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