principle of hplc:How Does High Performance Liquid Chromatography Work?/what is Detector,Chromatogram?

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].

Note that high-pressure tubing and fittings are used to interconnect the pump, injector, column, and detector components to form the conduit for the mobile phase, sample, and separated compound bands.

hplc system

Figure E: High-Performance Liquid Chromatography [HPLC] System

The detector is wired to the computer data station, the HPLC system component that records the electrical signal needed to generate the chromatogram on its display and to identify and quantitate the concentration of the sample constituents (see Figure F). Since sample compound characteristics can be very different, several types of detectors have been developed. For example, if a compound can absorb ultraviolet light, a UV-absorbance detector is used. If the compound fluoresces, a fluorescence detector is used. If the compound does not have either of these characteristics, a more universal type of detector is used, such as an evaporative-light-scattering detector [ELSD]. The most powerful approach is the use multiple detectors in series. For example, a UV and/or ELSD detector may be used in combination with a mass spectrometer [MS] to analyze the results of the chromatographic separation. This provides, from a single injection, more comprehensive information about an analyte. The practice of coupling a mass spectrometer to an HPLC system is called LC/MS.

HPLC Waters Alliance

Figure F: A Typical HPLC [Waters Alliance] System

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.]

After a few minutes [lower image], during which mobile phase flows continuously and steadily past the packing material particles, we can see that the individual dyes have moved in separate bands at different speeds. This is because there is a competition between the mobile phase and the stationary phase for attracting each of the dyes or analytes. Notice that the yellow dye band moves the fastest and is about to exit the column. The yellow dye likes [is attracted to] the mobile phase more than the other dyes. Therefore, it moves at a faster speed, closer to that of the mobile phase. The blue dye band likes the packing material more than the mobile phase. Its stronger attraction to the particles causes it to move significantly slower. In other words, it is the most retained compound in this sample mixture. The red dye band has an intermediate attraction for the mobile phase and therefore moves at an intermediate speed through the column. Since each dye band moves at different speed, we are able to separate it chromatographically.

chromatographic column bands

Figure G: Understanding How a Chromatographic Column Works – Bands

What Is a Detector? As the separated dye bands leave the column, they pass immediately into the detector. The detector contains a flow cell that sees [detects] each separated compound band against a background of mobile phase [see Figure H]. [In reality, solutions of many compounds at typical HPLC analytical concentrations are colorless.] An appropriate detector has the ability to sense the presence of a compound and send its corresponding electrical signal to a computer data station. A choice is made among many different types of detectors, depending upon the characteristics and concentrations of the compounds that need to be separated and analyzed, as discussed earlier.

What Is a Chromatogram? A chromatogram is a representation of the separation that has chemically [chromatographically] occurred in the HPLC system. A series of peaks rising from a baseline is drawn on a time axis. Each peak represents the detector response for a different compound. The chromatogram is plotted by the computer data station [see Figure H].

chromatogram separation representation

Figure H: How Peaks Are Created

In Figure H, the yellow band has completely passed through the detector flow cell; the electrical signal generated has been sent to the computer data station. The resulting chromatogram has begun to appear on screen. Note that the chromatogram begins when the sample was first injected and starts as a straight line set near the bottom of the screen. This is called the baseline; it represents pure mobile phase passing through the flow cell over time. As the yellow analyte band passes through the flow cell, a stronger signal is sent to the computer. The line curves, first upward, and then downward, in proportion to the concentration of the yellow dye in the sample band. This creates a peak in the chromatogram. After the yellow band passes completely out of the detector cell, the signal level returns to the baseline; the flow cell now has, once again, only pure mobile phase in it. Since the yellow band moves fastest, eluting first from the column, it is the first peak drawn.

