April 19, 2023 1

Introduction to LC/GC Instrumentation

CM3007 Introduction to Separation Science

Analytical ChemistryLecture #1

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Milestones in Chromatography

1903 Tswett - plant pigments separated on chalk columns

1931 Lederer & Kuhn - LC of carotenoids 1938 TLC and ion exchange 1950 reverse phase LC 1954 Martin & Synge (Nobel Prize) 1959 Gel permeation 1965 instrumental LC (Waters)

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Basis of Chromatography

Definition:

Cs = Cm K

Mechanism - selective retardation caused by interactions with bonded phase of stationary phase

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Chromatogram

Detector signal vs. retention time or volume

time or volume

Det

ecto

r S

igna

l 1 2Presence of severalPeaks separated inTime/volume evidencesSeparation of components

Peak area Concentration

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Definitions

Mobile phase - phase that moves through chromatograph– In LC - eluent– In GC - carrier gas

Stationary phase - column; phase that is stationary in chromatograph

Bonded phase - reactive groups imparted to stationary phase in order to achieve selectivity

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Types of Chromatography

Classification by mobile phase:– Gas - Gas chromatography (GC)

» 1951 Martin and James (fatty acids)

– Liquid - Liquid chromatography (LC)» 1964 Horvath (Yale) instrument

» 1966 Horvath and Lipsky (nucleic acid components)

– Supercritical fluid - Supercritical fluid chromatography (SFC)

» 1958 Lovelock (Yale)

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Purpose of Chromatography

Identify components in mixtures

Isolate component in mixture

Demonstrate level of purity– Quantitation

» internal standard

analytical

preparative orsemi-preparative

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Instrumentation for LC

Strong/weak eluent Pump (up to 6000 psi) Injector Column Detector {Fraction Collector} Computer

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Instrumentation for GC

Carrier gas– N2, He, H2

Injector Column Detector Computer

oven

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Modes of LC Separation Adsorption or normal phase

Reverse phase

Hydrophobic interaction

Size exclusion

Ion Exchange

Affinity

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Modes of LC Separation

Adsorption or Normal phase– Stationary phase more polar than mobile phase

» column: hydroxyapatite (Ca10(PO4)6(OH)2

» mobile phase: hexane, CH2Cl2, THF, CH3OH

» gradient elution

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Modes of LC Separation (cont’d) Reverse phase (RPC)

– Stationary phase hydrophobic and mobile phase hydrophilic

» column: silica, polystyrene covalently modified with alkyl chain 3-18 C’s

» mobile phase: buffered water + organic solvent (propanol CH3CN, CH3OH)

» gradient elution

Affinity (AC) – Based on specific and selective interactions between

enzymes or antibodies and ligands

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Modes of LC Separation (cont’d)

Hydrophobic interaction (HIC)– Stationary phase hydrophobic and mobile

phase hydrophilic» stationary phase: low density of short alkyl chains

or phenyl

» mobile phase: decreasing concentration of high salt solution (3M NH4SO4)

» gradient elution

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Modes of LC Separation (cont’d) Size Exclusion (Gel Permeation) (SEC)

– Chemical interactions between sample and stationary phase undesirable; separation accomplished by sieving mechanism with porous stationary phase

» stationary phase: varies - silica

» mobile phase: low ionic strength (100 mM) buffer or dilute acid (0.1% TFA) with CH3CN or isopropanol (20-50%)

» isocratic elution

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Modes of LC Separation (cont’d)

Ion-Exchange (IEC)– Ion exchange interactions between cationic or

anionic analyte and stationary phase bearing opposite charge

» stationary phase: polystryrene, silica modified with functional groups such as quaternary amines

» mobile phase: buffer containing increasing concentration of salt (NaCl, MgCl2, K3PO4, NH4SO4)

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Ion Exchange Strong - charge of strong ion exchanger is

essentially independent of pH– sulfonic acids (cation)– quaternary amines (anion)

