Introduction to LCGC Instrumentation

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Introduction to LC/GC Instrumentation

• Bioanalytical Chemistry
• Lecture 1

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Milestones in Chromatography

• 1903 Tswett - plant pigments separated on chalk
  columns
• 1931 Lederer Kuhn - LC of carotenoids
• 1938 TLC and ion exchange
• 1950 reverse phase LC
• 1954 Martin Synge (Nobel Prize)
• 1959 Gel permeation
• 1965 instrumental LC (Waters)

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Basis of Chromatography

• Definition Cs Cm K
• Mechanism - selective retardation caused by
  interactions with bonded phase of stationary phase

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Chromatogram

• Detector signal vs. retention time or volume

1
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Presence of several Peaks separated
in Time/volume evidences Separation of components
Detector Signal
time or volume
Peak area ? Concentration
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Definitions

• Mobile phase - phase that moves through
  chromatograph
• In LC - eluent
• In GC - carrier gas
• Stationary phase - column phase that is
  stationary in chromatograph
• Bonded phase - reactive groups imparted to
  stationary phase in order to achieve selectivity

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Types of Chromatography

• Classification by mobile phase
• Gas - Gas chromatography (GC)
• 1951 Martin and James (fatty acids)
• Liquid - Liquid chromatography (LC)
• 1964 Horvath (Yale) instrument
• 1966 Horvath and Lipsky (nucleic acid components)
• Supercritical fluid - Supercritical fluid
  chromatography (SFC)
• 1958 Lovelock (Yale)

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Purpose of Chromatography

• Identify components in mixtures
• Isolate component in mixture
• Demonstrate level of purity
• Quantitation
• internal standard

analytical
preparative or semi-preparative
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Instrumentation for LC

• Strong/weak eluent
• Pump (up to 6000 psi)
• Injector
• Column
• Detector
• Fraction Collector
• Computer

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Instrumentation for GC

• Carrier gas
• N2, He, H2
• Injector
• Column
• Detector
• Computer

oven
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Modes of LC Separation

• Adsorption or normal phase
• Reverse phase
• Hydrophobic interaction
• Size exclusion
• Ion Exchange
• Affinity

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Modes of LC Separation

• Adsorption or Normal phase
• Stationary phase more polar than mobile phase
• column hydroxyapatite (Ca10(PO4)6(OH)2
• mobile phase hexane, CH2Cl2, THF, CH3OH
• gradient elution

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Modes of LC Separation (contd)

• Reverse phase (RPC)
• Stationary phase hydrophobic and mobile phase
  hydrophilic
• column silica, polystyrene covalently modified
  with alkyl chain 3-18 Cs
• mobile phase buffered water organic solvent
  (propanol CH3CN, CH3OH)
• gradient elution
• Affinity (AC)
• Based on specific and selective interactions
  between enzymes or antibodies and ligands

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Modes of LC Separation (contd)

• Hydrophobic interaction (HIC)
• Stationary phase hydrophobic and mobile phase
  hydrophilic
• stationary phase low density of short alkyl
  chains or phenyl
• mobile phase decreasing concentration of high
  salt solution (3M NH4SO4)
• gradient elution

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Modes of LC Separation (contd)

• Size Exclusion (Gel Permeation) (SEC)
• Chemical interactions between sample and
  stationary phase undesirable separation
  accomplished by sieving mechanism with porous
  stationary phase
• stationary phase varies - silica
• mobile phase low ionic strength (100 mM) buffer
  or dilute acid (0.1 TFA) with CH3CN or
  isopropanol (20-50)
• isocratic elution

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Modes of LC Separation (contd)

• Ion-Exchange (IEC)
• Ion exchange interactions between cationic or
  anionic analyte and stationary phase bearing
  opposite charge
• stationary phase polystryrene, silica modified
  with functional groups such as quaternary amines
• mobile phase buffer containing increasing
  concentration of salt (NaCl, MgCl2, K3PO4, NH4SO4)