A little while later, the red band reaches the flow cell. The signal rises up from the baseline as the red band first enters the cell, and the peak representing the red band begins to be drawn. In this diagram, the red band has not fully passed through the flow cell. The diagram shows what the red band and red peak would look like if we stopped the process at this moment. Since most of the red band has passed through the cell, most of the peak has been drawn, as shown by the solid line. If we could restart, the red band would completely pass through the flow cell and the red peak would be completed [dotted line]. The blue band, the most strongly retained, travels at the slowest rate and elutes after the red band. The dotted line shows you how the completed chromatogram would appear if we had let the run continue to its conclusion. It is interesting to note that the width of the blue peak will be the broadest because the width of the blue analyte band, while narrowest on the column, becomes the widest as it elutes from the column. This is because it moves more slowly through the chromatographic packing material bed and requires more time [and mobile phase volume] to be eluted completely. Since mobile phase is continuously flowing at a fixed rate, this means that the blue band widens and is more dilute. Since the detector responds in proportion to the concentration of the band, the blue peak is lower in height, but larger in width.

principle of hplc:High Performance Liquid Chromatography (HPLC)

High performance liquid chromatography (HPLC) has become a very versatile and powerful separation and analytical method over the years. It is an advanced form of liquid chromatography (LC). Instead of introducing the solvent into the column and allowing it to drip down under the influence of gravity, in HPLC the sample is forced through the column under high pressures of nearly 400 atm, resulting in faster and more efficient separation. This technique is also called high pressure liquid chromatography.

The Principle of HPLC

HPLC follows the same basic principle as chromatography. Different components in the sample have varying affinities to the adsorbent material. This causes a difference in the flow rate for each component which leads to their separation as they come out of the column. The only difference is that the speed and sensitivity of HPLC is much higher than that of LC due to the application of a high pressure. The magnitude of pressure applied depends on several factors such as the length and diameter of the column, flow rate, size of particles in the stationary phase, and mobile phase composition.

The Components of HPLC

Columns: HPLC columns are normally made of stainless steel and are 50 - 300mm long with an internal diameter of 2 - 5mm. They are filled with the adsorbents (stationary phase) of particle size 3 – 10µm. Sample Injector: The sample is injected into the column by an injector which is capable of handling sample volumes in the range of 0.1 - 100mL under high pressures of up to 4000psi. Reservoir: The solvent or the mobile phase is placed in a glass reservoir. It is usually a blend of polar and non-polar liquids whose concentrations depend on the sample composition. Pump: The solvent in the mobile phase is aspirated by a pump from the reservoir and forced through the HPLC column and then the detector. Detector: The detector in a HPLC system is located at the end of the column and it detects the components of the sample that elute from the column. Different types of detectors such as fluorescence, mass-spectrometric, UV-spectroscopic, and electrochemical detectors are used. Data collection systems: The signal from the detector is received by recorders which are used to process, store, and reproduce chromatographic data. The data is interpreted and integrated by a computer which produces a user-friendly chromatograph.

The Technique of HPLC

The key steps in the HPLC separation technique are as follows:
  • Injection of the liquid sample into the column containing the stationary phase.
  • Individual sample components are forced down the tube by high pressure from the pump.
  • Components are separated under the influence of various chemical/physical interactions with the particles in the stationary phase.
  • The separated analytes are identified by the detector present at the end of the column.
  • The detector measures the concentration of the components.
  • Data from the detector is processed and a chromatogram is produced.

The Applications of HPLC

HPLC is widely used in the following applications:
  • Qualitative analysis - Separation of thermally unstable chemical and biological compounds, e.g., drugs (aspirin and ibuprofen), salts (sodium chloride), proteins (egg white or blood), organic chemicals (polystyrene and polyethylene), herbal medicines and plant extracts.
  • Quantitative analysis - To determine the concentration of a compound in a sample by measuring the height and area of the chromatographic peak.
  • Preparation of pure substances for clinical and toxicology studies and in organic synthesis. This is also called preparative chromatography.
  • Trace analysis – this is the analysis of compounds present in very low concentrations in a sample. This is very useful in pharmaceutical, toxicology, environmental, and biological studies.

Principle of HPLC:High Performance Liquid Chromatography (HPLC) : Principle, Types, Instrumentation and Applications

Chromatography is a technique to separate mixtures of substances into their components on the basis of their molecular structure and molecular composition. This involves a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Sample components that display stronger interactions with the stationary phase will move more slowly through the column than components with weaker interactions. This difference in rates cause the separation of variuos components. Chromatographic separations can be carried out using a variety of stationary phases, including immobilized silica on glass plates (thin-layer chromatography), volatile gases (gas chromatography), paper (paper chromatography) and liquids (liquid chromatography).

