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Ch 8 HPLC
Analytic and Quantitative Toxicology
26
Other
Undergraduate 4
12/10/2011
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Term
Advantages of HPLC
 Definition
Rapid & effiicent seperattions
-post column reactionability
can interface with many detectors
portable equipment
easy to automate
robust packings
Term
Disadvantages of HPLC
 Definition
expensive hardware, need expierienced operator
Efficieny influencesby many outside factors
always a possibility of co-elution
Term
Isocratic
 Definition
manner of carrying out HPLC in which the eluent composition is constant
Term
HPLC Column
 Definition
oBack-pressures in HPLC’s generally run up to 4,000PSI – so materials that can withstand such pressures are required as stationary phases
oTypically, stationary phase is silica with particle size ranging from 3-10uM
oAlso to withstand such pressures, the columns are usually constructed of stainless teel with porous frits at each end
oAlso available are columns enclose with either stainless steel or aluminum that are also lined with polyetheretherketone (PEEK) or glass
-Glass is more inert than stainless steel so less reactivity with materials inside column BUT cannot withstand very high pressure
oShorter in length compared to GC columns (<1m in length)
oTypically HPLCs are run at ambient temperatures
oHowever, it’s known that retention time can vary 1-2% with each 1C change in column temperature
oSince ambient temperatures within laboratories can vary 3-5C over a work period such changes would introduce unacceptable error into the analysis
Term
Eluent
 Definition
oPure water is extremely important!!
Reverse osmosis may give water suitable for isocratic separations
Deionized water is usually suitable for gradient separation
oEluent preparations must be reproducible if consistent separation and retention times are to be achieved solvent volumes
Should be measured carefully with appropriate apparatus and all liquids are carefully mixed and cooled if necessary
oEluent pH must be controlled
Using buffer of known pH
Adjusting the pH of the eluent just prior to making up to final volume
Adding known volume of an acid or base to a set volume solvent
oRemoval of particles from the eluent before use by filtration through a 0.45uM membrane
oDegassing the eluent to previous bubble formation into the tubing or column
Term
pumping
 Definition
oThe pump must deliver the eluent at a constant and reproducible flow rate independent of column back pressure
oGenerally, reciprocating action pumps are used with sapphire pistons
oThe pump is fitted with inlet and outlet check valves to maintain flow in one direction
Term
oPressure Fluctuations
 Definition
Reciprocating pumps will generally cause small but significant fluctuations in pressure that may disturb detector baseline stability
Most pumps have a method of reducing most of the pulsations:
•Coils of capillary tubing may be inserted between the pump and the injection system to minimize pulsation
•Sacrificial guard column with have a sim. effect
•A PTFE membrane with a reservoir of heptane may also be used to dampen pulsations
•Dual piston pumps working in either series or parallel will also dampen pulsations
Term
internal loop injector
 Definition
•Designed to deliver sample volumes of less than 1L.
•Use small hole or narrow space in a disk as a volume measure
Term
external loop injector
 Definition
•For larger volumes
•Use a narrow bore tubing for measure
Term
Bulk Property Detector
 Definition
•Measure a change in the physical property of the eluent (conductivity, light scattering) that’s effected or caused by the presence of the analyte
•Generally do not have sufficient sensitivity selectivity for use in analytical procedures
Term
Selective Detectors
 Definition
•Detectors that exploit chemical or physical properties of an analyte
•Useful for many forms of detection
Term
oUV Visible Absorption
 Definition
 Analyte must absorb in the 200-600nM range
 Instrument
• Either a low pressure mercury lamp as the light source
•Or a deuterium discharge lamp (200-400 nM) and tungsten lamp (400-700nM)
•Flow cell with volumes of 8uL or less
•Light path of 10mm – For microbore capillary HPLC cell volume is 0.1-0.3uL
Term
oFluorescent Detection
 Definition
Use mercury or xenon light source
Sensitivities of 10-100 fold increase compared to UV
Selectivity excellent for those analytes that fluorescence naturally
Analytes can be derivitized to enhance scope of detection
Detector can be double monochromator (highest sensitivity) double filter or a combination of monochromator (excitation) and filter (emission)
Can have a beam splitter to devert light to a reference cell
Term
Chemiluminescence Detection (CLD)
 Definition
potentially more sensitive than fluorescence because it does not require irradiation of the flow cell and hence there is no problem with light scattering and reduces signal-noise ratio
-simpler instrument
Term
Electrochemical detection
 Definition
Amperometer detection: a reactive detector in which the analytes are either oxidized or reduced at the surface of the detector
System has 3 electrodes:
•Working electrode – analytes are oxidized/reduced here
•Counter electrode – maintains the electric current
•Reference electrode – used as a reference
Term
oChemiluminescence Nitrogen Detection
 Definition
A gas-phase detector that exploits the reaction of nitric oxide and ozone to produce excited nitrogen dioxide with subsequent emission of light
