Gas Chromatography

1
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. 817 836
• Slayden p. 39 - 31
• 2nd Week
• Distillation of a Mixture
• Slayden p. 43 - 46
• 3rd Week
• Gas Chromatography and Refractive Index of
  Distillates from Distillation of Mixture
  Experiment
• Slayden p. 47

2
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)

3
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. We
  use DC200 from Dow Chemical
• Liquid phase is coated onto a support material,
  generally crushed firebrick

4
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

5
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

6
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

7
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
• Adjust Peak Areas for Thermal Response
• Calculate Total Area from Adjusted Areas
• Calculate Mole Fraction
• Calculate Mole Percentage

8
Gas Chromatography Acetates

• The Experiment (Cont)
• Groups Work in groups of three (2)
• Each group will obtain 2 copies of the
  chromatogram for the standard (equimolar) mixture
• Each Student will run their own unknown
• Samples
• The Standard (Equimolar) Mixture has 4 esters
• Ethyl Acetate, Propyl Acetate, Butyl Acetate,
  Hexyl Acetate
• The Unknowns have from 2 to 4 of the compounds in
  the standard mixture

9
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 section 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 Area, Adjusted Peak
  Areas, Total Peak area, Mole Fraction, and Mole
  are considered separate procedures

10
Gas Chromatography Acetates

• Data Summary Procedure Using complete
  sentences summarize, in paragraph form, all of
  the results obtained in the experiment
• Analysis Conclusion Section
• Develop a set of arguments to prove the identity
  of the unknown compounds in the unknown mixture
• Comment on the equivalency of the peak areas and
  equimolar content of the known mixture
• Why was it necessary to apply the Thermal
  Response Correction Factor to the measured peak
  areas?
• Chromatograms
• Copied chromatogram sets for each team member
  must be copied at the same scale, otherwise
  retention time computations will be wrong
• Tape the trimmed chromatograms to a blank sheet
  of paper and attach to end of report

11
Gas Chromatography Acetates

• Record Instrument readings (Place in GC procedure
  Results)
• Injection Port Temp
• Column Temp
• Detector Temp
• Gas Flow Rate 60 mL / min
• Chart Speed Generally 5 cm /min
• Moving Liquid Phase (DC-200)
• 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 B Port through the
  rubber septum
• Coordinating with chart recorder operator, inject
  the sample into the heated chamber, while
  simultaneously starting the chart recorder

For the instrument in the 407 lab, all three
temperatures are read from the single dial on the
front of cabinet
12
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 or
  10 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

13
Gas Chromatography Acetates

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

Note Disregard Air Peaks in all calculations
Retention Time Distances Mark Starting Point On
Chart (t 0) Draw vertical Line from Peak Top to
Base Line Measure Distance from Starting Point to
Base of Peak
14
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 by adjusting the peak areas using the
  Thermal Response algorithm described on slides
  17-24

15
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 the individual areas to get the total area
• Divide each area by total area to get mole
  fraction
• Multiply mole fraction by 100 to get adjusted
  mole
• See algorithm development on next slide
• Adjust the peak areas for non-linear thermal
  response using the algorithm described in slides
  17-28

16
Gas Chromatography Acetates

• Draw Baseline connecting peak bottoms
• Peak Area by the Triangulation Method
• Peak Area h w½
• Where h Peak Height from
  baseline
• w½ width of peak at
  ½ the peak height
• Adjust Peak Area for thermal response
• See discussion on following slides
• Total Adjusted Peak Area (TA) A B
• Mole Fraction (MF)
• A/TA B/TA
• Mole Percent MF x 100

17
Gas Chromatography Acetates

• Thermal Response Factor
• The areas of gas chromatogram peaks are
  proportional to the molar content of the mixture
• 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 cause deviations (non-linearity) in the
  relationship between peak area and molar content
• A correction factor called The Thermal Response
  Factor for a given compound can be established
  from the relative peak areas of an equimolar
  solution
• Equimolar mixtures contain compounds with the
  same molar content, i.e., the same number of
  moles
• Thus, equimolar mixtures should produce peaks of
  equal area, if the instrument response is linear

18
Gas Chromatography Acetates

• Thermal Response Ratios
• GC Peak Area Correction Factor (approach 1)
• The ratio of one peak area to another in a GC
  chromatogram should be proportional to the molar
  ratio of the components in the mixture
• The expression for modifying the Peak Areas for a
  non-linear area instrument response is
  constructed as follows
• Determine the area of each peak in an equimolar
  mixture
• Compute the ratio of one of the peaks selected as
  the basis for computation relative to each peak
  area

19
Gas Chromatography Acetates

• Thermal Response Correction Factor (cont)
• Multiply the area of each peak by the respective
  Thermal Response Factor (TRx)
• Compute the Total Adjusted Area
• Compute the Adjusted Mole Fraction
• Compute the Adjusted Mole Percent

