HighPerformance Liquid Chromatography HPLC

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High-Performance Liquid Chromatography (HPLC)

• Moving phase is liquid and stationary phase are
  coated solid particles
• As liquid carries the sample, different
  components are slowed to different degrees
  depending on their interaction with the
  stationary phase
• Major advantage over GC is it takes place at room
  temperature
• GC- needs to heat material. Any temperature
  sensitive material may be destroyed. Explosives
  are generally heat sensitive and therefore are
  more readily separated by HPLC

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Thin Layer Chromatography (TLC -1)

• Moving liquid phase, solid stationary phase
• TLC Procedure
• Sample is dissolved in a solvent
• Spotted onto the lower edge of the plate
• The plate is placed into a closed chamber with
  liquid
• The liquid slowly rises up by capillary action.
  Separation occurs as the components with the
  greatest affinity for the moving phase migrate
  faster
• Visualized UV fluorescence or developed with a
  chemical reagent spray ? color spots

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TLC -2
Q K

• Questioned sample (Q) must be developed alongside
  a standard or known (K) sample. If Q and K
  travel the same distance up the plate from the
  origins then they can be tentatively identified
  as the same
• ID cannot be considered definitive as other
  materials may have similar migration
• Distance traveled up can be assigned an Rf value
  distance traveled by the component divided by
  the distance traveled by the liquid phase. For
  example if the moving phase travels 10cm and spot
  8cm then Rf 8cm/10cm 0.8
• Rapid and sensitive down to 100ug
• Principal application is detection and
  identification of components in a complex mixture

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Pen Ink TLC Hands on exercise

• Draw a straight line with pencil 1 inch from the
  bottom of your plate paper towel
• Spot at least 8 different inks across the plate
  at ½ inch intervals- Label your plate with team
  name and pen ink (eg. red expo marker)
• Pour your solvent in to approximately ¼ inch
  depth
• Slowly drop your plate into the solvent
• Permit the front to move up at least 3 inches
• Remove the plate and let air dry
• Answer the following
• 1) Are there differences in migration?
• 2) Do you see any evidence of separation of dyes?
• 3) Are there any inks that do not migrate?
• 4) Based on your observations, which inks have
  the most affinity for the mobile phase? For the
  stationary phase?

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Electrophoresis

• Separation of materials according to migration
  rates on a stationary solid phase
• Uses electric potential across the stationary
  medium
• Medium may include starch or agarose coated on a
  glass plate of polymer in a capillary
• Substances possessing an electric charge migrate.
  The speed depends on size and charge
• Principal applications are the separation of
  mixtures of proteins and DNA

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Spectrophotometry Review

• Theory of Light- White light ROYGBIV
• Light is a wave - wavelength is inversely
  proportional to frequency- Visible light is only
  a small part of the electromagnetic spectrum
• Color visual indication of an objects ability
  to absorb some and reflect visible light
  components
• Different materials have different absorptions
• Absorption of UV, visible and IR are particularly
  applicable for identification of organic
  substances. How much? - Beers Law- Akc , A
  absorption cconcentration kproportionality

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Spectrophotometer

• Instrument used to measure and record the
  absorption spectrum of a chemical substance
• Components- 1. Radiation source
• 1- Radiation source (UV, vis, IR)
• 2. Monochromator or frequency selector
• 3. Sample holder
• 4. Detection to convert electromagnetic radiation
  into an electric signal (digitizer)
• 5. Recorder

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UV and Visible Spectrophotometry

• Measures the absorbance of UV and visible light
  as a function of wavelength or frequency
• UV spec of heroin has max absorption at 278nm
  providing materials probable identity
• Will not provide definitive result - other
  material may have a similar UV absorption

9
IR Spectrum

• IR specs provide far more complex patterns
• Different materials always have distinctively
  different IR spectra
• Each IR spectra is equivalent to a fingerprint
  of that substance and no other
• Fourier transform infrared spectrophotometer
  FT-IR
• Considered specific in itself for identification

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Mass Spectrometry (1)

• GC coupled to a MS overcomes limitation of GC
  (cannot produce specific identification alone)
• Material emerging from GC, enters a vacuum where
  they are bombarded by high energy electrons
  causing them to lose electrons and acquire a
  positive charge (ions).
• These ions are unstable and fragment
• Fragments pass through an electric field where
  they are separated according to their masses.
• No two substances produce the same fragmentation
  pattern under carefully controlled conditions.
• Very sensitive one millionth of a gram

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Mass Spectrometry (2)
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Mass Spectrometry (3)

• Sample first injected into a heated inlet port
  and carrier gas sweeps it into the GC column
• GC separates the mixture into its components
• Ion source filiment wire emits electrons striking
  the sample molecules causing them to fragment
  according to mass
• Detector counts the fragments passing thru the
  quadrupole Signal is small and must be amplified.
• Measures abundance of each fragment displaying
  the mass spectrum

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Summary 1

• Organic substances contain C. Inorganic ones
  comprise all others
• Choice of analytical techniques depends on
  substance category (organic vs inorganic) and the
  need for qualitative vs. quantitative
  determinations
• Qualitative relates just to the identity of the
  material whereas quantitative relates to the
  percent composition of components in a mixture
• Chromatography, spectrophotometry and mass spec
  are used by forensic scientists to identify or
  compare organic materials
• Chromatography is a means of separating and
  tentatively identifying the components of a
  mixture.
• Spectrophotometry is the study of the absorption
  of light by chemical substances
• Mass spectrometry characterizes by observing a
  substances fragmentation patterns after
  collision with high energy electrons

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Summary 2

• GC separates components of a mixture on the basis
  of their distribution between a moving (carrier)
  gas and a stationary phase which is a thin film
  of liquid contained in a column. The record of
  the separation is a chromatogram
• A direct connection between GC and MS allows
  components to flow into the MS (GC-MS).
  Fragmentation of each component produces a
  fingerprint pattern of the substance.
• HPLC separates compounds in a stationary phase
  and mobile liquid phase with temp sensitive
  compounds like explosives
• TLC uses a solid stationary phase and mobile
  liquid phase
• Electrophoresis uses electric potential to
  separate proteins and DNA of different size and
  charge on a gel-coated plate or polymer filled
  capillary
• Most labs use UV and IR spec to characterize
  chemical compounds. UV spec produces simple vs.
  IR complex spectra and distinctive spectra
  providing a fingerprint of the substance