the analytical separation of
  2-100s of components
• sample size is typically less than 0.1 mL of a
  solution of the component mixture) hundreds of
  different linings are available with different
  chemical (adsorbent) properties

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GAS CHROMATOGRAPHY HP 5890 Series II

• II. Gas Chromatography
• B. Instrument
• Detector Port
• Used to determine when a particular component
  (hopefully separated) is being eluted from the
  column and how much is present relative to the
  other components
• There are two major types
• Simple detectors (TCD/FID) indicate when and
  how much
• Spectroscopic detectors often are coupled with a
  TCD and give a spectrum of each component aiding
  in identification

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GAS CHROMATOGRAPHY HP 5890 Series II

• II. Gas Chromatography
• B. Instrument
• Detector Port
• Simple detectors
• Merely records the presence of a component in
  the exiting gas and the amount present and
  transmits the signal to the chart recorder or
  computer for analysis
• Thermal Conductivity Detector (TCD) We will use
  the thermal conductivity detector(next slide)
• Flame Ionization Detector (FID) H2 is used as
  the carrier gas as compounds leave the column
  they are burned (by O2 with H2) and the resultant
  ions counted, the more ions, the more compound is
  coming off the column at that instant
• 2. Spectroscopic Detectors
• Actually take a spectrum of the material leaving
  the column to identify the
• materials.
• Common examples
• GC-MS A mass spectrum is taken of each
  comp.
• GC-FTIR An infrared spectrum is taken
  of each component

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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography B. Instrument
Output The voltage output of the detector is
recorded by a computer or chart recorder,
usually as the amount of response at a given
time Each component shows up as a Gaussian
distribution centered about the components
retention time, called a peak
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography C. Analysis
Output A typical chromatogram from a packed
column
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography C. Analysis
Output A typical chromatogram from a
capillary column
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GAS CHROMATOGRAPHY HP 5890 Series II
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GAS CHROMATOGRAPHY HP 5890 Series II
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography C. Analysis
Qualitative What component is it? To figure
out the identity of each component, one must
compare retention times (tR)s versus known
standards or use a spectroscopic detection
technique As with melting points, many compounds
share similar retention times, however A common
technique is to spike a mixture with a suspected
component with a known standard
If peak grows with no loss in peak shape it is
probably isopentane
Suspect this peak is isopentane?
Add authentic isopentane to next injection
If peak distorts or splits it is definitely not
isopentane
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography C. Analysis
Quantitative How much of each component is
there? To figure out the amount of each
component, you calculate the area under each peak
(integration) The area of the peak is
proportional to the amount of component that
generated the response You will use the height x
width_at_1/2 height method to calculate the areas in
lab on modern computer based GCs this is done
automatically
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography C.
Analysis Quantitative How much of each
component is there? The individual areas are
totaled up and each individual component area
divided by the total This gives a relative
ratio of each component versus the other in the
mixture and an overall percentage composition
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography C. Analysis Column
Efficiency How good is the separation? If it
is not acceptable, what can be done to improve it
(column? pressure? temperature? sample size?)
Resolution Does the response
factor return to zero between each peak?
Do the peaks represent good Gaussian
distributions of the average component within a
peak? Or is there tailing?
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography C.
Analysis Theoretical Plates (n) As for
distillation, a theoretical plate indicates the
number of times a component goes through the
cycle that causes separation In the case of
GC, the number of times partitioning between
stationary and mobile phase occurs. From the
width of the peak vs. retention time, an
estimation of the column efficiency can be made
from the following equation
By dividing the length of the column by n, you
obtain the HEPT for the column
l
HETP ___
n
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GAS CHROMATOGRAPHY HP 5890 Series II
II. Gas Chromatography C. Analysis Column
Efficiency How good is the separation? If it
is not acceptable, what can be done to improve it
(column? pressure? temperature? sample size?)
Back to a theoretical perspective,
the Van Deemter equation describes the physical
factors that affect separation (remember HETP
the smaller the better)
B
Where u is the mobile phase flow velocity
HETP A ____ Cu
u
A is the multipath effect. In any real column,
the packing will not be uniform in size or
disposition. This causes some molecules in the
mobile phase to take a longer path widening
peaks. This is independent of flow velocity. B
describes molecular diffusion. Even though there
is a net pressure on the mobile phase, the
component still diffuses the longer it is in the
column. This is why this term is coupled with
the inverse of the flow velocity C is the
resistance to mass transfer. If the molecule
cannot equilibrate between the two phases,
separation does not occur. Sometimes too high of
a flow velocity will increase this term, as
pressure may force a component onto the
stationary phase.
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GC and Experiment 3
This is the retention time in minutes
All GC conditions are set up for this experiment
The integrator handles calculation of the area
under each peak and giving a relative of each
component