Chromatography Flashcards
considered the gold standard for most testing due to its high sensitivity and specificity
GC coupled with MS
used to separate complex mixtures on the basis of different physical interactions between the individual compounds and the stationary phase of the system
Chromatography
through which the mobile phase flows
stationary Phase
carries the complex mixture
mobile phase
holding the stationary phase
column
separated components
Eluate
Modes of Separation
adsorption
partition
steric exclusion
ion-exchange chromatography
known as liquid–solid chromatography
adsorption
based on the competition between the sample and the mobile phase for adsorptive sites on the solid stationary phase.
adsorption
The molecules that are most soluble in the mobile phase move
fastest
The stationary phase can be
acidic polar (silica gel)
basic polar (alumina)
nonpolar (charcoal)
T/F:
Liquid–solid chromatography is not widely used in clinical laboratories because of technical problems with the preparation of a stationary phase that has homogeneous distribution of absorption sites.
true
referred to as liquid–liquid chromatography
partition
based on relative solubility in an organic (nonpolar) solvent and an aqueous (polar) solvent
partition
partition (extraction) is performed in
separatory funnel
Polar molecules remain in the
aqueous solvent
nonpolar molecules are extracted in the
organic solvent
Modern partition chromatography uses ______________ that are chemically bonded to the support or high-molecular-weight polymers
pseudolipid stationary phase
a liquid–solid chromatography used to separate
solute molecules on the basis of size and shape
steric exclusion
enter the pores in the packing and are momentarily trapped
small molecules
excluded from the small pores and so move quickly between the particles
large molecules
partially restricted from entering the pores
Intermediate-sized molecules
hydrophilic beads that formed a gel when soaked in water
dextran
polyacrylamide
agarose
method that use hydrophilic beads of crosslinked dextran, polyacrylamide or agarose that formed gel when soaked in water
gel filtration
process using hydrophobic gel beads of polystyrene with a nonaqueous mobile phase
gel permeation chromatography
solute mixtures are separated by virtue of the magnitude and charge of ionic species
ion-exchange chromatography
stationary phase consist of large polymers of substituted benzene, silicates, or cellulose derivatives
resin
resin is soluble in water? T/F
false
made with exchangeable hydroxyl ions such as the diethylamine functional group
Anion-exchange resins
used to remove interfering substances from a solution, to concentrate dilute ion solutions, and to separate mixtures of charged molecules, such as amino acids.
Ion-exchange chromatography
thin layer sorbent that is uniformly coated on a glass or plastic plate
alumina
silica gel
cellulose
crosslinked dextran
The distance a component migrates, compared with the distance the solvent front moves
retention factor
most commonly used as a semiquantitative screening test.
thin layer chromatography
used to measure the absorbance of each developed spot
densitometer
uses pressure for fast separations, controlled temperature, in-line detectors, and gradient elution techniques.
high-performance liquid chromatography
it forces the mobile phase through the column at a much greater velocity
pumps
most widely used pump today that is used as a multihead
pump with two or more reciprocating pistons.
mechanical reciprocating pump
pump used for preoperative purposes
pneumatic pump
pump that is no longer commonly used
hydraulic amplifier pump
The stationary phase is packed into long stainless steel _____
columns
The most common material used for column packing
silica gel
The best and most widely used method for sample injector
loop injector
it has high reproducibility and are used at high pressures
loop injector
monitor the eluate as it leaves the column
detectors
used for spectral comparisons and compound identification and purity
photodiode array
used for spectral comparisons and compound identification and purity
photodiode array
common HPLC detector that measures current produced
when the analyte of interest is either oxidized or reduced at some fixed potential set between a pair of electrodes
amperometric/ electrochemical detector
used to record detector signal versus the time the mobile phase passed through the instrument
recorder
used to identify compounds when compared with standard retention times run under identical conditions
retention time
proportional to concentration of the compounds that
produced the peaks.
peak area
