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This is basic diagram of HPLC instrument. And we have already discussed it in basic evaluation. To hold mobile phase, there is solvent reservoir, then high pressure pump, between pump and column, there is injection system. Separated components are detected by detector and signals are transferred to data system. After detection , sample is discarded into waste.
High Pressure Liquid Chromatography The name “HPLC” originally referred to the fact that high pressure was needed to generate the flow required for liquid chromatography in packed columns. The early 1970’s saw a tremendous leap in technology. These new “HPLC” instruments could develop up to 6,000psi (400 bar) of pressure, and included improved detectors and columns. HPLC really began to take hold in the mid to late 1970’s. With continued advances in performance, the name was changed to High Performance Liquid Chromatography (HPLC). High Performance Liquid Chromatography (HPLC) is now one of the most powerful tools in analytical chemistry, with the ability to separate, identify and quantitate the compounds that are present in any sample that can be dissolved in a liquid.
HPLC system is highly sensitive and accurate and used to find out purity or impurity profile for the non volatile compound or high molecular weight compound.
HPLC is high pressure liquid chromatography or high performance liquid chromatography. In HPLC, separation is achieved under pressure so known as high pressure liquid chromatography and it is highly accurate and sensitive technique so also known as high performance liquid chromatography.
There are two types of HPLC, analytical and preparative. Analytical HPLC are again of two types, isocratic and gradient. Isocratic HPLC are widely used in QC while gradient HPLC are mainly used in R&D / ADL. Preparative HPLC are used for synthesis purpose. Analytical HPLC are used to find out purity or impurity profile. We will discuss the difference between isocratic and gradient HPLC later on.
There are mainly three separation techniques. Adsorption, Partition and Size exclusion Chromatography. Adsorption chromatography is subdivided into (1) Normal Phase (2) Reversed Phase (3)Ion exchange (4)Affinity and (5)Hydrophobic Interaction chromatography. In partition chromatography there are liquid liquid and gas chromatography. Size exclusion chromatography includes gel filtration, gel permeation and gel chromatography. Here we will discuss mainly normal phase and reversed phase chromatography. And we will give brief introduction of other chromatography.
In normal phase chromatography, silica gel is used as packing material and non polar solvent is used as mobile phase. In reversed phase chromatography, polar mobile phase and non polar packing material is used. In size exclusion chromatography, THF is used as mobile phase and porous polymer is used as stationary phase. In ion exchange and affinity chromatography, buffer solution is used for the elution of compounds.
First we will discuss adsorption chromatography. The principle of separation is adsorption. Separation of components takes place because of the difference in affinity of compounds towards stationary phase. This principle is seen in normal phase as well as reversed phase mode, where adsorption takes place.
This is the diagram of adsorption chromatography. Sample containing three different components, when injected into the column, they get adsorbed on to the surface of the packing material. Depending upon the interaction, components are separated and elute from the column. More interaction, more retention in the column and elute late. While components having less interaction with packing material, elute early from the column. This is shown diagrammatically over here. Component in red color have less interaction with stationary phase so elute first then component in black color and component in blue color is tightly interact with packing material so elute last.
In ion exchange chromatography, principle of separation is ion exchange, which is reversible exchange of functional groups. In ion exchange chromatography, an ion exchange resin is used to separate a mixture of similar charged ions. For cations, a cation exchange resin is used. For anions, an anion exchange resin is used.
In ion exchange chromatography, anion and cation exchange gels are used as stationary phase and buffer solution of different ph are used as mobile phase. Here separation depends on ionic properties of sample.
These are structures of cationic and anionic exchange gel.
Affinity chromatography uses the affinity of the sample with specific stationary phases. This technique is mostly used in the field of biotechnology, microbiology, biochemistry etc
Size exclusion chromatography In this type of chromatography, a mixture of components with different molecular sizes are separated by using gels. The gel used acts as molecular sieve and hence a mixture of substances with different molecular sizes are separated. Soft gels like dextran, agarose or polyacrylamide are used. Semi rigid gels like polystyrene, alkyl dextran in non aqueous medium are also used. The mechanism of separation is by steric and diffusion effects.
In size exclusion chromatography, components containing large particles elute first while components having small particles get trapped in porous packing material so elute later.
In partition chromatography, separation occurs between liquid stationary phase and sample molecules. Elution order is same as described in adsorption chromatography.
Till here , we have discussed about principles of separation and now we will see the instrumentation of HPLC. This is diagrammatical representation of HPLC .
