Column Chromatography

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Column Chromatography

• ????????? ????????????
• 2302244 ????????????? 2

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Column Chromatography

• ??????????????????????????????????????????????????
  ?????????????
• ?????????????????????????? Column Chromatography
• ???????? ????????????????????????????????????
• ????????????? ???????????????????????????????????
  ?????????????? (gravity)
• ????????? Column Chromatography
• Gas Chromatography (GC) ????????????????????????
• Liquid Chromatography (LC) ??????????????????????
  ?????

????????
column
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Gas Chromatography
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Gas Chromatography (GC)

• ??????????????????????????????????????????????????
  ??????? ??????????????????????????????????????????
  ??????????????????????????????????
• ??????????????????????????????????????????????????
  ????????????? ????????????????????????????????????
  ??????????? ??????? ???????? Gas Chromatography
  ????????????????????????????????????????????????
  (carrier gas)
• ?????????????????????????????? 2 ???? ???
• Gas-liquid chromatography (GLC)
• ???????????? (partitioning) ??????????????????????
  ???????????????????????????????????? solid
  support ???????????????????????????????
• Gas-solid chromatography (GSC)
• ?????????????????????????????????? (physical
  adsorption) ??????????????????????????????? ????
  molecular sieves, silica gel, alumina, activated
  carbon ???????

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???????????????????? GC
Injector
Detector
Carrier gas
Recorder and Data processing
Column
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???????????????????? GC

• ?????????????????? (GC) ??????????????????? 5
  ???? ???
• ?????? (carrier gas) - ???????????????????????????
  ???????????????
• ?????????????????? (injector) -
  ???????????????????????????????????????
• ??????? (column) - ?????????????? (oven)
  ???????????????????????????????
• ?????????????? (detector) - ??????????????????????
  ????????????????
• ??????????????? (recorder and data processing)

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?????? (Carrier Gas)

• ??????????????????????????????????????????????????
  ?? (chemically inert)
• ?????????????? ?????? He, N2, H2, ??? Air
• ????????????????????????? ????????????????????????
  ????????????? pressure regulator
• Inlet pressure 10-50 psi (above room pressure)
• ???????????????? (volumetric flow) ??????????????
  GC
• ?????? packed column 25-150 mL/min
• ?????? capillary column 1-25 mL/min

Bubble flow meter
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????????????? (carrier gas)

• ??????????????????????????????????????????????????
  ??????????? ??????????????????????????????????????
  ?????????????????????????
• ??????????????????????????? H ??????
  (??????????????????????????????)
  ????????????????????????????????
  ??????????????????????????????????????????????????
  ?????????????????
• ???????????????????????? Hopt ????????????????????
  ???? ???????????????????????????

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Choice of carrier gas
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????????????????????

• Carrier gas should contain less than 1ppm of
  oxygen, moisture, or other trace contaminants
• Carrier gas impurities can also contribute to
  detector noise
• Gas grade/Trap
• Longer column lifetime
• Less GC maintenance

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???????????

• Research grade
• 99.9999 total purity
• Total impurities lt 1ppm
• Complete analysis of all contaminants
• Ultra-Pure Carrier grade
• 99.9995 total purity
• total hydrocarbon lt 0.5ppm
• H2O, O2 each lt 1ppm
• UHP/Zero grade
• 99.999 total purity
• total hydrocarbon lt 0.5ppm
• H2O lt 3.5ppm, O2 lt 4ppm

Trace analysis lt 1 ppm
1-1000 ppm
gt 1
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Gas Purity
A. Molecular Sieve Trap B. Hydrocarbon Trap
C. Oxygen Trap
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Traps
Hydrocarbon Trap
Molecular Sieve Trap (moisture trap)
Oxygen Trap
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Sample Injection System

• Introduced as a plug of vapor with suitable size
• Slow injection or oversized samples cause band
  spreading and poor resolution
• Microsyringes
• Injection ports

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Injection port

• 50º C greater than boiling point of the least
  volatile component
• Sample size ?L
• Split mode (1100)
• Split/splitless mode
• Autosampler

Septum
Carrier gas
Septum purge
Heated injection port
Vent
Inlet liner
Column
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Vaporization Injectors

• Basic design
• a glass liner resides inside the heated, metal
  injector body
• Sample introduction
• rapidly vaporizes as of high temperature
• carried by the movement of carrier gas into the
  column

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Microsyringes/ Autosampler
Gas-tight syringe
Autosampler
Microsyringe
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Inlet Liners
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Accessories Vial, Septa and Caps
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GC Column
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Packed vs Capillary

