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- 2. Contents • What is chromatography? • HPLC ( high performance liquid chromatography) • Separation Modes of HPLC • Isoelectric Vs Gradient Elusion • The Stationary Phase • The mobile phase • Selecting a Stationary and mobile phase • The HPLC System • Main components of a HPLC system • Operating Procedure for HPLC • Temperature control in HPLC • Retention time • Application of HPLC • References
- 4. What is Chromatography? • Chromatography is a technique which separates components in a mixture due to the differing time taken for each component to travel through a stationary phase when carried through it by a mobile phase. • The stationary phase is fixed in place either in a column (a hollow tube made up of a suitable material, e.g. glass) or on a planar surface and the mobile phase moves over or through the stationary phase carrying with it the sample of interest.
- 5. How Separation is Achieved? • Figure shows how the separation is achieved. Mixture of component A and component B is introduced to the mobile phase. A and B are travelling at the same rate as the rate of flow of the mobile phase. • At time t1 they encounter the stationary phase. A has a slightly greater affinity for the stationary phase than B. This means that relative to B, A spends more time on the stationary phase and travels at a slower rate than B. • At time t2 A and B are beginning to separate. • At time t3 they are fully separated. • At time t4 there is further separation of A and B.
- 6. High Performance Liquid Chromatography (HPLC)
- 7. High Performance Liquid Chromatography (HPLC) • HPLC( high performance liquid chromatography) is a separation technique that involves: • Injection of a small volume of liquid sample into a tube packed with tiny particles, called the stationary phase where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure developed by a pump.
- 8. • These components are separated from one another by the column packing that involves various chemical and/ or physical interactions between their molecules and the packing particles. • These separated components are detected at the exit of this tube(column) by a flow-through device (detector) that measures their amount. An output from this detector is called a liquid chromatogram. • In principle, LC and HPLC work in the same way except the speed, efficiency , sensitivity and ease of operation of HPLC is vastly superior. HPLC is also known as high pressure liquid chromatography or high priced liquid chromatography.
- 9. Representation of High Performance Liquid Chromatography Injector Detector Column Solvents Mixer Pumps Waste Separation in based upon differential migration between the stationary and mobile phases. Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica Mobile Phase - carries the sample through the stationary phase as it moves through the column.
- 26. Separation Modes of HPLC • Key Point to Remember: • The correct selection of the column packing and the mobile phase are the most important factors in successful HPLC. • There are four major separation modes that are used to separate most compounds: 1. Partition or Reversed-phase chromatography 2. Adsorption or Normal-phase chromatography 3. Ion exchange chromatography 4. Size exclusion chromatography .Let's briefly look at each mode
- 27. Partition or Reversed-phase Chromatography • In partition or reversed-phase chromatography the stationary phase is non-polar organic species (e.g. C18, C8, etc) covalently bonded to the packing and the mobile phase is water (buffer) + water-miscible organic solvent (e.g. methanol, acetonitrile). • In a mixture of components to be separated those analytes which are relatively less polar will be retained by the non- polar stationary phase longer than those analytes which are relatively more polar. Therefore the most polar component will elute first. The attractive forces which exist are mainly non-specific hydrophobic interactions. The exact nature of these interactions is still under discussion.
- 28. • Partition or Reversed-phase Chromatography is the most widely used type of HPLC in pharmaceutical analysis. Over 90% of chromatographers use this mode. • The technique can be used for: o non-polar molecule o polar molecule o Ionizable molecules o and ionic molecules. • For samples containing a wide range of compounds, gradient elution is used: o One begins with a predominantly water-based mobile phase and then adds organic solvent as a function of time. o The organic solvent increases the solvent strength and elutes compounds that are very strongly retained on the RPC packing.
- 29. Adsorption or Normal-phase Chromatography • In adsorption or normal phase chromatography the stationary phase is polar (e.g. silica gel, cyanopropyl- bonded, amino-bonded, etc.) and the mobile phase is non-polar (e.g. hexane, iso- octane, methylene chloride, ethyl acetate). • In a mixture of components to be separated those analytes which are relatively more polar will be retained by the polar stationary phase longer than those analytes which are relatively less polar. Therefore the least polar component will elute first. The attractive forces which exist are mostly dipole-dipole and hydrogen bonding (polar) interactions.