As we know, main parts of HPLC system are solvent reservoir, high pressure pump, injector, column, detector and data system. We will discuss each part in detail.
This is the table describing main function of each part. RESERVOIR To hold solvent or Mobile Phase (Because it moves). PUMP used to generate and control the flow at a specified flow rate, typically in milliliters per minute. Sometimes termed as “Solvent delivery system” or “Solvent Manager”. INJECTOR To introduce (“inject”) the SAMPLE into the flowing mobile phase stream, which carries the sample into the HPLC COLUMN. Sometimes termed as “Sample Manager” or “Auto Sampler”. COLUMNS contains the chromatographic packing material needed to create the separation. This is called as Stationary Phase. DETECTOR needed to “see” the separated “compound bands” as they elute from the HPLC column. Mobile Phase exits the detector in to the waste.
First is mobile phase and solvent reservoir. A modern HPLC apparatus is equipped with one or more glass or stainless steel reservoirs, each of which contain 500 ml or more of solvent. Choice of mobile phase and its preparation is very important. Sample must be soluble in mobile phase Solvent must be filtered and degassed. If sample is not soluble in mobile phase then it may clot in the column and damage it. Different grades are available. AR/ LR/ GR/ HPLC/ GRADIENT/ SPECTROSCOPIC tec. Clean, high purity HPLC grade solvents and reagents should be used while preparing mobile phase.
Solvents should be filtered through a 0.45 micron filter. This is especially critical with salts and buffers. Also ensure that the filter material is compatible with the solvent used. The benefit of solvent filtering includes: Improved pump performance (including seals, check valves and plunger) Increase in injector life After filtering, store the solvent in a covered reservoir to prevent dust and debris from entering the solvent.
This is 0.45 micron solvent filter. It is made up of stainless steel and after regular interval of usage it can be cleaned by sonicating it in boiled and cooled water.
That solvent filter is put in solvent reservoir along with mobile phase as shown in this picture thus filtered mobile phase is passed into the system.
Solvent as well as sample must be filtered. These are sample filters. They are available in different types. Here filters made up of plastic, stainless steel and Polyethylene are shown. Filters in red and green color are made up of polyethylene and they are disposable.
Stainless steel and plastic filters can be opened like this and filter paper is placed and again closed.
Solvent filters are attached between needle and syringe.
It is often necessary to remove dissolved air from the mobile phase before the mobile phase is fed to the pump. This procedure is called mobile-phase degassing. The reason for degassing the mobile phase is that dissolved air tends to be released inside the HPLC system. This makes the operation of many HPLC pumps unreliable, leading to fluctuations in flow rate. Bubbles can also get trapped in the detector flow cell, causing problems with this module as well. Four approaches are commonly used to remove excess dissolved gas from mobile phase solvents: Vacuum, sonication, He-degas and in-line degas.
When pure solvents are mixed to make up the mobile phase, excess dissolved gas escapes to form bubbles . If the mobile phase reservoir is placed in an ultrasonic bath, the sound waves remove small bubbles which can escape more easily. This type of degassing works for premixed mobile phases by allowing the excess gas to escape before it enters the pump. It is not recommended for on-line mixing systems, in which bubbles form inside the HPLC system, because it cannot reduce the dissolved gas below the saturation level of the pure solvent. Sonication is often used in conjunction with vacuum degassing.
Vacuum degassing is very convenient because it can be combined with vacuum filtration of the mobile phase to remove both dissolved gas and particulate contamination. Like sonication, with which it is often combined, this technique works for premixed mobile phases, but is not recommended for on-line mixing.
Vacuum pump is attached to the flask and filtration kit containing 0.45 micron membrane filter is kept on to the mouth of flask. If 300 mm of vacuum pressure is used, then it filters 4 liters of solvent in approximately 8 min.
A stream of helium bubbles will sweep dissolved air out of liquids. Helium sparging is very effective; it can reduce the dissolved air. This makes helium sparging especially suitable for use with on-line mixing systems.
In many respects, this is the most convenient approach to degassing. There is in built facility for degassing. This only degas the solvent, not filter.
Improper solvent preparation leads to: Out gassing in pump head Air bubbles or particles trapped in the detector flow cell Mobile phase contamination Damage to pump, check valves and seals. Plugged in-line filters, frits, check valves or connecting tubing. Poor injection precision. High system back pressure. Flow related base line noise. Shifting retention times. Abnormal peak shapes. In correct qualitative/ quantitative results.