Term
oEvaporative Light Scattering Detection (ELSD)
 Definition
•In the ELSD effluent from the HPLC column is converted into a fine mist by a nebulized (mist) and the solvent is evaporated to leave a stream of solute particles into which a beam of light is directed through
•An optical detector set at an angle to measure the scattering light
•Detection is not dependent on any spectral properties rather on the presences of solute particles and hence such a detector is often called a universal detector
Term
oCharged Aersol Detection
 Definition
 Eluent passed through nebulizer and the solvent evaporation at ambient temperature
The CAD particles are charge by collision with a stream of positively charge nitrogen gas
High mobility ions are removed by a negative charged low voltage trap
Remaining analyte molecules transfer their charge to a collector – transferred chage is proportional to the analyte mass
Term
oRadioactive Detection
 Definition
Scintillation produces light via a matrix ionization caused by emitted radiation
•Fluid that will fluoresce when radioactive material is placed in it
•# of bursts of light are calculated
Light is amplified via a photomultiplier tube used for either 3H- or 14C- labeled substances
•3H easy to use for labeling but can provide false readings due to fallen hydrogens but are safer and cheaper
•14C are most sensitive and have a better signal to noise ratio but more expensive
Other suitable radionuclides include 32P or 37S
•32P used for nucleic acids
•37S and nitrogen radionuclides are used for proteins
Usually use beta radiations since small amounts of it can be detected
DO NOT GLOW IN THE DARK --- photomultiplier used to measured low level of light
Term
Reversed Phase Chrom.
 Definition
Typically uses a non-polar stationary phase
•Often silica modified by the additions of octadecyl, octyl, ethyl, methyl, or phenulpropyl [phenyl] siliyl moieties
•the longer the carbon chains, the more non-polar the silica will become
Mobile phase (eluent) is a polar solvent
•Typically is methanol or acetonitrile modified by adding water to adjust retention and selectivity
•Also tetrahydrofuran (THF) is used in such mixtures
To separate compounds are not soluble in the above solvents and water, THF-methanol mixtures are used
Term
Normal Phase Chromatography
 Definition
Chromatography of a non-polar mobile phase and a polar stationary phase
•Analytes are retained by adsorption onto the polar silanols at the silica surface
Eluents typically are alkanes such as heptane or alcohol modified alkanes
Alkanes are very non-polar
•Alcohols make these substances more polar
Term
oIon-Exchange Chromatography
 Definition
A process where an ionized analyte is attracted by the electrostatic forces to species of opposite charge on the stationary phase
When the analyte has a positive charge and the stationary phase has a negative charge, the technique is called cation-exchange chromatography
When the charges are reveres, it is known as anion-exchange chromatography
The prime component of the eluent is the counter ion that has the role of eluting sample components from the column in a reasonable time
In cation-exchange chromatography, the presence of other (+)-charged ions in the eluent decreases analyte retention
Term
oIon-Pair Chromatography
 Definition
Analyte ion is paired with an ion of an opposite charge added to the eluent to give a neutral species (ion-pair) that can be separated by reversed-phase chromatography
Ion-pair chromatography can be used with a C18-modified silica column
Typical ion-pairings include alkane sulfonates and alkane quaternary ammonium compounds
Because analytes and counter ions are ionic and thus hydrophilic
•Typical eluent solvents are methanol, acetonitrile-aqueous buffer mixtures
Term
oSize-Exclusion Chromatography
 Definition
 Based on the effective shape and size of molecules in the solution
• If organic solvents are used = Gel-permeation chromatography
• If aqueous solvents are used = Gel-filtration chromatography
 Stationary phase are porous particles with closed controlled pore size
 There is no interaction between analyte and surface of the stationary phase
 Analytes that are much smaller than the pore size are able to freely diffuse in the internal volume of he packing and elute in a volume equal to the total volume of the column
• Can elute quickly
 Molecules of analytes that are larger than the pore size elute in the void volume of the column
• Space around the pores
 Molecules of intermediate size will be able to penetrate some of he pores and will elute in a volume between the internal volume and the void volume depending on their size
Term
oIon-Pair Chromatography
 Definition
 Analyte ion is paired with an ion of an opposite charge added to the eluent to give a neutral species (ion-pair) that can be separated by reversed-phase chromatography
 Ion-pair chromatography can be used with a C18-modified silica column
 Typical ion-pairings include alkane sulfonates and alkane quaternary ammonium compounds
 Because analytes and counter ions are ionic and thus hydrophilic
• Typical eluent solvents are methanol, acetonitrile-aqueous buffer mixtures
Term
oAffinity Chromatography
 Definition
 Based on specific interactions such as enzyme-substrate or Ab-Ag interactions
• Very selective
• Recognizes molecule by its shape
 Molecular recognition mechanism is the basis of HPLC separations
Examples is immunoabsorbent separation
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