- 30. • Adsorption or Normal phase chormatography is used by about 10% of the liquid chromatographers. • The technique is useful for: o water-sensitive compounds o geometric isomers o cis-trans isomers o class separations o and chiral compounds.
- 31. Ion-Exchange Chromatography • In ion exchange, the column packing contains ionic groups (e.g. sulfonic, tetraalkylammonium) and the mobile phase is an aqueous buffer (e.g. phosphate, formate, etc.). • Ion exchange chromatographyis used by about 20% of the liquid chromatographers • The technique is well suited for: o The separation of inorganic and organic anions and cations in aqueous solution. o Ionic dyes, amino acids, and proteins can be separated by ion exchange.
- 32. Size –Exclusion Chromatography • In size-exclusion chromatography the sample molecule do not interact with the stationary phase, the separation is based on the size of the sample molecules. • Lower molecular weight species enter the pores on the column packing and higher molecular weight species do not fit in these pores and thus are not retained. • A range of stationary phases in different pore size is available and the selection of an appropriate one for a given separation is based on the molecular weight of the sample.
- 33. • The SEC technique is used by 10-15% of chromatographers, mainly for polymer characterization and for proteins. • There are two modes: – non-aqueous SEC [sometimes termed Gel Permeation Chromatography (GPC)] and – aqueous SEC [sometimes referred to an Gel Filtration Chromatography (GFC)].
- 34. Isocratic Vs Gradient Elusion Isocratic Elusion: A separation in which the mobile phase composition remains constant throughout the procedure is termed as isocratic elusion. • Best for simple separations • Often used in quality control applications that support and are in close proximity to a manufacturing process. Gradient Elusion: A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution. • Best for the analysis of complex samples • Often used in method development for unknown mixtures.
- 37. The Stationary Phase • It is the combination of a suitable stationary phase and mobile phase that enables the separation of a mixture and thus the analysis of the components in the mixture. • HPLC is characterised by the use of very small particles of stationary phase which are fixed in place in a HPLC column, often made of a material such as stainless steel. A typical column is shown in Figure.
- 38. Parameters to Describe a HPLC Column 1. Packing/matrix 2. Bonded phase 3. Particle size 4. Pore size 5. Length 6. Diameter 7. Hardware 8. Manufacturer
- 39. 1-Packing/Matrix of the HPLC column • The finely divided material with which the column is packed is usually silica. It can be used as stationary phase in adsorption chromatography or a bonded phase is attached for use in partition chromatography. • Silica is the most common packing material used in HPLC columns . It is physically robust (durable, strong, long-lasting) and chemically stable in virtually all solvents and at low pH. • Silica consists of silicon atoms bridged three-dimensionally by oxgen atom. The free silanol (Si-OH) groups are most active and most of the chromatographic properties of the silica surfaces are related to interactions with these. • Other polar statioany phase packing include alumina and graphitised carbon.
- 40. 2-Bonded Phase • Reversed phase bonded phases These are non-polar groups, attached to the matrix surface by covalent bonding. The most common bonded phases are alkyl groups. C18 is the most popular, referred as octyldecylsilane (ODS). The silane will contain a group that can react with the silica surface (e.g Cl).
- 41. • Normal phase bonded phases The bonded phases that are used for normal phase chromatography are polar. Typical example are cyano (CN), amino (NH2) and diol bonded phases. • Bonded phases for size exclusion HPLC Silica based size-exclusion chromatography packings have chemicals bonded to the surface to deactivate the silica but these bonded phases do not interact with the analyte molecules since the separation is based only on size. • Chiral stationary phases A large number of drugs contain chiral centers and often only one enantiomer is pharmacologically active. Therefore an analytical technique which separate the enantiomer is required. To use of HPLC for this separation involves the use of a chiral stationary phase. A large number of different phases are available and the selection of a suitable one will depend on the analyte.
- 42. 3-Particle size • The development of finely divided stationary phase resulted in the development of HPLC as an analytical technique. The magnitude of these particles size is typically in the range 1.5 to 10 microns. • Most packings have a distribution of particle sizes, this is inherent in the manufacturing process. A narrow distribution gives better results and thus the high performance column tend to have narrow distribution of particle size. • Over the past few years technology required to both produce and utilize sub 2 micron particles has been commercialized. Reduction in particle size gives benefits in efficiency and faster analysis but also results in higher operating pressures, this means that instrumentation is required which can operate at ultra high pressure.
