1. dia

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Chromatographs
column
eluent tank
injector
pump
detector
PC
CHROMATOGRAM

• Qualitative
• Quantitative information

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GasChromatography (GC) 1952 A.T. James A.J.P.
Martin

• High performancy
• Qualitative Quantitative information
• Complicated samples
• Separation

• 1956 van Deemter kinetic theory
• M. Golay capillary columns

MOBILE PHASE GAS STATIONARY PHASE solid or
liquid on solid support (GSC, GLC)
COLUMN ELUTION TECHNIQUE

• Base of separation
• Boiling point (vaporization)
• Structure

GASCHROMATOGRAPHY analysis in vapor phase
12 billion organic compounds 50 000
evaporative without destruction

• Evaporization depends on
• Molar mass
• polarity

Thermal stability
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GASCHROMATOGRAPHY (GC)
Sample introduction to the mobile
phase gas/vapor

• Sample can be
• gas
• liquid vaporization
• solid dissolution in liquid

Pressure and flow regulators
Gas tank
Gas cleaner
Gaschromatograph (GC)
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GASCHROMATOGRAPH (GC)
Gas tank
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Eluent gas

• Depending on the type of detector
• H2
• Ar
• N2
• He

Name sign ppm
pure 2.5 99,5 5000
pure 3.0 99.9 1000
Very pure 3.5 99,95 500
Very pure 4.0 99,99 100
Very pure 4.5 99,995 50
Very pure 5.0 99,999 10
Very pure 6.0 99,9999 1
Ultra pure 7.0 99,99999 0,1
Reductor valve Type depends on the quality and
pressure of the gas
Flow-rates
Inside apparatus Pressure and flow controllers
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Sample introduction

1. Injection in a very short time
2. Vapor/gas phase
3. Mixible with eluent gas

volume 0,1 ?l-1 ml
Liquid vaporization 100-10000 X volume increase
Syringe For gas liquid sample
six-port valve
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FLASH INJECTOR

1. Samle introduction
2. Vaporization
3. Inlet to column

Packed columns greater diameter greater sample
volume Capillary columns small sample volume
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Flash injector
vaporization
Injection

1. Sample vaporization
2. Liquids 100 1000 X volume increase
3. Mixing with eluent

1. Stick needle into the septum
2. Push the syringe piston
3. Remove syringe

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Quick injection
Slow injection
Eluent gas moves the sample to the column.
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• SPLIT
• SPLITLESS
• ON-COLUMN
• PTV

Type of injectors
Split-injector
Carrier gas
Septum wash
split-gas
Split/splitless ratio determines amount of
sample moving to the column
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2001
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Splitless injector
Purge Off
Purge On
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On-column
PTV (Programmed Temperature Vaporizer)
Injection directly to the column
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Columns
Capillary
polyimid, 350 oC
quarz
d
Stationary phase
microbore d lt 150 ?m standard capillary 150 ?m
lt d lt 500 ?m widebore d gt 500 ?m
Adsorption mechanism PLOT (Porous Layer Open
Tubular)
Distribution mechanism WCOT Wall Coated
OT SCOT Support Coated OT
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Interaction between stationary phase and sample
Active side silanol groups

• tailing
• Non-symmetric peaks

desactivation sylil reagents
Quartz surface
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Stationary phases I.

• Thermal stability
• No bleeding
• Known chemical structure
• Chemical inertnees
• Low price

Adsorbents (GSC) porous, with large special suface
modified adszorbents Based on carbon or silicagel

• inorganic adsorbents
• silicagel
• aluminium-oxide
• zeolits (molekulasziták)

• Organic adsorbents
• active carbon
• polymers

Analytes Hydrocarbons with small molar mass, He,
Ne, Ar, Kr, Xe
(PLOT)
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Stationary phases II. (GLC)
(absorption dissolution of gas and liquids in
liquids)
Polymers WCOT polymers on the surface of
capillary)
Relative small number 12-15
substituted polysiloxans (silicons) long lifetime
R substituents on polysiloxans
Thermal stability up 250-300 ?C
Methyl -CH3
Phenyl

• Substituents
• Methyl
• phenyl
• Cianopropyl
• Trifluoropropyl

Cianopropyl -CH2CH2CH2CN
Trifluoropropyl -CH2CH2CF3
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• methyl-phenyl
• cianopropyl-phenyl
• etc.

substitution how much of Si atoms
100 metil 5 fenil 95 metil
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Polyethyleneglycols (PEG)
Special separation

