05: separations and liquid chromatography

chromatography

technique of carrying out many separation steps for the separation of compounds from a mixture

partition chromatography

solute dissolved in liquid phase coated on surface of solid support

adsorption chromatography

solute adsorbed on surface of stationary phase

UV shadowing

based on the compound absorbing UV light, which prevents excitation of a phosphor that has been mixed in with the silica

stains

change color based on reaction with the compounds; for example, oxidation (permanganate), acid-base reactions (bromocresol green), complexation (iron chloride), and reactions that form new covalent bonds with a noncomitant color change

HPLC pump

forces mobile phase through the column at high pressure

injector

introduces a reproducible volume of sample onto the column

column

effects the separation of analytes

detector

produces a measurable signal when analytes elute from the column

inlet and inline solvent filter

removes particulate matter and air bubbles from the mobile phase

pre-column filter

removes particulate matter introduced with the sample

guard column

removes particulate matter and chemical components that would irreversibly bind to the analytical column

back pressure regulator

prevents bubbles from forming in the detector cell as the mobile phase exits the column

HPLC column

stainless steel column packed under very high pressure with the stationary phase

analytical column

• used for trace analysis and detection

• diameters between 1-10 mm and lengths on the order of 10-100 mm

• particle sizes < 10 μm

preparative and semi-preparative columns

• used for purification of synthesized compounds (laboratory scale)

• diameters and particle sizes are typically about an order of magnitude larger

diode array

permits the acquisition of full spectra as analytes elute from the column (or acquisition of several wavelengths in parallel)

fluorescence detector

very sensitive but often requires derivatization of non-fluorescent analytes

refractive index detector

universal detector, but not very sensitive

evaporative light scattering detector

useful with almost all non-volatile analytes

charged aerosol detector

useful with almost all analytes

electrochemical detector

voltametric measurements on eluent

HPLC-mass spectrometry

both sensitive and powerful, providing information about molecular structure and identity while also permitting quantitative analysis

dead time (t_m)

time required for the mobile phase to travel the length of the column

retention time (t_r)

time required for the analyte to elute from the column

retention factor

measure of the relative amount of time an analyte spends in the stationary phase

resolution

degree to which two peaks are separated in a chromatogram

theoretical plate

conceptual representation of a separation step

isocratic elution

same mobile phase composition is used throughout the separation

gradient elution

mobile phase composition is gradually changed during the separation