• Length 2-50 m or more
• Stainless steel, glass, fused silica, or Teflon

Open-tubular Column (capillary column)
Packed Column
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Packed Column

• Glass or metals
• 2-3 m long, 2-4 mm i.d.
• Densely packed with packing materials or solid
  support coated with thin layer of stationary
  liquid phase
• Diatomaceous earth
• Size 60-80 mesh (250-170 ?m) or 80-100 mesh
  (170-149 ?m)

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Open Tubular Column

• Better resolution efficient mass transfer
  between gas and SP
• Tubing fused silica, glass, copper, stainless
  steel

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Types of Open Tubular Column
Solid support coated with liquid phase
Porous Adsorbent
Liquid phase
Wall-coated Open Tubular (WCOT)
Support-coated Open Tubular (SCOT)
Porous Layer Open Tubular (PLOT)
FSOT Fused-silica open tubular column
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Characteristics
Megabore column
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Stationary Phases

• Low volatility, thermal stability, chemical
  inertness
• Provide k and ? within a suitable range
• consider the polar characteristics of the
  analytes and select SP of similar polarity
• Like dissolves like

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Stationary Phases

• Solid phase
• Most uses for separation of low MW compounds and
  gases
• Common SP silica, alumina, molecular sieves such
  as zeolites, cabosieves, carbon blacks
• Liquid phase
• Over 300 different phases are widely available
• grouped liquid phases
• Non-polar, polar, intermediate and special phases
• Polymer liquid phase

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Stationary Phase Polymers

• Siloxane

• Arylene

• Polyethylene glycol

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Liquid phases

• Non-polar phase
• Primarily separated according to their
  volatilities
• Elution order varies as the boiling points of
  analytes
• Common phases dimethylpolysiloxane,
  dimethylphenylpolysiloxane
• Polar phase
• Contain polar functional groups
• Separation based on their volatilities and
  polar-polar interaction
• Common phases polyethyleneglycol
• Intermediate phase

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Bonded and Cross-linked SP
Polymer chains
Cross-linking
Bonding
Fused silica tubing surface

• Bonded and cross-linked SP provides long term
  stability, better reproducibility and performance.

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Common stationary phase coating for capillary
column
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(No Transcript)
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Column Dimensions

• Column Length 10 60 m
• Column Internal Diameter 0.10 0.53 mm
• Stationary Phase Film Thickness 0.10 0.25 mm

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Detectors
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Purpose of Detector

• - Monitor the carrier gas as it emerges from the
  column
• - Generate a signal in response to variation in
  its composition due to eluted components.

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Ideal Detector

• Adequate sensitivity 10-8 10-15 g solute/s
• Good stability and reproducibility
• Linear response to analytes
• Temperature range from room temperature to at
  least 400oC
• Short response time
• High reliability and ease of use
• Similarity in response
• Nondestructive of sample
• Low background noise and ease of operation

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Classification of Detectors

• Concentration vs. Mass flow rate
• Selective vs. Universal
• Destructive vs. Nondestructive

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Concentration vs. Mass Flow

• Concentration-dependent detectors TCD, ECD
• normally non-destructive
• can be used in series
• make-up gas lower the response
• Mass flow dependent detectors FID, NPD, FPD
• signal related to rate of solute molecules enter
  the detector
• destructive
• unaffected by make-up gas

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Selective vs. Universal

• Universal detectors TCD
• Detecting all solutes
• Beneficial for qualitative screening
• Selective detectors ECD,NPD,FPD
• Responds to particular types of compounds
• common chemical or physical property
• Enhances sensitivity for trace analysis

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GC Detector
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Flame Ionization Detector (FID)

• Selectivity
• Compounds with C-H bonds
• A poor response for some non-hydrogen containing
  organics (e.g. hexachlorobenzene)
• Fewer ions or none in flame
• carbonyl, alcohol, halogen, and amine
• Insensitive toward noncombustible gases such as
  H2O, CO2, SO2 and NOx

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FID Assembly
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Mass Spectrometer Detector (MSD)
MS with EI ion source, a quadrupole mass analyzer
and a continuous-dynode electron multiplier.
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MSD Assembly
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Hyphenated GC-MS
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Liquid Chromatography

• Partition liquid chromatography
• Adsorption liquid chromatography
• Ion or Ion exchange chromatography
• Size exclusion chromatography
• Affinity chromatography
• Chiral chromatography

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Liquid Chromatography (LC)

• ??????????????????????????????????????????????????
  ?????????????????????????????????????????
• Liquid Chromatography ????????????????????????????
  ?? ????????
• Classical LC ???? Column LC
• ?????????????????????? (??????????????? 100-250
  mm) ???????????????????????????????????? 1-5
  ?????????
• High Performance Liquid Chromatography (HPLC)
• ?????????????????????? (??????????????? 5-50 mm)
  ???????????????????????????????????? 1-5
  ?????????
• ??? high pressure pump ???????????????????????????
  ????????????