Pumps are used to pass mobile phase through the column at high pressure and at controlled flow rate. Ideal pump should have following characteristics: It should generate pressures up to 6000 psi It should give flow rate ranging from 0.1 to 10 ml/min Flow control and flow reproducibility should be of ± 0.5% It should be composition resistant and give a pulse free output. Mobile phase change should be easy.
Pumps are categorized into Single head reciprocating piston pump (Dual Piston Pump) Syringe pump and Diaphragm pump Depending upon elution technique, they are categorized into ISOCRATIC ELUTION GRADIENT ELUTION
There are different parts in pump. Pressure gauge (Dampener) To reduce pulsations Piston For suction of mobile phase from solvent reservoir and its withdrawal to column Check valves Piston in co ordination with check valve maintain flow rate Purge valve For purging of system. If there is any bubble in mobile phase and it is passed into the system then there are chances of damage of piston, check valves, column etc. so purging is done in which some of the mobile phase is bypassed and then it is allowed to pass into the system.
This is the picture of piston along with seal. Piston is made up of stainless steel, borosilicate or Safire glass. Motor driven piston moves through this seal and gives accurate flow.
If mobile phase is not filtered/ contaminated, particle gets trapped between seal and plunger resulting in scratches on to the surface of plunger and gives non reproducible flow rate.
This is the picture of check valve. Depending upon the design of pump, there are two or more than two check valves are present. In check valve, there is a ball and seat arrangement which is explained in next slide.
As seen over here, ball and seat are fitted in the holder.
If mobile phase is not filtered/ contaminated, particle gets trapped between ball and seat resulting in non reproducible flow rate. It can be cleaned by sonicating it.
Most commercial HPLC pumps are based on a reciprocating piston design, as shown here. Single head reciprocating piston pump The twin piston pumps with short stroke are among the most commonly used pumps for HPLC. Both pump heads are switched in series, whereby the piston in the first pump head delivers a specific volume per stroke.
An excentric disk presses piston 1 to the right and displaces the solvent. The double ball sapphire valves ensure that the solvent stream can flow in only one direction. The second piston is used to produce a nearly complete pulsation damping. With the twin piston pump, a pressure of 40 MPa is achieved. During filling stroke, check valve A gets opened to facilitate suction of mobile phase from reservoir.
During pumping stroke, valve A gets closed and check valve B is opened so mobile phase is transferred into the column and thus pumping of solvent continues.
Advantages of this type of pumps are: Modest cost Pressure up to 8000 psi External reservoir and Simple mechanical design The main disadvantage of this type of pump is sinusoidal pressure pulsations and seal and valve maintenance is necessary.
A more efficient way to provide a constant and almost pulse free flow is the use of dual-headed reciprocating pumps. Both pump chambers are driven by the same motor through a common cam; this common drive allows one piston to pump while the other is refilling. As a result, the two flow-profiles overlap each other significantly reducing the pulsation downstream of the pump; this is visualized over here. so we can obtain smooth flow.
Syringe Type Pumps are most suitable for small bore columns because this pump delivers only a finite volume of mobile phase before it has to be refilled. These pumps have a volume between 250 to 500 mL. The pump operates by a motorized lead screw that delivers mobile phase to the column at a constant rate. The rate of solvent delivery is controlled by changing the voltage on the motor.
Syringe pumps give pulse free flow. We can obtain pressure up to 10000 psi and there is a convenient flow control. But it is expensive and solvent change is difficult.
In a diaphragm pump, a diaphragm is hydraulically moved backwards and forwards rapidly by a simple piston pump. The cavity on the other side of the diaphragm is connected to two non-return valves operating in opposition. When the diaphragm moves back, solvent is drawn through one non-return valve into the cavity from the solvent reservoir and when the diaphragm moves forward the solvent is expelled through the other non-return valve to the column. The driving piston is situated on the other side of the diaphragm and uses a separate solvent supply. Consequently, the piston never comes into contact with the actual mobile phase.
Types of HPLC pumps based on elution techniques: ISOCRATIC: A separation that employs a single solvent of constant composition is termed as isocratic elution. GRADIENT: A separation that employs two (or more) solvent systems that differ significantly in polarity is termed as gradient elution. Frequently separation technique is greatly enhanced by gradient elution. Here the proportion of the solvent is varied in programmed way, sometimes continuously and sometimes in a series of steps. Modern HPLC equipment is often equipped with devices that introduce solvents from two or more reservoirs into a mixing chamber at rates that vary continuously.
