Title: Thin Layer Chromatography
1Thin Layer Chromatography Gas Chromatography
- By
- Heather Roden
- Denisse Iacobucci
- Joe Holak
2Forensic Analysis using Thin Layer Chromatography
- Ink analysis
- Determines the specific chemicals
- Uses organic solvents
- Results are compared to a database of pen ink
3Forensic 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
4Forensic 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 -
5Forensic Analysis using Thin Layer Chromatography
- Organic acid analysis
- Separation of carboxylic acids
- Organic acids are in textile, food preservatives,
and medical agents
6TLC 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
7TLC 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
8TLC procedure
- Add filter paper
- Place in solvent
- Sealed container
9How 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
10Calibration/Standards TLC
- No calibration
- Standards
- Compare to other known substances
- Rf value
11Solvents
- Choose a solvent depending on the polarity of the
compound - Least Polar
- More polar
Petroleum ether Cyclohexane Toluene Chloroform Acc
tone Ethanol Methanol
12Solvents
- 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.
13Visualization
- 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
14Visualization
A plate under a UV light to display the compounds
after they were developed
15Interpretation
Calculating the Rf value
16Calculate Rf Value
17Rf 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
18Rf Value
- The Rf value is not informative
- What affects the Rf value?
- Temperature
- Solvent
- Thickness and amount of spot
- Other compounds
19Results
- Multiple spots from one sample can be achieved.
20Pros for TLC
- Sensitivity
- Speed
- Inexpensive
21Cons for TLC
- Too little of sample
- Too much of sample
- Subjective
22Gas Chromatography
23Forensic 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
24Forensic Analysis using Gas Chromatography cont
- Urine Analysis
- Cocaine
- Major cause of crime in the United States
25Forensic 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
26Forensic 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
27Forensic 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
28Materials
- 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)
29Gas Chromatography Procedures
30GC ProcedureInjector site
- Inject sample
- Control temperatures to vaporize sample
- Mix with carrier gas
- Enters column
31GC 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
32GC 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
33Standards for GC
- Calibration typically preformed with a known
volatile compound/mixture - Attenuation controlling the height of the
peaks.
34Results
- 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
35Calculate Rt
Speed of the chart paper is 2 cm per second
36Results
- 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)
37Results
- 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
38Results
- For quantitative measurement integrate the peak.
39Results
- 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
40Calculate Area
41Percentage of Compounds
- A percentage can be calculated by dividing the
area of the peak by the total area and
multiplying by 100
42Ratio
- With the areas, a ratio can be calculated
- A ratio of 1.881 of compound B to compound A
43Pros for GC
- Speed
- Resolution
- Qualitative analysis
- Quantitative analysis
44Pros for GC cont
- Sensitivity
- Simplicity
- Inexpensive
45Cons for GC
- Can be slow
- Quantitative analysis
- Destructive
- Volatility
46References
- 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
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Aerograph, 1968. - NT Curriculum Project, University of Wisconsin
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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
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