Thin Layer Chromatography

1
Thin Layer Chromatography Gas Chromatography

• By
• Heather Roden
• Denisse Iacobucci
• Joe Holak

2
Forensic Analysis using Thin Layer Chromatography

• Ink analysis
• Determines the specific chemicals
• Uses organic solvents
• Results are compared to a database of pen ink

3
Forensic Analysis using Thin Layer Chromatography

• Dye analysis
• Fibers
• Significant evidence
• Use thin layer chromatography to determine the
  different dyes in the fiber
• See how the colors elute

4
Forensic Analysis using Thin Layer Chromatography

• Pesticide analysis
• Pesticides are a hazard to the environment
• Many deaths are the results of poisoning from
  pesticides
• Pesticides are classified by their use or
  chemical type
• Determination of organophosphorus compounds in
  pesticide

5
Forensic Analysis using Thin Layer Chromatography

• Organic acid analysis
• Separation of carboxylic acids
• Organic acids are in textile, food preservatives,
  and medical agents

6
TLC ProceduresPlate preparation

• Mix the absorbent, water and a binder such as
  calcium sulfate
• Silica gel, paper and alumina
• Spread a thin layer of absorbent on an unreactive
  hard surface
• Glass, plastic, thick aluminum
• Heat in oven at 110C for 30 mins to activate
  and dry the plate

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TLC Procedure

• Place a small amount of solvent in a beaker
• In pencil, draw a straight line across the plate
  about 1 cm from the end of the plate
• Place a drop of sample solution on the line

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TLC procedure

• Add filter paper
• Place in solvent
• Sealed container

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How TLC works

• Sample solution is dissolved by solvent
• The solution sample will travel at different
  distances based on solubility, polarization, size
• Silica gel
• Polar substances do not move far
• Non polar substances move farther up the plate

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Calibration/Standards TLC

• No calibration
• Standards
• Compare to other known substances
• Rf value

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Solvents

• Choose a solvent depending on the polarity of the
  compound
• Least Polar
• More polar

Petroleum ether Cyclohexane Toluene Chloroform Acc
tone Ethanol Methanol
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Solvents

• The solvent can be a mixture of compounds but the
  polar solvent properties will over take the
  non-polar one.
• 10-30 Methly tert-butyl ether, MTBE, in hexane,
  C6H14, works well
• 10-30 Methylene chloride, CH2Cl2, in hexane,
  C6H14, for a less polar mixture
• 10-30 Acetone, CH3COCH3, in Methylene chloride,
  CH2Cl2, for a more polar mixture
• Trial and error is the best way to approach which
  solvent to use.

13
Visualization

• Destructive visualization
• Spray plate with H2SO4, and then bake in the oven
  at 110ºC for 15-20 minutes. Compound is destroyed
  but all spots will be visible
• Nondestructive visualization because of the use
  of a UV light the sample will not be destroyed.
  Although, not all of the spots on the plate will
  be visible.
• Long wave UV
• Short wave UV
• Semi-destructive visualization

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Visualization
A plate under a UV light to display the compounds
after they were developed
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Interpretation
Calculating the Rf value

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Calculate Rf Value
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Rf Value

• The Rf value needs to be between 0.0 and 1.0
• If the value is over 1.0 or less than 0.0, the
  calculation is wrong (you goofed)
• If the Rf value is greater than 0.8 or lower than
  0.2 the values are hard to interpret, thus
  creating a larger error
• The best Rf values are 0.3 to 0.6

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Rf Value

• The Rf value is not informative
• What affects the Rf value?
• Temperature
• Solvent
• Thickness and amount of spot
• Other compounds

19
Results

• Multiple spots from one sample can be achieved.

