High-Performance Liquid Chromatography Flashcards
Uses pressure for fast separations, controlled temperature, in-line detectors, and gradient elution techniques.
High-Performance Liquid Chromatography
Basic components of High-Performance Liquid Chromatography
- Pumps
- Columns
- Sample Injectors
- Detectors
- Recorders
Forces the mobile phase through the column at a much greater velocity than that accomplished by gravity flow columns and includes pneumatic, syringe, reciprocating, or hydraulic amplifier pumps
Pumps
widely used pump which uses a multi-head pump with two or more reciprocating pistons
Mechanical Reciprocating pump
are used for preoperative purposes, hydraulic amplifier pumps are no longer commonly used
Pneumatic pumps
o Can be put in an oven and heated to enhance the rate of partition
o Fine, uniform column packing results in much less band broadening but requires pressure to force the mobile phase through.
Columns
In HPLC Columns, what phase when packed into long stainless steel columns?
Stationary phase
Most common material used for column packing is _____ ___. It is very stable and can be used in different ways. It can be used as solid packing in liquid– solid chromatography or coated with a solvent, which serves as the stationary phase
silica gel
is now popular; the stationary phase is nonpolar molecules (e.g., octadecyl C-18 hydrocarbon) bonded to silica gel particles
Reversed-phase HPLC
can be used to separate ionic, nonionic, and ionizable samples
Reversed-phase column
used to produce the desired ionic characteristics and pH for separation of the analyte
buffer
small syringe can be used to introduce the sample into the path of the mobile phase that carries it into the column
Sample Injectors
best and most widely used method. Have high reproducibility and are used at high pressures
Loop injector
Monitor the eluate as it leaves the column and, ideally, produce an electronic signal proportional to the concentration of each separated component
Detectors
detect absorbances of visible or UV light are most commonly used
Spectrophotometers
also used for spectral comparisons and compound identification and purity. These detectors have been used for drug analyses in urine
Photodiode array (PDA) and other rapid scanning detectors
o Unlike gas chromatography/MS, which requires volatilization of targeted compounds, liquid chromatography (LC)/PDA enables _______ injection of aqueous urine samples
direct
Another common HPLC detector, which measures current produced when the analyte of interest is either oxidized or reduced at some fixed potential set between a pair of electrodes.
amperometric or electrochemical detector
Used to record detector signal versus the time the mobile phase passed through the instrument, starting from the time of sample injection
Recorders
In High-Performance Liquid Chromatography, the graph is called a
chromatogram
proportional to concentration of the com- pounds that produced the peaks
Peak area
When the elution strength of the mobile phase is constant throughout the separation, it is called
isocratic elution
o The late-eluting compounds may have long retention times, producing broad bands resulting in decreased sensitivity.
o an HPLC technique that can be used to overcome this problem.
Gradient elution
