Gas chromatography.

1
Gas Chromatography

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

2
Gas Chromatography Acetates

• Gas Chromatography, Refractive Index
  Distillation
• The next two (2) experiments introduce Gas
  Chromatography and Simple Fractional
  Distillation.
• They are then tied together along with the
  Refractive Index technique in a third experiment.
  
• This Week
• Gas Chromatography Acetates
• Pavia ? p. 837 855
• Slayden ? p. 45-47
• 2nd Week
• Distillation of a Mixture
• Slayden ? p. 41 - 27
• 3rd Week
• Gas Chromatography and Refractive Index of
  Distillates from Distillation of Mixture
  Experiment
• Slayden p. 39

3
Gas Chromatography Acetates

• Gas Chromatography
• Uses
• Separation and analysis of organic compounds
• Testing purity of compounds
• Determine relative amounts of components in
  mixture
• Compound identification
• Isolation of pure compounds (microscale work)
• Similar to column chromatography, but differs in
  3 ways
• Partitioning process carried out between Moving
  Gas Phase and Stationary Liquid Phase
• Temperature of gas can be controlled
• Concentration of compound in gas phase is a
  function of the vapor pressure only.
• GC also known as Vapor-Phase Chromatography (VPC)
  and Gas-Liquid Partition Chromatography (GLPC)

4
Gas Chromatography Acetates

• Gas Chromatograph
• Microliter Syringe
• Heated injection port with rubber septum for
  inserting sample
• Heating chamber with carrier gas injection port
• Oven containing copper, stainless steel, or glass
  column.
• Column packed with the Stationary Liquid Phase ?
  a non-volatile liquid, wax, or low melting
  solid-high boiling hydrocarbons, silicone oils,
  waxes or polymeric esters, ethers, and amides
• Liquid phase is coated onto a support material,
  generally crushed firebrick

5
Gas Chromatography Acetates

• Principals of Separation
• Column is selected, packed with Liquid Phase, and
  installed.
• Sample injected with microliter syringe into the
  injection port where it is vaporized and mixed
  into the Carrier Gas stream (helium, nitrogen,
  argon).
• Sample vapor becomes partitioned between Moving
  Gas Phase and Stationary Liquid Phase.
• The time the different compounds in the sample
  spend in the Vapor Phase is a function of their
  Vapor Pressure.
• The more volatile (Low Boiling Point / Higher
  Vapor Pressure) compounds arrive at the end of
  the column first and pass into the detector

6
Gas Chromatography Acetates

• Principals of Detection
• Two Detector Types
• Thermal Conductivity Detector (TCD) (we use this)
• Flame Ionization
• TCD is electrically heated Hot Wire placed in
  carrier gas stream
• Thermal conductivity of carrier gas (helium in
  our case) is higher than most organic substances.
  
• Presence of sample compounds in gas stream
  reduces thermal conductivity of stream
• Wire heats up and resistance decreases.
• Two detectors used one exposed to sample gas and
  the other exposed to reference flow of carrier
  gas.
• Detectors form arms of Wheatstone Bridge, which
  becomes unbalanced by sample gas.
• Unbalanced bridge generates electrical signal,
  which is amplified and sent to recorder

7
Gas Chromatography Acetates

• Factors Affecting Separation
• Boiling Points of Components in Sample
• Low boiling point compounds have higher vapor
  pressures.
• High boiling point compounds have lower vapor
  pressures requiring more energy to reach
  equilibrium vapor pressure, i.e., atmospheric
  pressure.
• Boiling point increases as molecular weight
  increases.
• Flow Rate of Carrier Gas
• Choice of Liquid Phase
• Molecular weights, functional groups, and
  polarities of component molecules are factors in
  selecting liquid phase.
• Length of Column
• Similar compounds require longer columns than
  dissimilar compounds. Isomeric mixtures often
  require quite long columns

8
Gas Chromatography Acetates

• The Experiment
• Purpose Introduce the theory and technique of
  gas chromatography.
• Identify a compound by it retention time.
• From the relationship between peak area and
  mole content calculate the mole fraction and
  mole percent of a compound in a mixture.
• Approach
• Obtain chromatograph of a known equimolar mixture
  of four (4) esters.
• (Ethyl, Propyl, Butyl, Hexyl Acetate)
• Obtain chromatograph of unknown mixture (one or
  more compounds in the known mixture).
• Determine Retention Times.
• Calculate Peak Areas
• Calculate Total Area
• Calculate Mole Fraction
• Calculate Mole Percentage

