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Chromatographs
eluent tank pump injector column
detector PC CHROMATOGRAM
• Qualitative &
• Quantitative information
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GasChromatography (GC)1952: A.T. James & A.J.P. Martin
MOBILE PHASE: GASSTATIONARY PHASE: solid or liquid on solid support (GSC, GLC)
COLUMNELUTION TECHNIQUE
GASCHROMATOGRAPHY: analysis in vapor phase
High performancyQualitative & Quantitative informationComplicated samplesSeparation
Base of separation: 1. Boiling point (vaporization)2. Structure
Evaporization depends on:•Molar mass•polarity
Thermal stability
~12 billion organic compounds~ 50 000: evaporative without destruction
•1956: van Deemter: kinetic theory•M. Golay: capillary columns
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GASCHROMATOGRAPHY (GC)Sample introduction to the mobile phase:
gas/vapor
Sample can be: 1. gas2. liquid: vaporization3. solid: dissolution in liquid
Gas tank
Gas cleaner Gaschromatograph (GC)
Pressure and flow regulators
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GASCHROMATOGRAPH (GC)
Gas tank
PC
Flow controller
column
injector detectorcleaner
Pressure controllerthermostate
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Eluent gas Depending on the type of detector:•H2
•Ar•N2
•He
Reductor valve:Type depends on the quality and
pressure of the gas
Inside apparatus:Pressure and flow controllers
Flow-rates
Name sign % ppm
pure 2.5 99,5 5000
3.0 99.9 1000
Very pure 3.5 99,95 500
4.0 99,99 100
4.5 99,995 50
5.0 99,999 10
6.0 99,9999 1
Ultra pure 7.0 99,99999 0,1
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Sample introduction
1. Injection in a very short time2. Vapor/gas phase3. Mixible with eluent gas
volume0,1 l-1 ml
Liquid vaporization: 100-10000 X volume
increaseSyringeFor gas & liquid sample
„six-port” valve
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Septum (rubber)
Eluent gas inlet
Heating block(25 – 300 oC)
liner (glass)
column
FLASH INJECTOR
Packed columns: greater diameter: greater sample volumeCapillary columns: small sample volume
1. Samle introduction2. Vaporization3. Inlet to column
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Flash injectorInjection vaporization
1. Sample vaporization2. Liquids: 100 – 1000 X volume increase3. Mixing with eluent
1. Stick needle into the septum2. Push the syringe piston 3. Remove syringe
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Eluent gas moves the sample to the column.
solvent
Quick injection
Slow injection
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Type of injectors•SPLIT•SPLITLESS•ON-COLUMN•PTV
Carrier gasSeptum wash
split-gas
Split/splitless ratio: determines amount of sample moving to the column
Split-injector
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200:1
5:1
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Splitless injector
Purge Off
Purge On
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On-column
Injection directly to the column
PTV(Programmed Temperature Vaporizer)
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polyimid, 350 oC
quarz
Stationary phase
microbore: d < 150 m standard capillary: 150 m < d < 500 m widebore: d > 500 m
d
Columns
Adsorption mechanism: PLOT
(Porous Layer Open Tubular)
Distribution mechanism:WCOT: Wall Coated OT
SCOT: Support Coated OT
Capillary
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SiOH
SiOH
SiOH
SiOH
SiOH
SiOH
Quartz surface
•„tailing”•Non-symmetric peaks
desactivation: sylil reagents
Si-O-Si(CH3)3
Si-O-Si(CH3)3
Si-O-Si(CH3)3
SiOH
Interaction: between stationary phase and sample
Active side: silanol groups
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Stationary phases I.