High perfomance Liquid Chromatography

High performance liquid chromatography (HPLC) is basically a highly improved form of column liquid chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster. All chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation.

Types of HPLC

There are following variants of HPLC, depending upon the phase system (stationary) in the process :
  1. Normal Phase HPLC: This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase and non-polar mobile phase. Therefore, the stationary phase is usually silica and typical mobile phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these. Polar samples are thus retained on the polar surface of the column packing longer than less polar materials.
  2. Reverse Phase HPLC: The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. It works on the principle of hydrophobic interactions hence the more nonpolar the material is, the longer it will be retained.
  3. Size-exclusion HPLC: The column is filled with material having precisely controlled pore sizes, and the particles are separated according to its their molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later.
  4. Ion-Exchange HPLC: The stationary phase has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.

Instrumentation of HPLC

High-performance-liquid-chromatography-hplc As shown in the schematic diagram in Figure above, HPLC instrumentation includes a pump, injector, column, detector and integrator or acquisition and display system. The heart of the system is the column where separation occurs.
  1. Solvent Resorvoir : Mobile phase contents are contained in a glass resorvoir. The mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid components whose respective concentrations are varied depending on the composition of the sample.
  2. Pump : A pump aspirates the mobile phase from the solvent resorvoir and forces it through the system’s column and detecter. Depending on a number of factors including column dimensions, particle size of the stationary phase, the flow rate and composition of the mobile phase, operating pressures of up to 42000 kPa (about 6000 psi) can be generated.
  3. Sample Injector : The injector can be a single injection or an automated injection system. An injector for an HPLC system should provide injection of the liquid sample within the range of 0.1-100 mL of volume with high reproducibility and under high pressure (up to 4000 psi).
  4. Columns : Columns are usually made of polished stainless steel, are between 50 and 300 mm long and have an internal diameter of between 2 and 5 mm. They are commonly filled with a stationary phase with a particle size of 3–10 µm. Columns with internal diameters of less than 2 mm are often referred to as microbore columns. Ideally the temperature of the mobile phase and the column should be kept constant during an analysis.
  5. Detector : The HPLC detector, located at the end of the column detect the analytes as they elute from the chromatographic column. Commonly used detectors are UV-spectroscopy, fluorescence, mass-spectrometric and electrochemical detectors.
  6. Data Collection Devices : Signals from the detector may be collected on chart recorders or electronic integrators that vary in complexity and in their ability to process, store and reprocess chromatographic data. The computer integrates the response of the detector to each component and places it into a chromatograph that is easy to read and interpret.

Applications of HPLC

The information that can be obtained by HPLC includes resolution, identification and quantification of a compound. It also aids in chemical separation and purification. The other applications of HPLC include :
  • Pharmaceutical Applications 1. To control drug stability. 2. Tablet dissolution study of pharmaceutical dosages form. 3. Pharmaceutical quality control.
  • Environmental Applications 1. Detection of phenolic compounds in drinking water. 2. Bio-monitoring of pollutants.
  • Applications in Forensics 1. Quantification of drugs in biological samples. 2. Identification of steroids in blood, urine etc. 3. Forensic analysis of textile dyes. 4. Determination of cocaine and other drugs of abuse in blood, urine etc.
  • Food and Flavour 1. Measurement of Quality of soft drinks and water. 2. Sugar analysis in fruit juices. 3. Analysis of polycyclic compounds in vegetables. 4. Preservative analysis.
  • Applications in Clinical Tests 1. Urine analysis, antibiotics analysis in blood. 2. Analysis of bilirubin, biliverdin in hepatic disorders. 3. Detection of endogenous Neuropeptides in extracellular fluid of brain etc.

HPLC instrumentation and chromatography principle

HPLC instrumentation and chromatography principle This HPLC chromatography lecture explains the HPLC principle and instrumentation.