intro to chromatography

chromatography

method of separating the components of a complex mixture by differential movement through a two phase system

gas chromatography

separation technique with a gas mobile phase

only in a column

Where can gas chromatography be carried out?

in a column or plane

Where can liquid chromatography be carried out?

liquid chromatography

separation technique with a liquid mobile phase

the difference in the electronegativities of the two atoms making the bond

The polarity of a covalent bond can be judged by determining…?

stationary phase

the substance which is fixed in place for the chromatography procedure

mobile phase

the phase which moves in a definite direction

distribution constant (Kc)

the controlling factor in the partition equilibrium for a solute between the mobile and statioary phase

retention time

the characteristic time it takes for a particular analyte to pass through the system (inlet to detector) under a set of conditions

dead time (tM)

the time taken to pass through void volume of column; how long mobile phase not retained by stationary phase takes to get through the column

volatile and thermal stable

analytes for GC must be…?

carrier gas

gas in GC responsible for carrying the solute through the column

thermal conductivity and flame ionization detectors

Two most common detectors for GC

mobile phase supply system, pump and programmer, sample valve, column and thermostat, detector, data acquisition system

6 basic liquid chromatograph units

stationary phase is polar; mobile phase is nonpolar

normal phase chromatography

stationary phase is nonpolar; mobile phase is polar

reversed phase chromatography

nonpolar

For normal phase chromatography the most _____ compounds elute first.

polar

For reversed phase chromatography the most _____ compounds elute first

gel permeation chromatography

another name for size exclusion chromatography

size exclusion chromatography

separates molecules base on their size (no adsorption and very fast)

larger molecules

size of molecules that elute first in SEC

proteins

SEC is common when separating and purifying…

affinity chromatography

involves the use of packing which has been chemically modified by attaching a compound with a specific affinity for the desired molecules

affinity matrix

packing material used in affinity chromatography

inert and easily modified

two things that an affinity matrix must be

agarose

most common substance used in affinity matrix

mechanism of affinity chromatography

desired molecules adsorb to the ligands on the matrix until a solution of high salt concentration is passed through the column causing the desorption of the molecules from the ligands and their elution from the column

cation exchange chromatography

stationary phase carries a negative charge (affinity for positively charged molecules)

anion exchange chromatography

stationary phase carries a positive charge (affinity for negatively charged molecules)

mechanism of ion exchange chromatography

charged molecules in the liquid phase pass through a column until a binding site in the stationary phase appears; the molecules will not elute from the column until a solution of varying pH or ionic strength is passed through

supercritical fluid chromatography

intermediate chromatographic technique that utilizes extreme conditions of temperature and pressure in such a way that the mobile phase remains a supercritical fluid

a supercritical fluid

carrier in supercritical fluid chromatography

high solubilities, large densities, and large diffusivities and viscosities

compared to liquids, supercritical fluids have…

first

compounds with a greater affinity for the mobile phase will elute…

overloading the column

can cause a fronting peak shape

increased adsorption

can cause a tailing peak shape

co-elution

can cause a doublet peak shape

TF=b/a

equation for tailing factor

retention factor (k’)

time from injection to elution; often used to describe the migration rate of an analyte on a column

selectivity factor (alpha)

describes the separation of two species on the column (always greater than 1)

sharper peaks and higher resolution

greater number of theoretical plates gives…

sharper peaks and greater efficiency

lower height equivalent to a theoretical plate (HETP) value…

plate number = (retention time / standard deviation)²

equation for plate number

HETP = column length / plate number

equation for HETP

plate number, selectivity factor, and retention factor

three terms that must be maximized to obtain high resolution

reducing the size of the stationary phase particles

to increase the number of theoretical plates, the HETP can be reduced by…