Thermal stabilityNo „bleeding”
Known chemical structureChemical inertnees
Low priceAdsorbents (GSC)
porous, with large special suface
Organic adsorbents: •active carbon •polymers
inorganic adsorbents:•silicagel•aluminium-oxide•zeolits (molekulasziták)
modified adszorbents:Based on carbon or silicagel
Analytes: Hydrocarbons with small molar mass, He, Ne, Ar, Kr, Xe
(PLOT)
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Stationary phases II.(GLC)
Polymers: WCOT: polymers on the surface of capillary)
Relative small number: 12-15
substituted polysiloxans (silicons): long lifetime
n
R R
RR
Si O Si O
R: substituents on polysiloxans
Thermal stability: up 250-300 C
Substituents::MethylphenylCianopropylTrifluoropropyl
Methyl: -CH3 Phenyl:
Cianopropyl: -CH2CH2CH2CN
Trifluoropropyl: -CH2CH2CF3
(absorption: dissolution of gas and liquids in liquids)
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CH3
CH3
CH3 CH
3
CH3 CH
3
Si
Si
Si
Si
O
O
O
O •methyl-phenyl•cianopropyl-phenyl•etc.
substitution: how much % of Si atoms
100 % metil5 % fenil & 95 % metil
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Polyethyleneglycols(PEG)
CH2HO O CH2 O Hn Disadvantage:
•Lower thermal stability•„oxygen-sensitivity”
Special separation
Carbowax
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Polarity of stationary phase: •Structure of stationary phase•Quality of functional groups•Number of functional groups
Apolar stationary phases:• 100 % methyl• 5 % phenyl
Midium polar phases:•35 % phenyl•50 % phenyl
Polar phases:•cyanopropyl•PEG
Selectivity depends on: the interaction between stationary phase and analyte
Interactions depend on: •Quality of analytes•Structure of stationary phase
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thermostate
column
Thermostate
•Large temperature range -50 – 400 C•Programmable heating: 0- 40 oC/min•„cooling”
Type of working:
•Izotherm
•Programmable heating
t (min)
T (oC)
Decrease of analysis timeGood peak shape
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Detectors
Quantitative analysis: signal of detector is proportional with concentration of analytes in detector
universal: signal for every compoundsselective: signal for a groups of compoundsspecific: signal for special compounds
destruktivnon destruktiv
Dinamic range: change of concentration results a change in signal
linearity: T= mc (deviation < 5 %)
sensitivity: m (ratio of signal/concentration)
Limit of detection (LOD): signal to noise ratio: 3
Limit of quantitation (LOQ): signal to noise ratio: 10
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DetectorsThermal Conductivity Detector
(katharometer)
non destructívuniversal
W-filaments: 100-200 mA heating current
Wheatstone-bridge
dinamic range: 105
LOD: 5-50 ng
Carrier gas:H2, HeN2
Change of impedance
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Flame-ionization detector (FID)
hydrogen/air microburner with a pair of electrodes
Carrier gas: non ionizable gas: N2, Ar, He, H2
Organic compounds leaving the column are burning in burner jet, ions are forming
Ions result a small current
Carbon-detector: it is good for organics, except formic acid
destructívDinamic range: 105-106
LOD: 0,05-0,5 ng
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High Performance Liquid Chromatography (HPLC )
Sample: liquid
eluent tank injector pumpGas removal
column
thermostate
detector PC
Mobile phase: liquidStationary phase: adsorbent (LSC) or
liquid on a support(LLC) Column
Elution technique
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HPLC
pump
(thermostate)
Gas removal
detector
automated injector
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Eluent
Should (have) be:Low viscosityinert: no reaction with analytesChemical stabilityNo corrosionNo toxycityHigher boiling pointLow priceGood quality and purityCompatible with detectorUV-absortion: low
purity:HPLC grade
Water and buffers too !!!
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EluentAnalytes distributed between stationary and mobile phase:
interaction of analytes with both phases
Polarity of molecule & mobile phase & stationary phase
hexane
chloroform
tetrahidrofuran
acetonitrile
isopropanol
ethanol
methanol
water
Eluent strength: determined on silicagel on the bease of heat of adsorption of solvents
POLARITY
Mixed solvents: should be mixcible
izoeluotrope mixture: eluent strength is the same:k’, Rs: may change !!!
