27-11-2012, 05:03 PM
High Performance Liquid Chromatography
[attachment=41363]
Describing the 5 major HPLC components and their functions
Pump:
•The role of the pumpis to force a liquid (called the mobile phase) through the liquidchromatograph at a specific flow rate, expressed in milliliters per min (mL/min).
•Normal flow rates in HPLC are in the 1-to 2-mL/min range.
•Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-bar).
•During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).
Injector:
•The injectorserves to introduce the liquid sampleinto the flow stream of the mobile phase.
•Typical sample volumes are 5-to 20-microliters (μL).
•The injector must also be able to withstand the high pressures of the liquid system.
•An autosampleris the automatic version for when the user has many samples to analyze or when manual injection is not practical. 3.
Column:
•Considered the “heart of the chromatograph” the column’s stationary phase separates the sample componentsof interest using various physical and chemical parameters.
•The small particles inside the column are what cause the high backpressureat normal flow rates.
•The pump must push hard to move the mobile phasethrough the columnand this resistance causes a high pressurewithin the chromatograph.
Detector:
•The detectorcan see (detect) the individual molecules that come out (elute) from the column.
•A detector serves to measure the amount of those molecules so that the chemist can quantitatively analyze the sample components.
•The detector provides an output to a recorder or computer that results in the liquid chromatogram(i.e., the graph of the detector response).
Computer:
•Frequently called the data system, the computer not only controls all the modules of the HPLC instrument but it takes the signal from the detector and uses it to determine the time of elution (retention time) of the sample components (qualitative analysis) and the amount of sample (quantitative analysis).
HPLC Uses
HPLC is used for chemistry and biochemistry research analyzing complex
mixtures, purifying chemical compounds, developing processes for synthesizing
chemical compounds, isolating natural products, or predicting physical
properties. It is also used in quality control to ensure the purity of raw
materials, to control and improve process yields, to quantify assays of final
products, or to evaluate product stability and monitor degradation.
In addition, it is used for analyzing air and water pollutants, for
monitoring materials that may jeopardize occupational safety or health, and
for monitoring pesticide levels in the environment. Federal and state
regulatory agencies use HPLC to survey food and drug products, for identifying
confiscated narcotics or to check for adherence to label claims
How Does High Performance Liquid Chromatography Work?
The components of a basic high-performance liquid chromatography [HPLC] system are shown in the simple diagram in Figure E.
A reservoir holds the solvent [called the mobile phase, because it moves]. A high-pressure pump [solvent delivery system or solvent manager] is used to generate and meter a specified flow rate of mobile phase, typically milliliters per minute. An injector [sample manager or autosampler] is able to introduce [inject] the sample into the continuously flowing mobile phase stream that carries the sample into the HPLC column. The column contains the chromatographic packing material needed to effect the separation. This packing material is called the stationary phase because it is held in place by the column hardware. A detector is needed to see the separated compound bands as they elute from the HPLC column [most compounds have no color, so we cannot see them with our eyes]. The mobile phase exits the detector and can be sent to waste, or collected, as desired. When the mobile phase contains a separated compound band, HPLC provides the ability to collect this fraction of the eluate containing that purified compound for further study. This is called preparative chromatography [discussed in the section on HPLC Scale].
HPLC Operation
A simple way to understand how we achieve the separation of the compounds contained in a sample is to view the diagram in Figure G.
Mobile phase enters the column from the left, passes through the particle bed, and exits at the right. Flow direction is represented by green arrows. First, consider the top image; it represents the column at time zero [the moment of injection], when the sample enters the column and begins to form a band. The sample shown here, a mixture of yellow, red, and blue dyes, appears at the inlet of the column as a single black band. [In reality, this sample could be anything that can be dissolved in a solvent; typically the compounds would be colorless and the column wall opaque, so we would need a detector to see the separated compounds as they elute.]
[attachment=41363]
Describing the 5 major HPLC components and their functions
Pump:
•The role of the pumpis to force a liquid (called the mobile phase) through the liquidchromatograph at a specific flow rate, expressed in milliliters per min (mL/min).
•Normal flow rates in HPLC are in the 1-to 2-mL/min range.
•Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-bar).
•During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).
Injector:
•The injectorserves to introduce the liquid sampleinto the flow stream of the mobile phase.
•Typical sample volumes are 5-to 20-microliters (μL).
•The injector must also be able to withstand the high pressures of the liquid system.
•An autosampleris the automatic version for when the user has many samples to analyze or when manual injection is not practical. 3.
Column:
•Considered the “heart of the chromatograph” the column’s stationary phase separates the sample componentsof interest using various physical and chemical parameters.
•The small particles inside the column are what cause the high backpressureat normal flow rates.
•The pump must push hard to move the mobile phasethrough the columnand this resistance causes a high pressurewithin the chromatograph.
Detector:
•The detectorcan see (detect) the individual molecules that come out (elute) from the column.
•A detector serves to measure the amount of those molecules so that the chemist can quantitatively analyze the sample components.
•The detector provides an output to a recorder or computer that results in the liquid chromatogram(i.e., the graph of the detector response).
Computer:
•Frequently called the data system, the computer not only controls all the modules of the HPLC instrument but it takes the signal from the detector and uses it to determine the time of elution (retention time) of the sample components (qualitative analysis) and the amount of sample (quantitative analysis).
HPLC Uses
HPLC is used for chemistry and biochemistry research analyzing complex
mixtures, purifying chemical compounds, developing processes for synthesizing
chemical compounds, isolating natural products, or predicting physical
properties. It is also used in quality control to ensure the purity of raw
materials, to control and improve process yields, to quantify assays of final
products, or to evaluate product stability and monitor degradation.
In addition, it is used for analyzing air and water pollutants, for
monitoring materials that may jeopardize occupational safety or health, and
for monitoring pesticide levels in the environment. Federal and state
regulatory agencies use HPLC to survey food and drug products, for identifying
confiscated narcotics or to check for adherence to label claims
How Does High Performance Liquid Chromatography Work?
The components of a basic high-performance liquid chromatography [HPLC] system are shown in the simple diagram in Figure E.
A reservoir holds the solvent [called the mobile phase, because it moves]. A high-pressure pump [solvent delivery system or solvent manager] is used to generate and meter a specified flow rate of mobile phase, typically milliliters per minute. An injector [sample manager or autosampler] is able to introduce [inject] the sample into the continuously flowing mobile phase stream that carries the sample into the HPLC column. The column contains the chromatographic packing material needed to effect the separation. This packing material is called the stationary phase because it is held in place by the column hardware. A detector is needed to see the separated compound bands as they elute from the HPLC column [most compounds have no color, so we cannot see them with our eyes]. The mobile phase exits the detector and can be sent to waste, or collected, as desired. When the mobile phase contains a separated compound band, HPLC provides the ability to collect this fraction of the eluate containing that purified compound for further study. This is called preparative chromatography [discussed in the section on HPLC Scale].
HPLC Operation
A simple way to understand how we achieve the separation of the compounds contained in a sample is to view the diagram in Figure G.
Mobile phase enters the column from the left, passes through the particle bed, and exits at the right. Flow direction is represented by green arrows. First, consider the top image; it represents the column at time zero [the moment of injection], when the sample enters the column and begins to form a band. The sample shown here, a mixture of yellow, red, and blue dyes, appears at the inlet of the column as a single black band. [In reality, this sample could be anything that can be dissolved in a solvent; typically the compounds would be colorless and the column wall opaque, so we would need a detector to see the separated compounds as they elute.]