• Disadvantage
• Lower thermal stability
• oxygen-sensitivity

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

• Polar phases
• cyanopropyl
• PEG

• Midium polar phases
• 35 phenyl
• 50 phenyl

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

• Type of working
• Izotherm
• Programmable heating

T (oC)
thermostate

• Large temperature range -50 400 ?C
• Programmable heating 0- 40 oC/min
• cooling

t (min)
Decrease of analysis time Good peak shape
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Detectors
Quantitative analysis signal of detector is
proportional with concentration of analytes in
detector
universal signal for every compounds selective
signal for a groups of compounds specific
signal for special compounds
destruktiv non destruktiv
Dinamic range change of concentration results a
change in signal linearity T mc (deviation lt 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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Detectors
Thermal Conductivity Detector (katharometer)
Change of impedance
Wheatstone-bridge
W-filaments 100-200 mA heating current
Carrier gas H2, He N2
dinamic range 105 LOD 5-50 ng
non destructív universal
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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
destructív
Dinamic range 105-106 LOD 0,05-0,5 ng
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High Performance Liquid Chromatography (HPLC )
Mobile phase liquid Stationary phase adsorbent
(LSC) or liquid on a support(LLC)
Column Elution technique
Sample liquid
pump
Gas removal
eluent tank
injector
PC
detector
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HPLC
Gas removal
pump
automated injector
detector
(thermostate)
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Eluent

• Should (have) be
• Low viscosity
• inert no reaction with analytes
• Chemical stability
• No corrosion
• No toxycity
• Higher boiling point
• Low price
• Good quality and purity
• Compatible with detector
• UV-absortion low

purity HPLC grade Water and buffers too !!!
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Eluent
Analytes distributed between stationary and
mobile phase interaction of analytes with both
phases
Polarity of molecule mobile phase stationary
phase

• Change of polarity
• Change of quality of mobile phase
• Mixing of solvents

hexane chloroform tetrahidrofuran acetonitrile iso
propanol ethanol methanol water
Mixed solvents should be mixcible
Eluent strength determined on silicagel on the
bease of heat of adsorption of solvents
izoeluotrope mixture eluent strength is the
same k, Rs may change !!!
Izocratic elution fixed mobile phase
composition Gradient elution eluent strength is
increasing in time
Use of buffers adjusting of pH in the case of
analysis of ionisable components
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Pumps To carry of eluent

• Should be
• pressure (400 bar)
• Stable flow-rate
• Compatible with different solventsno corrosion
• Small hold-up volume
• No pulsation

Flow-rates in classical analytical HPLC 0,1-1,5
ml/min (0-5 ml/min)
Syringe-type pump
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Reciprocating pump
Pulsation double pistons (phase-deviation)
Volume 10-100 ?l Change of flow rate easy
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Gas removals Liquids contain dissolved gases

• Effect of gas bubbles
• In pump
• Pressure pulsation
• Different flow-rates
• Mechanical instability
• In detector
• Increased noise (retention time changes)

Remove of gas from the solvent

• Ultrasound
• cheap
• Non effective

• Vacuum
• Higher price
• effective

• He-purge
• Higher price
• effective

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Sample loading

1. Quick
2. 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 Phase RP
LC Reversed Phase
NPLC polarity of stationary phase gt polarity of
mobile phase RPLC polarity of stationary phase
lt polarity of mobile phase

• Material of column
• Stainless steel
• Glass
• PEEK (poly(ether-ether-ketone)

Packing regular spherical

• Size of column
• diameter 2-5 mm
• length 5-25 cm

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Modified silica gel
Modifying groups C18 octadecyl C18H37 C8
octyl C8H17 C4 buthyl C4H9 Amino
CH2CH2CH2NH2 Ciano CH2CH2CH2CN Phenyl C6H5
RPLC C18 stationary phase methanol/water
mobile phase NPLC silicagel stationary phase
hexane/alcohol mobile phase
Guard column avoid contamination of analytical
column
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Detectors
Quantitative analysis signal of detector is
proportional with concentration of analytes in
detector
universal signal for every compounds selective
signal for a groups of compounds specific
signal for special compounds
destruktiv non destruktiv
Dinamic range change of concentration results a
change in signal linearity T mc (deviation lt 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 spectrophotometer Application UV-Vis range
Lambeert-Beer A? e? c l
A lg I0/I
Cuvette quartz l5-10 mm
Light source UV deuterium lamp Vis wolfram lamp
Detector fotodiode
Most usable HPLC detector 190 nm lt ? lt 800 nm
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Dioda Array Detector (DAD)
polychromator
lence
Light source
cuvette
Diode array
Advantage Spectra and chromatogram at the same
time
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Paper and thin-layer chromatography
Planar arrangement
Stationary phase paper silica 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 solvent
• Place the plate to the closed container saturated
  with vapors of developing solvent
• Running of analytes based on capillary activity
• After 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 phaselike in Normal Phase
HPLC
Qualitative data retardation factor
(Rf) Quantitative data intensity of spots

• Advantages
• simple
• cheap

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