Liquid chromatography

Why LC

10% volatile enough for GC

non volatile use LC

Mobile phase affects separation

HPLC

Packed columns only

High pressure

Column efficiency

Increases as particle size decreases due to shorter diffusion distances

Smaller particles need higher pressure

Superficially porous particles

Non porous inner

Porous outer

Lower pressure required

25% less sample capacity

Frictional heating

Mobile phase heats up

Negative effects efficiency

UHPLC

Smaller particles

Higher pressure

Normal phase

Polar stationary phase- silica

Nonpolar mobile phase

Retention depends on polarity of mobile phase

More polar stronger mobile phase

Reverse phase

Nonpolar stationary phase

Polar mobile phase

Retention depends on polarity of mobile phase

Less polar stronger mobile phase

Hydrophilic Interaction LC

For small molecules too polar for reverse phase

Polar stationary phase

Polar mobile phase

Isocratic elution

Single solvent

1) Choose organic solvent

2) Change solvent polarity

3) Change stationary phase

4) Optimize column dimensions

Gradient elution

Solvent mixture

1) run wide gradient

2) eliminate before first peak and after last peak

3) reduce gradient time

DAD

UV absorption

Refractive index detector

1000x less sensitive

Beam of light is deflected

Cannot be used for gradient elution

ELSD

For low volatility analytes

Nebulized sample detected by light

Go from diode laser to photodiodes

Compatible with gradient elution

Charged aerosol detector

Nebulized

Measures charge of aerosol particles ionized from secondary gas

Electrochemical detector

For redox analytes

Measures current between working and auxiliary electrode

Fluorescence detector

Sensitive

For fluorescent compounds

Mass spectrometric detector

ESI, APCI

very sensitive

LC detectors

1) DAD

2) Refractive index

3) ELSD

4) Charged aerosol

5) Electrochemical

6) fluorescence

7) ms