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Ion Exchange

• Strong - charge of strong ion exchanger is
  essentially independent of pH
• sulfonic acids (cation)
• quaternary amines (anion)
• Weak - charge of weak ion exchanger is pH
  dependent
• carboxymethyl (CM) (cation)
• primary, secondary, and tertiary amines, e.g.,
  diethylaminoethyl (DEAE) (anion)

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Anion Exchange - Proteins

• Anion exchange - protein is acidicbonded phase
  eitherweak - DEAEstrong - quaternary amine

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Cation Exchange - Proteins

• Cation exchange - protein is basicbonded phase
  eitherweak - CMstrong - sulfonic

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Elution Approaches

• Isocratic - constant mobile phase composition
• Gradient - variable mobile phase composition
• step - change accomplished sharply at a defined
  point in time
• continuous - change accomplished gradually over
  time

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Modes of GC Separation

• Packed
• earliest
• Solid particles either porous or non-porous
  coated with thin (1 ?m) film of liquid
• 1 - 8 mm ID 1 - 10 m length

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Modes of GC Separation

• Capillary (open tubular)
• modern
• Inner wall modified with thin (1 ?m) film of
  liquid
• 0.1 - 0.5 mm i.d. 10 - 50 m length
• types
• porous layer open tubular (PLOT)
• wall-coated open tubular (WCOT)
• liquid coating

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GC Liquid Phase

• Low volatility
• High bp
• Chemically unreactive
• Examples
• 1-squalene
• Tetrahydroxyethylenediamine
• Carbowax (polyethylene glycol)

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Elution Analogies GC vs. LC

• Isothermal (GC) ? Isocratic (LC)
• Programmed temperature (GC) ? Gradient (LC)
• Raising column temperature (GC)
• Decreases retention time
• Sharpens peaks

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5 Properties of a Good Detector

• Sensitivity
• ?Response/ ?C
• Selectivity
• Universal or selective response
• selectivity - ability to distinguish between
  species
• Rapid response
• Linearity - concentration range over which signal
  proportional to concentration
• Stability with respect to noise (baseline noise)
  and time (drift)

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Detectors for LC

• UV-vis
• PMT (single ?)
• PDA (simultaneous multi- ? monitoring)
• Fluorescence
• Electrochemical
• Amperometry
• NMR
• microcoil NMR J. Sweedler (U. Illinois)

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Detectors for GC

• Electron capture (ECD)
• radioactive
• good for X-, NO2- and conjugated
• Thermal conductivity (TCD)
• change in resistance of heated wire
• Flame ionization (FID)
• destruction of combustible sample in flame
  produces measurable current
• Fourier transform infrared (FTIR)
• Mass spectrometry (MS)

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MS Components

• Ionization source
• Analyzer
• Differentiating characteristic m/z
• Ion detector

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Ionization Methods - Examples

• Electron capture (EC)
• 70 eV e- neutral molecule ? energetic molecular
  ion
• hard fragmentation
• Chemical ionization (CI)
• Reagent ion molecule ? molecular ion reagent
  ion
• Reagent ion He, OH- (water), CH5 or CH3 (CH4)
• soft less fragmentation

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Ionization Methods - Examples (contd)

• Electrospray (ESI)
• generation of ions by desolvation or desorption
  of charged liquid droplets
• Matrix Assisted Laser Desorption (MALDI)
• ionization facilitated by laser irradiation of
  sample dissolved in an organic matrix
• EX sinapinic acid

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Types of MS Analyzers

• Quadrupole - most common
• Ion trap
• Time of Flight (TOF)

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Two Operational Modes

• Scan
• Collect mass data over known range
• Slow
• Selective ion monitoring (SIM)
• Sample mass at predetermined values
• Fast

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Chromatogram
time of injection
tr
Detector Response
Retention Time
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Chromatogram - GC-MS vs. LC (UV)

• x-axis
• GC-MS - m/z
• LC - retention time or volume
• y-axis - detector response
• GC-MS - abundance
• LC - Abs