chromatographs eluent tank pump injector column detectorpc chromatogram qualitative &...
Post on 26-Dec-2015
215 Views
Preview:
TRANSCRIPT
Chromatographs
eluent tank pump injector column
detector PC CHROMATOGRAM
• Qualitative &
• Quantitative information
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
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
GASCHROMATOGRAPH (GC)
Gas tank
PC
Flow controller
column
injector detectorcleaner
Pressure controllerthermostate
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
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
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
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
Eluent gas moves the sample to the column.
solvent
Quick injection
Slow injection
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
200:1
5:1
Splitless injector
Purge Off
Purge On
On-column
Injection directly to the column
PTV(Programmed Temperature Vaporizer)
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
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
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)
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)
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
Polyethyleneglycols(PEG)
CH2HO O CH2 O Hn Disadvantage:
•Lower thermal stability•„oxygen-sensitivity”
Special separation
Carbowax
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
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
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
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
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
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
HPLC
pump
(thermostate)
Gas removal
detector
automated injector
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 !!!
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
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
Reciprocating pump
Volume: 10-100 lChange of flow rate: easy
Pulsation: double pistons (phase-deviation)
V
time
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:
Sample loading
1. Quick2. Sample should be mixable with eluent
Sample volume: 10-50 l
Micro syringe:
„Six-port” valve
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
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
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
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
Dioda Array Detector (DAD)
polychromator
Light source lencecuvette
Diode array
Advantage:Spectra and chromatogram at the same time
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
top related