20
Pros for TLC

• Sensitivity
• Speed
• Inexpensive

21
Cons for TLC

• Too little of sample
• Too much of sample
• Subjective

22
Gas Chromatography
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Forensic Analysis using Gas Chromatography

• Determination of explosives
• If a forensic scientist can identify the type of
  explosive then he/she could possibly identify the
  source
• Ammonium nitrate
• Water analysis

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Forensic Analysis using Gas Chromatography cont

• Urine Analysis
• Cocaine
• Major cause of crime in the United States

25
Forensic Analysis using Gas Chromatography cont

• Urine analysis cont
• Amphetamines
• Uses similar procedure as cocaine
• Quinine in horse urine
• Prohibited in race horses
• Need to use mass spectrometer with gas
  chromatography

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Forensic Analysis using Gas Chromatography cont

• Blood analysis
• Concentration of alcohol
• Used if death is thought to be the result of
  intoxication
• Used for breath analysis
• Must be confirmed by gas chromatography in the
  United Kingdom and Europe

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Forensic Analysis using Gas Chromatography cont

• Gasoline analysis
• 10,000 fires are intentionally set each year
• Used to check for arson
• Fire debris evidence is collected in paint cans
  to keep the vapors inside

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Materials

• Microliter syringe
• Capillary tube
• A tank of He/N gas
• Depending on the instrument used
• Vaporization chamber
• Oven
• Detector
• Electric/chart recorder
• Integrators (if available)

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Gas Chromatography Procedures
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GC ProcedureInjector site

• Inject sample
• Control temperatures to vaporize sample
• Mix with carrier gas
• Enters column

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GC Procedurecolumn

• Packed columns
• 1.5 10 m long
• 2-4 mm diameter
• Glass, stainless steel
• Solid packing
• Capillary columns
• 25-60 m long
• Less 1 mm diameter

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GC Procedure/ TheoryDetector

• Flame ionization detector
• Destructive
• H and air is mixed with sample
• Ignited
• Collector electrode
• Thermal conductivity detector
• Universal
• Nondestructive
• Non-selective vs selective detectors

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Standards for GC

• Calibration typically preformed with a known
  volatile compound/mixture
• Attenuation controlling the height of the
  peaks.

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Results

• Retention time (Rt)
• The time required for the compound to pass
  through the column
• Rt is a characteristic value and independent of
  the presence of any other compound

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Calculate Rt
Speed of the chart paper is 2 cm per second

• Peak A
• Peak B

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Results

• Qualitative
• If the sample is suspected to be a certain
  compound, the sample can be spiked with said
  compound. In the read out, if there is no new
  peak for the spiked compound, the sample and
  the compound are the same.
• Quantitative
• In the read out, the area under the curve is the
  amount of the compound (integrate the peak)

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Results

• Quantitative measurement
• Two Type of Peaks
• Asymmetric peak
• Cut the area under the peak out of the read out
  and mass the weight. For this to be accurate the
  GC must be calibrated.
• Symmetric peak
• Measure the height and the width at half of the
  height

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Results

• For quantitative measurement integrate the peak.

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Results

• An easy way to calculate the area under the curve
  without integral calculus is to measure the
  height and the width at half of the height

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Calculate Area

• Peak A
• Peak B

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Percentage of Compounds

• A percentage can be calculated by dividing the
  area of the peak by the total area and
  multiplying by 100

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Ratio

• With the areas, a ratio can be calculated
• A ratio of 1.881 of compound B to compound A

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Pros for GC

• Speed
• Resolution
• Qualitative analysis
• Quantitative analysis

44
Pros for GC cont

• Sensitivity
• Simplicity
• Inexpensive

45
Cons for GC

• Can be slow
• Quantitative analysis
• Destructive
• Volatility

46
References

• Barnes Mark, "Gas Chromatography The Modern
  Analytical Tool". Practicing Oil
  Analysis Magazine. July 2002
• Bell, Charlse Jr., et. al. Organic Chemistry
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  Brooks/Cole. United States. 2001.
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  matography/thinlayer.htmlgt.
• "Gas Chromatography." Sheffield Hallam
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• Globalspec. The Engineering Search Engine. 2008.
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  /Hamilton/Syringes_Microliter_Syringes_05_uL_500_u
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  Positive Ion, High Mass Calibration of Gas
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  tent/ApplicationNotes/APP_GasChromaIonHighMassCali
  brationGCMS.pdfgt
• Thin Layer Chromatography. 16 Aug 2007. 14 Jan
  2008 lthttp//www.chem.ucla.edu/bacher/General/30B
  L/tips/TLC1.htmlgt.
• Zubrick, James. The Organic Chem Lab Survival
  Manual A Student's Guide to Techniques. Troy,
  NY John Wiley Sons, 1984.
• Scott, Raymond P.W. Introduction to Analytical
  Gas Chromatography. New York Marcel Dekker, 1998
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  York Marcel Dekker, 1999.