20
Gas Chromatography Acetates

• Thermal Response Ratios
• 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 As/Ai (s2) 1.44 1.00 1.44 1.44 1.32 1.09 1.44 1.24 1.16 1.44 1.47 0.98
Unknown Mixture Measured Peak Area 2.14 2.18 2.12 1.54
Unknown Mixture Adjusted Areas 2.14 1.00 2.14 2.18 1.32 2.88 2.12 1.24 2.63 1.54 1.47 2.26
Total Adjusted Area ? 2.14 2.88 2.63
2.26 9.91 Mole Fraction EtAc 2.14 /
9.91 0.216 Mole Fraction ProAc 2.88 / 9.91
0.291 Mole Fraction Bu Ac 2.63 / 9.91
0.265 Mole Fraction HexAc 2.26 / 9.91
0.228
21
Gas Chromatography Acetates

• Thermal Response Ratios
• GC Peak Area Correction Factor (alternate
  approach)
• The ratio of one peak area to another in a GC
  chromatogram should be proportional to the molar
  ratio of the components in the mixture
• If the peaks of an equimolar mixture do not have
  the same area, the relationship between the area
  of a peak and the mole fraction of the compound
  in the mixture is incorrect and would have to be
  adjusted by some factor
• The Thermal Response Factor (TR) is determined
  from an Equimolar Mixture

22
Gas Chromatography Acetates

• The derivation that follows utilizes ratios
  between any two compounds in a mixture, one of
  which will be designated as the basis for
  computation
• Assuming an equimolar mixture of 4 acetates
• Ethyl Acetate, Propyl Acetate, Butyl Acetate,
  Hexyl Acetate
• In the equation development below, the subscript
  i will be used to designate the compounds in a
  mixture
• i(1,2,3,4) Ethyl(1), Propyl(2), Butyl(3),
  Hexyl(4)
• In the derivation and examples that follow, Ethyl
  Acetate will be used as the basis for the
  calculations (designated by subscript (s), 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

23
Gas Chromatography Acetates

• Thermal Response Ratios (Cont)
• The following expression equates corrected area
  ratios to an adjustment of the molar ratios
• The area ratio (mole ratio) of each component (i)
  is shown relative to the selected base of
  computation compound (s) in the mixture
• If the equation is rearranged to indicate an
  adjustment to the measured areas
• Note the subscripts relative to the TR factor

24
Gas Chromatography Acetates

• Compute the TRs/TRi ratios from the measured peak
  areas from the standard equimolar mixture
• For an equimolar mixture molei/moles 1
• Thus, substitution in equation 2 gives
• Again note the relative position of the
  subscripts
• From equation (3), each individual TRs/TRi ratio
  is calculated from the peak areas of the standard
  equimolar mixture

25
Gas Chromatography Acetates

• Thermal Response Ratios (Cont)
• Adjusting the Peak Areas of the Unknown 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 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
• The new Mole is computed by multiplying the
  mole fraction by 100

.
26
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 As/Ai (s2) 1.44 1.00 1.44 1.44 1.32 1.09 1.44 1.24 1.16 1.44 1.47 0.98
Unknown Mixture Measured Peak Area 2.14 2.18 2.12 1.54
Unknown Mixture areai/areas (s2) 2.14 1.00 2.14 2.18 1.02 2.14 2.12 0.99 2.14 1.54 0.72 2.14
Apply TRs/Tri correction factor to measured area
ratios using equation 2
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 ? 1.32
1.34 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 ? 1.47 1.06 ?
moli/mol2 1.00 1.34 1.23 1.06
4.63 ? mole EtAc 1.00 / 4.63 100
21.6 ? mole ProAc 1.34 / 4.63 100
28.9 ? mole BuAc 1.23 / 4.63 100
26.6 ? mole HexAc 1.06 / 4.63 100
22.9
27
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 As/Ai (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 areai/areas (s2) 2.14 1.00 2.14 2.18 1.01 2.14 2.12 0.99 2.14 1.54 0.72 2.14
Apply TRs/Tri correction factor to measured area
ratios using equation 2
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
28
Gas Chromatography Acetates
Thermal Response Ratios (Cont) Ex. 3 - Assumes
the unknown is missing Ethyl Acetate and
Propyl Acetate (S3) is used as basis for
calculations
ProAc (3) BuAc (4) HexAc (6)
Standard Equimolar Mixture Measured Peak Area 186 208 210
Standard Equimolar Mixture TRs/TRi As/Ai (s3) 186 1.0 186 186 0.89 208 186 0.89 210
Unknown Mixture Measured Peak Area 2.18 2.12 1.54
Unknown Mixture areai/areas (s3) 2.18 1.0 2.18 2.12 0.97 2.18 1.54 0.71 2.18
Apply TRs/Tri correction factor to measured area
ratios using equation 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 HexAc 0.63 / 2.50 100 25.2