- 43. 4-Pore size • Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics, especially for larger analytes. For example, a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside. 5-Length • The length of the column influences the extent of the separation of the components in the mixture. • A longer column enables more separation. The time taken to perform the separation will increase as the length increases since it takes longer to travel through the column. • Also as the column length increases the backpressure experienced will also increases. • Another problem with increasing the column length is that the spread of the components of the mixture in the column will increases, this will result in difficulties during detection and quantification.
- 44. 6-Diameter The internal diameter of the column usually measured in mm. 7-Hardware The material used to construct the external tubing and end fitting of the column • Stainless steel (the most popular; gives high pressure capabilities) • Glass (mostly for biomolecules) • PEEK (Polyether ether ketone) polymer (biocompatible and chemically inert to most solvents) 8-Manufacturer The name of manufacturer of the column.
- 45. The Mobile Phase
- 46. The Mobile Phase • The mobile phase for HPLC is the liquid phase which is continually flowing through the stationary phase and which carries the analyte through with it. The composition of the mobile phase which is used is dependent on both the stationary phase and the nature of the compounds being analysed. • The different properties of solvents define whether they are suitable for use as a mobile phase either under reversed phase or normal phase conditions. • The most common solvents used for HPLC are listed below in order of increasing polarity: n-hexane, methylene chloride, chloroform, methyl-t-butyl ether, tetrahydrofuran (THF), isopropanol (IPA), acetonitrile (MeCN or ACN), methanol (MeOH), water.
- 47. • A blend of two (or more) of these solvents is used as the mobile phase in a HPLC analysis. The proportions of the different solvents in the blend act to adjust the polarity of the mobile phase. This is combined with a suitable stationary phase to achieve the separation of a mixture. Ideally, the components in the mixture will be separated fully and will all elute within a practical time scale. • By convention, chromatographers usually refer to the strong solvent in a mobile phase as the 'B' solvent and the weak solvent as the 'A' solvent. Generally, solvent strength is related to polarity, with non-polar solvents being 'strong' solvents for reversed phase HPLC and polar solvents being 'strong' for normal phase HPLC.
- 48. • A binary mixture is a mixture of two solvents and is the most common type of mobile phase. However ternary mixtures, where three solvents are blended, are also used. The choice of the solvents in the mobile phase, and the proportions of each, will be selected during method development. • The most important property of the solvent is its ability to interact with both the stationary phase and the analytes in the mixture, resulting in the desired separation. However, there are other important properties that need to be considered. An ideal solvent will be readily available in high purity, relatively inexpensive, safe to use routinely, and compatible with the entire HPLC system including the detector.
- 49. Mobile Phase Preparation 1-Quality All reagents and solvents should be of the highest quality it should contain no impurities. De-ionized water often contains trace levels of organic compounds and therefore it is not recommended for HPLC use. Ultra pure water is generated by passing de-ionized water through an ion exchange bed. 2-Buffer All buffers should be prepared freshly. Buffer reagents can contain a stabilizing agent. They may affect the chromatographic behavior of the buffer solution so reagents that are free of stabilizer are preferred. 3-Filtration All HPLC solvents should be filtered through a 0.45 micrometer filter before use. This removes any particulate matter that may cause blockage. After filtration, the solvents should be stored in a covered reservoir to prevent contamination with dust.
- 50. 4-Degassing Before the freshly prepared mobile phase is pumped around the HPLC system , it should be thoroughly degassed to remove all dissolved gasses. Dissolved gas can be removed from solution by: • bubbling with Helium • sonication • vaccum filtration If the mobile phase is not degassed, air bubbles can form high pressure in the system resulting in system instability and spurious baseline. The most efficient form of degassing is bubbling with helium or another low solubility gas.
- 51. Selecting a Stationary and Mobile Phase
- 52. Selecting a Stationary and mobile phase • The selection of a suitable column and the mobile phase for an analysis of a mixture of components is very difficult. • This decision is made during HPLC method development, which is the process of selecting a suitable column and mobile phase for a given separation. • The best choice will depend on both the nature of the analyte and the aim of the analysis.