9
Gas Chromatography Acetates

• The Experiment (Cont)
• Groups Work in groups of three (3).
• Each group will use the same standard
  chromatogram.
• Each Student will run their own unknown
• Samples
• The Standard has 4 esters
• Ethyl Acetate, Propyl Acetate, Butyl Acetate,
  Hexyl Acetate
• The Unknowns have 1 to 4 of the compounds in the
  standard

10
Gas Chromatography Acetates

• The Report
• The Gas Chromatograph instrument settings and the
  processing of the samples to get the
  chromatograms are considered one (1) procedure.
• When multiple samples or sub-samples are
  processed with the same procedure, it is not
  necessary to set up a separate procedure for each
  sample. Setup a suitable template in Results to
  report all of the results obtained.
• Thus, the process to obtain Gas Chromatograms of
  the Known mixture of 4 acetates and the
  Unknown mixture utilize the same procedure.
• The computation of the Peak Areas and the Total
  Peak area are considered separate procedures.
• The computation of Mole Fraction and Mole are
  considered separate procedures

11
Gas Chromatography Acetates

• Summarize in paragraph form, all of the results
  obtained in the experiment. Use a logical
  organization and order of the results.
• The Conclusion for the Gas Chromatography of
  Acetates experiment must present arguments, using
  applicable results, that support the
  identification of compounds in the Unknown
  mixture.
• The known mixture was an equimolar mixture of
  four (4) acetates. Comment on the equivalency of
  the peak areas in the known mixture and the
  application of the Thermal Response Correction
  Factor to adjust the peak areas and the computed
  mole percent of the unknown mixture

12
Gas Chromatography Acetates
Note all three temperatures are the same

• Record Instrument readings
• Injection Port Temp
• Column Temp
• Detector Temp
• Gas Flow Rate (65 mL / min)
• Chart Speed (5 cm / min)
• Injecting the Sample
• Sample is injected into the B port with the
  microsyringe
• The Microsyringe is fragile and expensive BE
  CAREFUL
• Mark Starting Point on chart short vertical
  line
• Insert needle fully into rubber septum or until
  resistance is met maybe a ¼ inch remains.
• Inject sample quickly and remove needle.
• Start chart recorder simultaneously with sample
  injection

13
Gas Chromatography Acetates

• Determine the Retention Time
• The period following injection that is required
  for a compound to pass through the column to the
  point where the detector current is maximum, i.e.
  maximum pen deflection or maximum peak height.
• For a given set of constant conditions (carrier
  gas, flow rate of carrier gas, column
  temperature, column length, liquid phase,
  injection port temperature), the retention time
  of any compound is always constant.
• Retention Time is similar to the Retardation
  Factor, Rf in Thin Layer Chromatography.
• Compute Retention Time from the Chart Speed (5
  cm/min) and the distance on the chart from the
  time of injection to the point on the chart where
  the perpendicular line drawn from the peak height
  intersects the base line

14
Gas Chromatography Acetates

• Determination of Retention Time
• Since Velocity (v) Distance / Time d / t
• Ret Time (t) Distance(cm) / Velocity(cm/min)
  d / v

Starting Point On Chart
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Gas Chromatography Acetates

• Quantitative Analysis
• The area under a gas chromatograph peak is
  proportional to the amount (moles) of the
  compounds eluted.
• The molar percentage composition of a mixture can
  be approximated by comparing the relative areas
  of the peaks in the chromatogram.
• This approach assumes that the detector is
  equally sensitive to all compounds and its
  response is linear
• This assumption is usually not valid and will be
  addressed in the Thermal Response Adjustment
  section starting on slide 17

16
Gas Chromatography Acetates

• Triangulation Method of Determining Area Under
  Peak
• Multiply the height of peak (in mm) above the
  baseline by the width of the peak at half the
  height.
• Baseline is a straight line connecting side arms
  of the peak. Best if peaks are symmetrical.
• Add areas to get total.
• Divide each area by total area to get mole
  fraction
• See next slide