This is isocratic mode of HPLC where mobile phase composition remains same throughout the run.
There is some change purposely incorporated during the particular sample run to achieve a better and faster separation. In case of pumping mobile phase, the composition of the mobile phase is continuously varied during the particular run.
This diagram shows how we can change the mobile phase composition during the run. There are different solvent reservoir bottles and for each there is an individual pump. Flow is set for particular solvent and then both solvents are mixed in mixing chamber. Program is given for solvent A , 0.5 ml/min and for solvent B 1 ml/min.
After run of few minutes, program is changed for solvent A, from 0.5 ml/min to 1 ml/ min and for solvent B, from 1 ml/min to 0.5 ml/min. Thus mobile phase composition is changed. (change of polarity during run)
This is graphical representation of isocratic and gradient run. In isocratic, mobile phase composition remains same throughout the run so we have straight line while in gradient, there is continuous increase in ratio of particular solvent.
This graph shows how gradient is beneficial over isocratic? When sample containing 7 components is run on isocratic mode, it takes almost 90 minutes to elute all components while same sample is run on gradient mode, we get seventh peak within 40 minutes so run time is reduced and sharp peaks are observed compared to isocratic run.
Difference between isocratic and gradient Isocratic Gradient Least Complex More complexity Composition remains constant Composition varies with time 40% Water/60%Methanol Bottle A: 100% Water Premixed Solvents Bottle B: 100 Methanol Automated Solvent Blending 0 % B to 100% B over 10 minutes Must wash column with high strength solvent after prolonged use Must re-equilibrate column before re-injecting Compound co-elution more frequent No compound co-elution difficult to get resolution of compounds Dwell volume effects
The sample introduction device such as injector to introduce the sample in a flow of mobile phase at high pressure. The valve injection through fixed or variable loop is a common way of introducing the sample. The Rheodyne valve is the mostly used devise. There are two models available, 7125 and 7725i. Difference between 7125 and 7725i is that, in 7725i remote system is given. And autosamplers are fully automatic device.
Samples are injected into the HPLC via an injection port. There are various means of injecting the sample. Manual Injectors Samples are loaded by the analyst Auto samplers Loads and inject samples; limited pre-injection treatment of samples (e.g., dilution or addition of aliquots of a standard) Sample Managers Integrates sample pre-treatment and injection with other system parameters such as the selection of the column, analytical conditions and processing method. Now let&apos;s turn to the LC column: the most important part of the LC system. We&apos;ll look at the different kinds of columns that are used in HPLC, how to describe individual columns, and how to care for the column so as to prolong its useful life.
The injection port of an HPLC commonly consists of an injection valve and the sample loop. The sample is typically dissolved in the mobile phase before injection into the sample loop. The sample is then drawn into a syringe and injected into the loop via the injection valve. A rotation of the valve rotor closes the valve and opens the loop in order to inject the sample into the stream of the mobile phase.
There are two positions in injection valve. Load and Inject. In load position, mobile phase is constantly passed into the column but it does not pass into the loop. Syringe is filled with sample and injected in this position so loop is filled and excess sample is discarded in the waste. When injection valve is rotated from load to inject position, mobile phase now passed into the loop and enters into the column carrying sample. This is how sample is injected into the column. Here green arrow shows flow of mobile phase and red arrow indicates flow of sample.
In modern HPLC systems, the sample injection is typically automated. In auto samplers, sample carousel are filled with sample and then program is given. Sample is injected into the system through motor driven syringe. Then syringe is automatically rinsed and another sample is injected. Thus sample injection continues. This is fully automated device.
Now let&apos;s turn to the LC column: the most important part of the LC system. We&apos;ll look at the different kinds of columns that are used in HPLC, how to describe individual columns, and how to take care of the column so as to prolong its useful life.
We will see that exactly how sample gets separated into the column. When sample is injected, it forms band inside the column. Then components in sample gets separated depending upon their interaction with stationary and mobile phase and form individual band. Blue analyte interacts with stationary phase strongly and so form narrow band and will elute last. While yellow analyte band interacts more with mobile phase, forms somewhat diffuse band and not retained on the column for a longer time.
Columns are available in different types. First we will discuss about column hard ware. Then we will discuss each column type. Like, analytical column, preparative column, precolumns, guard columns and micro bore columns.
