Ch28 HPLC

Isocratic Runs

concentration of solvent stays same and peak width depends on diffusion processes only therefore peak width increases linearly w/ tR

Gradient Runs

peak width depends on diffusion processes and gradient focusing on column head b/c mobile phase constantly changing therefore pushing peak

Gradient- Initial band Focusing

solute trapped at top by weak mobile phase + strongly retained analyte, narrow band starting zone. run starts when solvent strength increases

Gradient- Band Compression

Solute band travels, back velocity stronger+faster b/c mobile phase constantly changing (w/ increasing solvent strength) while front velocity sees slightly weaker solvent therefore squeezes band keeping peaks narrow

Peak Capacity (P)

Number of peaks (n) that can be separated in given time w/ given resolution, depends on column length and particle size
P=1+tG/W
W=width avg.
tG=gradient pal

What is HPLC used for?

Nonvolatile chemical and biological compounds, organic compounds (polymers), proteins, thermally unstable compounds

Why is UV light used for quantitative determination of substances separated by HPLC?

Everything absorbs at some quantity in UV and is proportional to quantity of substance

Common HPLC Detectors

Spectroscopic, refractive index, fluorescent

UV Absorption Detection

UV beam directed @ flow cell passing thru sensor, compound may absorb beam therefore altering light energy falling on sensor

Spectroscopic detection MS

Ionizes elute and measures fragments, produces fingerprint of compound

Refractive Index Detection

Measure of molecule's ability to deflect light in mobile phase relative to a reference flow cell, universal detector, but not very sensitive, ONLY CAN BE USED FOR ISOCRATIC analysis

Fluorescence Detection

Offers higher sensitivity and selectivity, allows quantify and identifying compounds in complex matrices at extremely low conc., BUT LIMITED TO MOLECULES THAT FLUORESCE

End Capping

Fully reacting silica surface, removing silanols+their acidity, increasing coverage

Monomeric phases in silica are…

More reproducible and easier to control, but suffer at higher temperatures

Chiral Stationary Phases

Intxn btwn chiral analytes in normal phase to separate enantiomers