17
Gas Chromatography Acetates

• Peak Area by the Triangulation Method
• Peak Area h w½
• Where h Peak Height
• w½ width of peak at
  ½ the peak height
• Total Peak Area(TA) A B
• Mole Fraction(MF) A/TA, B/TA
• Mole Percent MF x 100

18
Gas Chromatography Acetates

• Thermal Response Factor
• Equimolar mixtures each compound in the mixture
  has the same number of moles should produce
  chromatograms in which all peaks have the same
  area
• Compounds with different functional groups or
  widely varying molecular weights do not all have
  the same thermal conductivity. This can cause
  the instrument to produce response variations,
  which result in unequal peak areas.
• A correction factor called The Thermal Response
  Factor for a given compound is determined from
  the relative peak areas of an equimolar solution

19
Gas Chromatography Acetates

• Thermal Response Calculations For GC Analysis
• For the analysis of the Alkyl Acetates in the
  table below, the subscript i stands for the
  number of carbons in the alkyl group, so that i
  2, 3, 4, 6 for this experiment. Thus, subscript
  2 stands for Ethyl Acetate subscript 3 stands
  for Propyl Acetate, etc.
• Note In the table below any of the compounds can
  be used as the basis for the calculations, i.e.,
  s can be 2, 3, 4, or 5 for our mixture of 4
  Acetates.
• From the Chromatogram of your Standard Equimolar
  Mixture of Alkyl Acetates fill in the blanks
  below.

20
Gas Chromatography Acetates

• Thermal Response Ratios a correction factor
• The area ratios under the GC peaks are
  proportional, but not equal, to the molar ratios
  of the components in the mixture.
• Let TR be the thermal response of a component,
  generally
• The thermal response ratio of the different
  components, TRx/TRy, a constant, could be
  simplified to a single term, but we will continue
  to express it as a ratio

.
21
Gas Chromatography Acetates

• Thermal Response Ratios (Cont)
• For the analysis of the Alkyl Acetates in the
  equations below, the subscript i stands for the
  number of carbons in the alkyl group, so that
• i 2(Ethyl), 3(Propyl), 4(Butyl), 5(Hexyl)
• In the derivation and examples that follow, Ethyl
  Acetate will be used as the basis for the
  calculations, but any of the other compounds
  could also be used, such as in the case where the
  unknown mixture does not contain any Ethyl Acetate

22
Gas Chromatography Acetates

• Thermal Response Ratios (Cont)
• The mole ratio of each component relative to
  Ethyl Acetate in the unknown mixture is
  calculated from
• Since we will need the TRs/TRi ratios from the
  above equation, they will be calculated from the
  areas under the peaks in the standard equimolar
  mixture
• For an equimolar mixture molei/moles 1
• Thus, substitution in equation 2 gives

23
Gas Chromatography Acetates
.

• Thermal Response Ratios (Cont)
• Adjusting the Peak Areas of the Unknown Mixture
• From equation (3), each individual TRs/TRi ratio
  is calculated from the peak areas in the GC trace
  of the standard equimolar mixture
• Using each TRs/TRi ratio, the mole ratio of each
  component in the unknown mixture, relative to the
  base compound, is calculated from equation (2)
• The Molei/Moles values from equation 2 now
  represent the adjusted peak areas, and thus are
  proportional to the molar content of the unknown
  mixture
• The adjusted Molei/Moles values are summed
• The new Mole Fractions are computed by dividing
  each Molei/Moles value by the total

24
Gas Chromatography Acetates

• Thermal Response Ratios (Cont)
• Example Ethyl Acetate (S2) is used as basis
  for calculations

