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Thin Layer Chromatography

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Title: 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

7
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

8
TLC procedure
  • Add filter paper
  • Place in solvent
  • Sealed container

9
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

10
Calibration/Standards TLC
  • No calibration
  • Standards
  • Compare to other known substances
  • Rf value

11
Solvents
  • Choose a solvent depending on the polarity of the
    compound
  • Least Polar
  • More polar

Petroleum ether Cyclohexane Toluene Chloroform Acc
tone Ethanol Methanol
12
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

14
Visualization
A plate under a UV light to display the compounds
after they were developed
15
Interpretation
Calculating the Rf value

16
Calculate Rf Value
17
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

18
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
23
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

24
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

26
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

27
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

28
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)

29
Gas Chromatography Procedures
30
GC ProcedureInjector site
  • Inject sample
  • Control temperatures to vaporize sample
  • Mix with carrier gas
  • Enters column

31
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

32
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

33
Standards for GC
  • Calibration typically preformed with a known
    volatile compound/mixture
  • Attenuation controlling the height of the
    peaks.

34
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

35
Calculate Rt
Speed of the chart paper is 2 cm per second
  • Peak A
  • Peak B

36
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)

37
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

38
Results
  • For quantitative measurement integrate the peak.

39
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

40
Calculate Area
  • Peak A
  • Peak B

41
Percentage of Compounds
  • A percentage can be calculated by dividing the
    area of the peak by the total area and
    multiplying by 100

42
Ratio
  • With the areas, a ratio can be calculated
  • A ratio of 1.881 of compound B to compound A

43
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
    Laboratory with Qualitative Analysis. Thompson
    Brooks/Cole. United States. 2001.
  • Clark, Jim. "Thin Layer Chromatography." 2007. 14
    Jan 2008 lthttp//www.chemguide.co.uk/analysis/chro
    matography/thinlayer.htmlgt.
  • "Gas Chromatography." Sheffield Hallam
    University. 14 Jan 2008 lthttp//teaching.shu.ac.uk
    /hwb/chemistry/tutorials/chrom/gaschrm.htmgt.
  • Globalspec. The Engineering Search Engine. 2008.
    lthttp//www.globalspec.com/FeaturedProducts/Detail
    /Hamilton/Syringes_Microliter_Syringes_05_uL_500_u
    L/33774/0?deframe1gt
  • McNair, H.M., and E.J. Bonelli. Basic Gas
    Chromatography. 4th. Berkeley, CA Varian
    Aerograph, 1968.
  • NT Curriculum Project, University of Wisconsin
    Madison. 1995. 1/20/2008. lt http//inst.sfcc.edu/c
    hemscape/mainmenu.htmgt
  • Patkin, Adam J. Chromatographic Technique for
    Positive Ion, High Mass Calibration of Gas
    chromatograph and Mass Spectroscopy. Perkin
    Elmer. April 1999. lthttp//las.perkinelmer.com/Con
    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
  • Fried, Bernard. Thin-layer Chromatography. New
    York Marcel Dekker, 1999.
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