- 53. The HPLC System
- 54. The HPLC System Instrumentation is required to enable the flow of the mobile phase through the stationary phase and also to convert the separated components into meaningful information. A typical configuration of a HPLC system is shown in Figure:
- 55. Main components of a HPLC system 1. Pump The role of the pump is to force mobile phase through the liquid chromatograph 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 elusion) or an increasing mobile phase composition (gradient elusion). 2. Injector The injector serves to introduce the liquid sample into 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 autosampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical.
- 56. 3. Column Considered the "heart of the HPLC" the column's stationary phase separates the sample components of interest using various physical and chemical parameters. The small particles inside the column are what cause the high backpressure at normal flow rates. The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatograph. 4. Detector There are many detection principles used to detect the compounds eluting from an HPLC column. The most common are: Spectroscopic Detection Refractive Index Detection Fluorescence Detection
- 57. Spectroscopic Detection i-Ultraviolet (UV) Absorption • An ultraviolet light beam is directed through a flow cell and a sensor measures the light passing through the cell. • If a compound elutes from the column that absorbs this light energy, it will change the amount of light energy falling on the sensor. • The resulting change in this electrical signal is amplified and directed to a recorder or data system. • An UV spectrum is sometimes also obtained which may aid in the identification of a compound or series of compounds.
- 59. ii-Mass Spectroscopy (MS) • A MS detector senses a compound eluting from the HPLC column first by ionizing it then by measuring it's mass and/or fragmenting the molecule into smaller pieces that are unique to the compound. • The MS detector can sometimes identify the compound directly since its mass spectrum is like a fingerprint and is quite unique to that compound.
- 60. Refractive Index (RI) Detection • The ability of a compound or solvent to deflect light provides a way to detect it. • The RI is a measure of molecule's ability to deflect light in a flowing mobile phase in a flow cell relative to a static mobile phase contained in a reference flow cell. • The amount of deflection is proportional to concentration. • The RI detector is considered to be a universal detector but it is not very sensitive.
- 62. Fluorescence Detection • Compared to UV-Vis detectors fluorescence detectors offer a higher sensitivity and selectivity that allows to quantify and identify compounds and impurities in complex matrices at extremely low concentration levels (trace level analysis). • Fluorescence detectors sense only those substances that fluoresce
- 64. 5.Computer The computer 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).
- 66. Operating Procedure for HPLC Start-up 1. Switch on the computer. 2. Switch on the detector and HPLC pump. 3. Double click on the HPLC software icon on the desktop. 4. Check there is sufficient elution solvents. Please top-up if there is insufficient. Use only HPLC grade solvents to prevent clogging of the system. 5. If HPLC has not been in use for a while, air bubbles in the system are needed to purge out. Turn the knob on HPLC a quarter and press Purge. The purging process will take 10 minutes. 6. Once the purging is finished, turn the knob back. 7. Attach the column in the correct flow direction. 8. Open the required method file or build a new method file. Ensure that the wavelength, flow rate, elution time and elution proportion are correct. 9. Monitor the baseline by clicking the Baseline icon in the program. Check tubing connection, ensure that there is no leakage. 10. Once the baseline is flat, the system is ready for sample injection.
- 67. Sample preparation and injection 1. Prepare the sample in the appropriate solvent. Ensure there is no particulate by filtering the sample solution through a membrane. 2. Wash the syringe thoroughly with the appropriate solvent (at least three times). 3. Wash the syringe with the sample solution (at least three times). 4. Draw up the sample. Ensure there is no bubble in the syringe. 5. Click the Single Run icon once ready. 6. Insert the syringe and lift up the injection valve anticlockwise (LOAD position). 7. Inject the sample and turn down the injection valve clockwise (INJECT position). The elution begins. 8. Withdraw the syringe and wash it with the appropriate solvent (at least three times). After the run is finished, flush the system with the appropriate solvent for at least 10 minutes.