The basic design criteria for columns involve the ability to withstand back pressure, properly contain the particles, provide a well-controlled flow path for the sample and individual compound “bands”, and be chemically inert relative to the separation. Typical materials of construction are stainless steel, “PEEK,” an engineered plastic, and glass. By way of review, first consider the column hardware (the metal casing of the column and its connectors)
We will be most concerned with the column END FITTINGS and FRITS. (Apart from the column packing inside the column, these parts of the column are most susceptible to damage from improper use). The column packing is held inside the column by the frits, and the frits are held onto the column end by the end fittings. This picture gives detailed outline of column hard ware and its arrangement in the column.
As we have seen in previous slide that frits are held onto the column end by the end fittings. If mobile phase and sample is not properly filtered then particulate matters clot on the frit which restrict the smooth flow of mobile phase so back pressure is increased. In such case, end fittings can be opened as shown in this picture.
Then frit is removed and can be cleaned by sonicating it in boiled and cool water and again fitted on to the column.
HPLC Column holds the stationary phase for separating the components of the sample. These are the analytical columns. The size of the particles of column packing is very important, and this should always be specified. Most columns come with particles of 5-micron (mm) or 10 micron although 3, 7, 15 and 20 micron particles are also used. The column length and width should also be noted. Columns of different lengths like 50, 100, 125, 150, 250 and 300 mm are available. Generally outer diameter is 4.6 mm but if length of the column is 300 mm then column with outer diameter of 4.0 mm is used.
From the outside, all you can tell about the column is how long and how wide it is. Apart from that, its particle size, type of packing material, part no, batch no, serial no and direction of flow are also mentioned.
Usually information about the column is contained on the column label or the written certificate that comes with the column known as COA(certificate of analysis) Be sure that you are using the right column before you start running an LC procedure. What we have been talking about so far applies mainly to the column that does the main separation job: the analytical column. However there are other kinds of columns used in LC, and other components of the LC system designed to protect the analytical column. Let&apos;s talk about these next.
A PRECOLUMN is used to protect the column against high-pH mobile phases; it is located between the pump and the sample injector. The precolumn is packed loosely with ordinary silica, which does not have to be of HPLC-quality. The role of the precolumn is to precondition the mobile phase in order to minimize chemical attack by the mobile phase on the column packing of the analytical column. Precolumns are packed by the operator of the LC system. If a precolumn is used, it should be opened and checked from time to time to make sure that all of the silica has not dissolved or been used up.
The GUARD COLUMN is a short column positioned between the sample injector and the inlet of the analytical column, or between an in-line filter and the analytical column inlet. It is packed with material similar to that contained in the analytical column, for example C18. The job of the guard column is to pick up or retain sample impurities that could be irreversibly adsorbed onto the analytical column - thereby damaging its performance. Mainly used when dirty samples are to be injected. It is cheap and can be refilled.
Do not get confused between pre column with guard column. Pre column is placed between pump and injector. While guard column is placed between injector and analytical column.
Preparatory Columns- These columns are utilized when the objective is to prepare bulk (milligrams) of sample for laboratory preparatory applications. A preparatory column usually has a large column diameter which is designed to facilitate large volume injections into the HPLC system. The inside width of these columns can be 1 or 2 cm or wider. Microbore and small-bore columns are also used for analytical and small volumes assays. A typical diameter for a small-bore column is 1-2 mm. However, besides the advantage of smaller sample and mobile phase volume, there is a noted increase in mass sensitivity without significant loss in resolution.
This shows difference in peak resolution when same amount of sample is injected in microbore and analytical column. In microbore column, sharp peaks are observed and have better sensitivity than analytical column. Microbore columns are used when sample available is very small in amount.
In HPLC, retention is mainly based on normal phase and reversed phase chromatography which depends on polarity. Other retention mechanisms are based on electrical charge and molecular size.
Normal and reversed phase chromatography is mainly based on POLARITY. In ion exchange chromatography, separation is achieved on the basis of ionic properties of sample. In size exclusion chromatography, sample mixture is separated based on size of the molecules of the sample.
Polarity plays very important role in separation. A simple rule describes this behavior for polarity-based retention mechanisms: “Like Attracts Like, and Opposites are Not Attracted When We Use Polarity”. – Just of opposite to magnetism. Polar will attract Polar (like), and repel Non-Polar (opposites). Non-Polar will attract Non-Polar (like), and repel Polar (opposites). Same Principle is applied while selecting column stationary phase & mobile phase for compound retention.