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Liquid Chromatography (LC)

• ?????? HPLC ?????????????????????? ????????????
• Partition liquid chromatography
• Based on a partitioning of solutes between mobile
  phase and stationary phase (solute diffuses into
  the interior of stationary phase)
• Adsorption liquid chromatography
• Based on an adsorption of solutes onto the
  surface of solid stationary phase which is called
  adsorbent.
• Ion or Ion exchange chromatography
• Based on an exchange between solute ions in
  mobile phase and ions of like charge associated
  with the stationary phase surface
• Size exclusion chromatography
• Based on a physical sieving process
• Affinity chromatography
• Based on specificity of analyte-stationary phase
  interactions
• Chiral chromatography
• Based on a chiral selector

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Partition chromatography

• Bonded phase packing organosilane
• Normal phase
• Highly polar SP, relatively non-polar MP
• Least polar component is eluted first increasing
  polarity of MP decreases elution time
• Reversed phase
• Non-polar SP, relative polar MP
• Most polar component eluted first increasing
  polarity of MP increases elution time

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Normal phase
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Reversed Phase
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???????????????????? HPLC
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???????????????????? HPLC

• ??????? HPLC ??????????????????? 6 ???? ???
• ??????????????????????? (Reservoir)
• ??????????? (pumping system) ???????????????????
  ?????????
• ?????????????????? (injector)
  ????????????????????????????????
• ??????? (column)
• ?????????????? (detector) ??????????????????????
  ????????????????
• ??????????????? (recorder and data processing)

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?????????????

• ??????????????????????????????????????????????????
  ?????????????
• ??????????????????????????????????????????????????
  ??????????????????????????????????????????????????
  
• ???????????????????????????????????
• ?????????????????????????????
• ?????????????? degasser ??????????????????????????
  ???
• ??? purge ?????????????? ???? ???????
  ????????????????????????????
• ????????????????????? HPLC ?? 2 mode
  ???????????????????????????????
• Isocratic ??????????????????????????????????????
  ?????????????
• Gradient elution ???????????????????????????????
  ??????????????????????????????????????????????????
  ??? (stepwise)

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Pumping system

• Generate pressure up to 6000 psi
• Pulse free output
• Flow rates 0.1-10 mL/min
• Flow reproducibility
• Resistance to corrosion
• Screw-driven syringe pump
• Reciprocating pump
• Pneumatic pump

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Pumping System

• ???????????????? HPLC ??? ????????????????
  (reciprocating pumps)
• ??????????????? ??????? (piston)
  ??????????????????????????????????????????????????
  ??????????????????????????????????????????????????
  ?????????????????????????????????????????????????
  ?????? (reservoir) ????????????????? check valve
• ??????????????????????????????????????????????????
  ?????????????????

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Reciprocating pump
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Sample Injection System

• ??????? microsampling valve
• Sampling loop 5-500 ?L

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Autosampler
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Columns ?????? HPLC

• Stainless steel tubing
• 10-30 cm, 2-5 mm i.d., Packing 3-10 ?m particle
  size, 40,000 60,000 plates/m
• Micro column
• 3-7.5 cm, 1-4.6 mm i.d., 3 or 5 ?m particle size,
  100,000 plates/m
• Silica particles coated with thin organic films
  which are chemically or physically bonded to the
  surface, alumina particles, porous polymer,
  ion-exchange resin

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HPLC Columns
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Guard Column

• Positioned ahead of analytical column
• Remove particulate matter and contaminants
• Increase the life time of the analytical column

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Detector

• Low dead volume minimize extra column band
  broadening
• UV-Vis
• Fluorescence
• Electrochemical
• Refractive index
• Conductivity
• Mass Spectrometry
• FT-IR

From column
Detector
Light Source
Flow Cell
To waste
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UV-Vis Detectors
Diode-Array UV-Vis
UV-Vis
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Fluorescence Detector
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Refractive Index
Reflective detector
Deflection detector