Izocratic elution: fixed mobile phase compositionGradient elution: eluent strength is increasing in time
Use of buffers: adjusting of pH in the case of analysis of ionisable components
Change of polarity: •Change of quality of mobile phase•Mixing of solvents
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PumpsTo carry of eluent
Flow-rates in classical analytical HPLC: 0,1-1,5 ml/min (0-5 ml/min)
Should be:•pressure (400 bar)•Stable flow-rate•Compatible with different solvents:no corrosion•Small hold-up volume•No pulsation
Syringe-type pump
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Reciprocating pump
Volume: 10-100 lChange of flow rate: easy
Pulsation: double pistons (phase-deviation)
V
time
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Gas removalsLiquids: contain dissolved gases
Ultrasound:•cheap•Non effective
Vacuum:•Higher price•effective
Effect of gas bubbles:In pump:
•Pressure pulsation•Different flow-rates•Mechanical instability
In detector:
•Increased noise (retention time changes)
He-purge:•Higher price•effective
Remove of gas from the solvent:
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Sample loading
1. Quick2. Sample should be mixable with eluent
Sample volume: 10-50 l
Micro syringe:
„Six-port” valve
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Columns
Function: separation
Liquid chromatography:
NP LC: Normal PhaseRP LC: Reversed Phase
NPLC: polarity of stationary phase > polarity of mobile phase
RPLC: polarity of stationary phase < polarity of mobile phase
Material of column:•Stainless steel•Glass•PEEK (poly(ether-ether-ketone)
Size of column: •diameter: 2-5 mm•length: 5-25 cm
Packing:regularspherical
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Modified silica gel
SiO2
OH
OH
OH
OHOH
Modifying groups:C18: octadecyl: C18H37
C8: octyl: C8H17
C4: buthyl: C4H9
Amino: CH2CH2CH2NH2
Ciano: CH2CH2CH2CNPhenyl: C6H5
Guard column: avoid contamination of analytical column
RPLC: C18 stationary phase & methanol/water mobile phaseNPLC: silicagel stationary phase& hexane/alcohol mobile phase
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Detectors
Quantitative analysis: signal of detector is proportional with concentration of analytes in detector
universal: signal for every compoundsselective: signal for a groups of compoundsspecific: signal for special compounds
destruktivnon destruktiv
Dinamic range: change of concentration results a change in signal
linearity: T= mc (deviation < 5 %)
sensitivity: m (ratio of signal/concentration)
Limit of detection (LOD): signal to noise ratio: 3
Limit of quantitation (LOQ): signal to noise ratio: 10
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UV-Vis spectrophotometerApplication: UV-Vis range
Lambeert-Beer:A = ε c l
Light source:UV: deuterium lampVis: wolfram lamp
rés
fényforrás
monocromator
splitter
DETECTOR
Reference side
Measuring side
cuvetteI0
I0 I0
I
Detector:fotodiode
Cuvette: quartzl=5-10 mm
Most usable HPLC detector190 nm < < 800 nm
A = lg I0/I
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Dioda Array Detector (DAD)
polychromator
Light source lencecuvette
Diode array
Advantage:Spectra and chromatogram at the same time
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Paper and thin-layer chromatography
Planar arrangement
Stationary phase: papersilica gel or aluminium-oxide on a glass plate
Evaluation of chromatogram:Dropping liquid sample on the one edge of the plate with capillary Evaporation (drying) the solventPlace the plate to the closed container saturated with vapors of developing solventRunning of analytes: based on capillary activityAfter development of chromatogram, remove plate from container and dry it Locating analytes on the plate: spraying with chemical reagents, like iodine, sulfuric acid or UV-light
Selection of mobile phase:like in Normal Phase HPLC
Qualitative data: retardation factor (Rf)Quantitative data: intensity of spots
Advantages: •simple•cheap
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