- 68. Temperature control in HPLC Reasons for temperature control : Reproducibility • Retention in HPLC is temperature dependent • If temperature varies , then it is difficult to assign peaks to specific compounds in chromatogram and the peak areas/heights may vary. Solubility • Certain chemical compounds may have low solubility in the HPLC mobile phase • If they are injected into the flow stream they may precipitate or other difficulties may arise. Stability • Certain chemical compounds especially biological compounds such as enzymes or proteins may not be stable at room temperature or higher. The temperature needs to be much lower down to 4 ͦc.
- 69. Retention time • The time taken for a particular compound to travel through the column to the detector is known as its retention time. • This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound. • Different compounds have different retention time. For a particular compound, the retention time will vary depending on: 1. The pressure used (because that affects the flow rate of the solvent) 2. The nature of the stationary phase (not only what material it is made of, but also particle size) 3. The exact composition of the solvent 4. The temperature of the column.
- 71. 1-Separation and analysis of non-volatile or thermally-unstable compounds • HPLC is optimum for the separation of chemical and biological compounds that are non-volatile Typical non-volatile compounds are – Pharmaceuticals like aspirin, ibuprofen, or acetaminophen – Salts like sodium chloride and potassium phosphate – Organic chemicals like polymers – Many natural products such as ginseng, herbal medicines, plant extracts – Thermally unstable compounds such as enzyme • If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in gasoline, etc.), gas chromatography is a better separation technique.
- 72. 2-Qualitative analysis • The aim of qualitative analysis is to answer the question 'What' is in the sample? • Two discrete situations exist for qualitative analysis: – the sample components are known and peaks within the chromatogram need to be assigned to the known components – the sample is a completely unknown which you are attempting to characterize • In the former case, it might be possible to inject standards of the pure compound, and assign the peaks in the chromatogram based on the retention time of the standard. Having a selective detector, such as diode-array UV or Fluorescence detector, which assists in identification by producing spectra or a specific response, can assist in peak assignment. • In the second case, it may be necessary to employ detectors that can be used to aid in identification, such as mass spectrometers.
- 73. • Perhaps the most straightforward way to assign peaks within the chromatogram of a sample solution is to inject standard solutions under identical analytical conditions. By comparing the retention factor (k) and response of the peak in the chromatogram of the standard solution, with the sample chromatogram, peaks may be tentatively assigned. • It is important that the concentration of the standard solution is matched to the sample solution as closely as possible. This avoids peak mis-assignment due to peak shape effects.
- 74. Using reference standards for peak identification
- 75. 2-Qualitative analysis • After the peaks have been integrated and identified, the next step in the analysis is quantification. Quantification uses peak areas or heights to determine the concentration of a compound in the sample. • The external standard (ESTD) procedure is the basic quantification procedure in which both standard and unknown sample are analysed under the same conditions. • The result (usually peak height or peak area measured using a data system) from the unknown sample is then related to that of a standard, to calculate the amount in the sample. • The ESTD procedure uses absolute response factor. The response factor is normally calculated as: • The sample amount can be calculated as:
- 77. 4-Preparation of Pure Compound(s) • By collecting the chromatographic peaks at the exit of the detector, • and concentrating the compound (analyte) by removing/evaporating the solvent, • a pure substance can be prepared for later use (e.g. organic synthesis, clinical studies, toxicology studies, etc.). This methodology is called preparative chromatography
- 78. 5-Trace analysis A trace compound is a compound that is of interest to the analyst but it's concentration is very low, usually less than 1% by weight, often parts per million (ppm) or lower; • the determination of trace compounds is very important in pharmaceutical, biological, toxicology, and environmental studies since even a trace substance can be harmful or poisonous; • in a chromatogram trace substances can be difficult to separate or detect; • high resolution separations and very sensitive detectors are required.
- 79. References • An Introduction to HPLC for Pharmaceutical Analysis by Oona Mcpolin. • HPLC Basics: Fundamentals of Liquid Chromatography (HPLC) Courtesy of Agilent Technologies, Inc. • Standard Operating Procedure for SHIMADZU Prominence HPLC (http://medchem.science.nus.edu.sg) • High performance liquid chromatography by Veronika R.Meyer. • Practical HPLC method development by Lioyd R.Synder. • Remington (the science and practice of pharmacy) 21st edition. • High performance liquid chromatography in biotechnology by Agnes Henschen. • Quantitative & Qualitative HPLC www.chromacademy.com