Chromatographs
eluent tank
detector
pump
PC
injector
column
CHROMATOGRAM
• Qualitative
&
• Quantitative information
GasChromatography (GC)
1952: A.T. James & A.J.P. Martin
•1956: van Deemter: kinetic theory
•M. Golay: capillary columns
High performancy
Qualitative & Quantitative information
Complicated samples
Separation
MOBILE PHASE: GAS
STATIONARY PHASE: solid or liquid on solid support (GSC, GLC)
COLUMN
ELUTION TECHNIQUE
Base of separation:
1. Boiling point (vaporization)
2. Structure
GASCHROMATOGRAPHY: analysis in vapor phase
~12 billion organic compounds
~ 50 000: evaporative without destruction
Evaporization depends on:
•Molar mass
•polarity
Thermal stability
GASCHROMATOGRAPHY (GC)
Sample introduction to the mobile phase:
gas/vapor
Sample can be:
1. gas
2. liquid: vaporization
3. solid: dissolution in liquid
Pressure and flow
regulators
Gas tank
Gas cleaner
Gaschromatograph (GC)
GASCHROMATOGRAPH (GC)
injector
detector
cleaner
PC
column
thermostate
Pressure controller
Flow controller
Gas tank
Eluent gas
Name
pure
Very pure
Ultra pure
sign
%
ppm
2.5
99,5
5000
3.0
99.9
1000
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
7.0
99,99999
0,1
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
Sample introduction
1. Injection in a very short time
2. Vapor/gas phase
3. Mixible with eluent gas
Syringe
For gas & liquid sample
volume
0,1 l-1 ml
Liquid vaporization:
100-10000 X volume
increase
„six-port” valve
FLASH INJECTOR
Septum (rubber)
1. Samle introduction
2. Vaporization
3. Inlet to column
Eluent gas inlet
Heating block
(25 – 300 oC)
liner (glass)
column
Packed columns: greater diameter: greater sample volume
Capillary columns: small sample volume
Flash injector
Injection
1. Stick needle into the septum
2. Push the syringe piston
3. Remove syringe
vaporization
1. Sample vaporization
2. Liquids: 100 – 1000 X volume increase
3. Mixing with eluent
Quick injection
solvent
Slow injection
Eluent gas moves the sample to the column.
Type of injectors
Split-injector
•SPLIT
•SPLITLESS
•ON-COLUMN
•PTV
Carrier gas
Septum wash
split-gas
Split/splitless ratio: determines amount of sample moving to the column
5:1
200:1
Splitless injector
Purge Off
Purge On
On-column
PTV
(Programmed Temperature Vaporizer)
Injection directly to the column
Columns
Capillary
polyimid, 350 oC
quarz
d
Stationary phase
microbore: d < 150 m
standard capillary: 150 m < d < 500 m
widebore: d > 500 m
Adsorption mechanism:
PLOT
(Porous Layer Open Tubular)
Distribution mechanism:
WCOT: Wall Coated OT
SCOT: Support Coated OT
Interaction: between stationary phase and sample
Active side: silanol groups
SiOH
SiOH
•„tailing”
•Non-symmetric peaks
SiOH
SiOH
SiOH
SiOH
Quartz surface
desactivation: sylil reagents
Si-O-Si(CH3)3
Si-O-Si(CH3)3
SiOH
Si-O-Si(CH3)3
Stationary phases I.
Thermal stability
No „bleeding”
Known chemical structure
Chemical inertnees
Low price
Adsorbents (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)
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
Si
R
R: substituents on polysiloxans
Thermal stability: up 250-300 C
R
O
Si
O
R
Substituents::
Methyl
phenyl
Cianopropyl
Trifluoropropyl
n
Methyl: -CH3
Phenyl:
Cianopropyl: -CH2CH2CH2CN
Trifluoropropyl: -CH2CH2CF3
O
CH 3
Si CH
3
O
Si
O
CH 3 Si CH
3
O
CH 3 Si CH 3
•methyl-phenyl
•cianopropyl-phenyl
•etc.
substitution: how much % of Si atoms
100 % metil
5 % fenil & 95 % metil
Polyethyleneglycols
(PEG)
HO
CH2
O
CH2
O
H
n
Carbowax
Special separation
Disadvantage:
•Lower thermal stability
•„oxygen-sensitivity”
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
Thermostate
Type of working:
•Izotherm
•Programmable heating
column
T (oC)
thermostate
•Large temperature range -50 – 400 C
•Programmable heating: 0- 40 oC/min
•„cooling”
Decrease of analysis time
Good peak shape
t (min)
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 < 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
Detectors
Thermal Conductivity Detector
(katharometer)
Change of impedance
Wheatstone-bridge
W-filaments: 100-200 mA heating current
non destructív
universal
dinamic range: 105
LOD: 5-50 ng
Carrier gas:
H2, He
N2
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
High Performance Liquid Chromatography (HPLC )
Mobile phase: liquid
Stationary phase: adsorbent (LSC) or
liquid on a support(LLC)
Column
Elution technique
Sample:
liquid
eluent tank
Gas removal
column
thermostate
pump
detector
injector
PC
HPLC
Gas removal
pump
automated injector
detector
(thermostate)
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 !!!
Eluent
Analytes 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
P
O
L
A
R
I
T
Y
Change of polarity:
•Change of quality of mobile phase
•Mixing of solvents
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
Pumps
To carry of eluent
Should be:
•pressure (400 bar)
•Stable flow-rate
•Compatible with different solvents:no 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
Reciprocating pump
Pulsation: double pistons (phase-deviation)
Volume: 10-100 l
Change of flow rate: easy
V
time
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
Sample loading
1. Quick
2. Sample should be mixable with eluent
Sample volume: 10-50 l
Micro syringe:
„Six-port” valve
Columns
Function: separation
Liquid chromatography:
NP LC: Normal Phase
RP 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:
regular
spherical
Modified silica gel
OH
OH
SiO2
OH
OH
OH
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
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 < 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
UV-Vis spectrophotometer
Application: UV-Vis range
Lambeert-Beer:
A = ε c l
Measuring side
splitter
cuvette
rés
fényforrás
I0
I
I0
I0
Reference side
monocromator
Light source:
UV: deuterium lamp
Vis: wolfram lamp
Detector:
fotodiode
Most usable HPLC detector
190 nm <  < 800 nm
D
E
T
E
C
T
O
R
Cuvette:
quartz
l=5-10 mm
A = lg I0/I
Dioda Array Detector (DAD)
polychromator
Light source
lence
cuvette
Diode array
Advantage:
Spectra and chromatogram at the same time
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 phase:like in Normal Phase HPLC
Qualitative data: retardation factor (Rf)
Quantitative data: intensity of spots
Advantages:
•simple
•cheap