Weak - charge of weak ion exchanger is pH dependent– carboxymethyl (CM) (cation)– primary, secondary, and tertiary amines, e.g.,

diethylaminoethyl (DEAE) (anion)

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Anion Exchange - Proteins

Anion exchange - protein is acidicbonded phase eitherweak - DEAEstrong - quaternary amine

N+(CH2CH3)2

N+(CH2CH3)2

N+(CH2CH3)2

N+(CH2CH3)2

N+(CH2CH3)2

Resin

Protein

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Cation Exchange - Proteins

Cation exchange - protein is basicbonded phase eitherweak - CMstrong - sulfonic

Resin

Protein

COO-

COO-

COO-

COO-

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Elution Approaches

Isocratic - constant mobile phase composition

Gradient - variable mobile phase composition– step - change accomplished sharply at a defined

point in time– continuous - change accomplished gradually

over time

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Modes of GC Separation

Packed – earliest– Solid particles either porous or non-porous

coated with thin (1 m) film of liquid– 1 - 8 mm ID; 1 - 10 m length

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Modes of GC Separation

Capillary (open tubular) – modern– Inner wall modified with thin (1 m) film of liquid– 0.1 - 0.5 mm i.d.; 10 - 50 m length– types:– porous layer open tubular (PLOT)– wall-coated open tubular (WCOT)

» liquid coating

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GC Liquid Phase

Low volatility High bp Chemically unreactive Examples:

– 1-squalene– Tetrahydroxyethylenediamine– Carbowax (polyethylene glycol)

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Elution Analogies: GC vs. LC

Isothermal (GC) Isocratic (LC)

Programmed temperature (GC) Gradient (LC)– Raising column temperature (GC)

» Decreases retention time

» Sharpens peaks

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5 Properties of a Good Detector Sensitivity

Response/ C Selectivity

– Universal or selective response» selectivity - ability to distinguish between species

Rapid response Linearity - concentration range over which

signal proportional to concentration Stability with respect to noise (baseline

noise) and time (drift)

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Detectors for LC

UV-vis– PMT (single )– PDA (simultaneous multi- monitoring)

Fluorescence Electrochemical

– Amperometry NMR

– microcoil NMR: J. Sweedler (U. Illinois)

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Detectors for GC Electron capture (ECD)

– radioactive

– good for X-, NO2- and conjugated

Thermal conductivity (TCD)– change in resistance of heated wire

Flame ionization (FID)– destruction of combustible sample in flame produces

measurable current Fourier transform infrared (FTIR) Mass spectrometry (MS)

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MS Components

Ionization source

Analyzer– Differentiating characteristic m/z

Ion detector

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Ionization Methods - Examples

Electron capture (EC)– 70 eV e- + neutral molecule energetic molecular

ion

– hard; fragmentation Chemical ionization (CI)

– Reagent ion + molecule molecular ion + reagent ion

– Reagent ion = He, OH- (water), CH5+ or CH3

+ (CH4)

– soft; less fragmentation

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Ionization Methods - Examples (cont’d)

Electrospray (ESI)– generation of ions by desolvation or desorption

of charged liquid droplets Matrix Assisted Laser Desorption

(MALDI)– ionization facilitated by laser irradiation of

sample dissolved in an organic matrix– EX: sinapinic acid

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Types of MS Analyzers

Quadrupole - most common

Ion trap

Time of Flight (TOF)

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Two Operational Modes

Scan– Collect mass data over known range– Slow

Selective ion monitoring (SIM)– Sample mass at predetermined values– Fast

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Chromatogram

Retention Time

Det

ecto

r R

espo

nse

time of injectiontime of injection ttrr

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Chromatogram - GC-MS vs. LC (UV)

x-axis – GC-MS - m/z– LC - retention time or volume

y-axis - detector response– GC-MS - % abundance– LC - Abs