EtAc (2) ProAc (3) BuAc (4) HexAc (6)
Standard Equimolar Mixture Measured Peak Area 1.44 1.09 1.16 0.98
Standard Equimolar Mixture TRs/TRi (s2) 1.44 1.00 1.44 1.44 1.33 1.09 1.44 1.24 1.16 1.44 1.48 0.98
Unknown Mixture Measured Peak Area 2.14 2.18 2.12 1.54
Unknown Mixture moli/mols (s2) 2.14 ? 1.00 1.00 2.14 2.18 ? 1.33 1.35 2.14 2.12 ? 1.24 1.23 2.14 1.54 ? 1.48 1.07 2.14
Unknown Mixture molei percent 21.5 29.0 26.5 23.0
EtAc / EtAc mol2 / mol2 area2 /
area2 ? TR2 / TR2 2.14 / 2.14 ? 1.00
1.00 ProAc / EtAc mol3 / mol2 area3 /
area2 ? TR2 / TR3 2.18 / 2.14 ? 1.33
1.35 BuAc / EtAc mol4 / mol2 area4 /
area2 ? TR2 / TR4 2.12 / 2.14 ? 1.24 1.23
HexAc / EtAc mol6 / mol2 area6 / area2 ?
TR2 / TR6 1.54 / 2.14 ? 1.48 1.07 ?
moli/mol2 1.00 1.35 1.23 1.07
4.65 ? mole EtAc 1.00 / 4.65 100
21.5 ? mole ProAc
1.35 / 4.65 100 29.0 ? mole BuAc 1.23
/ 4.65 100 26.5 ? mole HexAc 1.07 /
4.65 100 23.0
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Gas Chromatography Acetates
Thermal Response Ratios (Cont) Example 2
Ethyl Acetate (S2) is used as basis for
calculations
EtAc (2) ProAc (3) BuAc (4) HexAc (6)
Standard Equimolar Mixture Measured Peak Area 128 186 208 210
Standard Equimolar Mixture TRs/Tri (s2) 128 1.00 128 128 0.69 186 128 0.62 208 128 0.61 210
Unknown Mixture Measured Peak Area 2.14 2.18 2.12 1.54
Unknown Mixture moli/mols (s2) 2.14 ? 1.00 1.00 2.14 2.18 ? 0.69 0.70 2.14 2.12 ? 0.62 0.61 2.14 1.54 ? 0.61 0.44 2.14
Unknown Mixture molei percent 36.4 25.4 22.2 16.0
EtAc / EtAc mol2 / mol2 area2 / area2 ? TR2
/ TR2 2.14 / 2.14 ? 1.00 1.00 ProAc /
EtAc mol3 / mol2 area3 / area2 ? TR2 /
TR3 2.18 / 2.14 ? 0.69 0.70 BuAc /
EtAc mol4 / mol2 area4 / area2 ? TR2 /
TR4 2.12 / 2.14 ? 0.62 0.61 HexAc /
EtAc mol6 / mol2 area6 / area2 ? TR2 /
TR6 1.54 / 2.14 ? 0.61 0.44 ?
moli/mol2 1.00 0.70 0.61 0.44
2.75 ? mole EtAc 1.00 / 2.75 100
36.4 ? mole ProAc 0.70 / 2.75 100
25.4 ? mole BuAc 0.61 / 2.75 100
22.2 ? mole HexAc 0.44 / 2.75 100 16.0
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Thermal Response Ratios (Cont) Ex. 3 - (Assumes
the unknown is missing Ethyl Acetate and
Propyl Acetate (S3) is used as basis for
calculations)
EtAc (2) ProAc (3) BuAc (4) HexAc (6)
Standard Equimolar Mixture Measured Peak Area 128 186 208 210
Standard Equimolar Mixture TRs/Tri (s3) 186 1.45 128 186 1.0 186 186 0.89 208 186 0.89 210
Unknown Mixture Measured Peak Area 0.0 2.18 2.12 1.54
Unknown Mixture moli/mol2 (s3) 0.0 2.18 1.0 2.18 2.12 0.87 2.18 1.54 0.63 2.18
Unknown Mixture molei percent 0.0 40.0 34.8 25.2
ProAc / EtAc mol3 / mol3 area3 / area3 ?
TR3 / TR3 2.18 / 2.18 ? 1.00 1.00 BuAc
/ EtAc mol4 / mol3 area4 / area3 ? TR3 /
TR4 2.12 / 2.18 ? 0.89 0.87 HexAc /
EtAc mol6 / mol3 area6 / area3 ? TR3 /
TR6 1.54 / 2.18 ? 0.89 0.63 ?
moli/mol3 1.00 0.87 0.63 2.50 ? mole
ProAc 1.00 / 2.50 100 40.0 ? mole
BuAc 0.87 / 2.50 100 34.8 ? mole
PexAc 0.63 / 2.50 100 25.2
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Thank you