Here structure of polar and non-polar compounds are shown. Polar bonds are easily breakable while non-polar bonds are not easy to break. Polar compounds are soluble in polar solvents while non-polar compounds are soluble in non-polar solvents.
The stationary phase in HPLC refers to the solid support contained within the column over which the mobile phase continuously flows. Particles of pure silica which are not bonded, are the most polar. Column manufacturers can change the polarity by chemically bonding a modifier to the silica particle surface. For example, by bonding C18 (or “OctaDecylSilane, ODS”) to the surface, the particle can be made to be very non-polar . C18 indicates 18 carbon chain. And if CN group is attached to silica then polarity is slightly reduced. Thus silica is most polar and as we go towards the ODS, there is decrease in polarity of stationary phase. Same is for mobile phase. The chromatographer carefully chooses the polarity of the mobile phase and stationary phase to develop the competition needed for the HPLC separation of the sample compounds. If stationary phase is polar then mobile phase should be non polar or vice versa. Water is most polar and hexane is non polar. In between other solvents lies depending upon its polarity range.
Chemicals have unique characteristics due to its structure. They can be POLAR or NON-POLAR. Depending on the structure of the molecule and its’ electron charge distribution, molecules will be “Very Polar” and some will be “Very Non-Polar”. Distribution of different sample analytes are shown over here based on their degree of polarity.
In normal phase chromatography, mobile phase is non polar and stationary phase is polar. As a mobile phase hexane, iso propanol and acetone can be used. And as a stationary phase, with silica, amino, cyano or hydroxyl groups can be attached.
As we have seen previously that silica is most polar and in normal phase, polar stationary phase is used. This is structure of silica gel. It is believed that silanol radicals on the surface of silica gel act as the active site and the sample is separated.
In Normal Phase Chromatography, The more polar component, the more stronger, it will be adsorbed and so retained longer. Polar (specific but nonionic) interactions of analyte with polar adsorption sites (SiOH, -NH2, -CN, Diol) cause its retention. Analytes with larger number of polar functional group are retained longer. Structural isomers are often separated
In reversed phase chromatography, interaction is hydrophobic. Here stationary phase is non polar and mobile phase is polar. So as a stationary phase C18 or C8 types of columns are used and as a mobile phase polar solvents like methanol, water, ACN or buffer solutions are used. Non polar samples can be analyzed in reversed phase chromatography.
This is the structure of silica-C18 packing material in which with silica, hydrocarbons having 18 carbon atoms are attached.
In Reversed Phase Chromatography, there is non polar interactions of analyte with hydrophobic adsorbent surface. The more polar component, retained less and so elute early. Analytes with larger hydrophobic part are retained longer. Structural isomers are not separated.
This is chart of solvents describing their degree of polarity.
The Polar sample compounds will be attracted to the Polar Stationary Phase and slow down. The Non-Polar compounds in the sample will be attracted to the Non-Polar Mobile Phase and move faster to create the separation. Remember that in Normal Phase chromatography, the Stationary Phase is Polar, therefore, a Polar compound(s) (Yellow) will be retained, and the Non-Polar(s) (Blue), will elute early.
Here, the sample compounds, which are Non-Polar, will be attracted to the Non-Polar Stationary Phase and slow down, while the Polar compounds in the sample will be attracted to the Polar Mobile Phase and move faster. Remember, that in Reversed-Phase chromatography, the Stationary Phase is Non-Polar, therefore the Non Polar compound(s) (Blue) will be retained, and the Polar(s) (Yellow) will elute early.
In normal phase chromatography, stationary phase is polar and mobile phase is non polar so non polar compound elute first while in reversed phase chromatography, stationary phase is non polar and mobile phase is polar so polar compound will elute first.
This is summary of different modes of chromatography.
This table describes the properties of normal phase and reversed phase chromatography.
Now we will see how can we calculate the polarity index of mobile phase and we can change it by changing the composition of mobile phase to get better result. Calculate polarity index of mobile phase having composition of 60 methanol, 20 water, and 20 ACN. By using this equation we can calculate polarity index of any mobile phase.
Sometimes it is necessary to analyze sample under specific temperature so column is placed in the oven compartment. Some samples give sharp peak when operated at specific temperature. We can see that when sample containing benzene, anthracene, pyrene and benz(a)pyrene is run at 60 ° C , then it gives sharp peaks in less time compared to 40 ° C and 20° C. so we can say that maintaining temperature is also very important parameter in HPLC.
As we know, there are two different types of HPLC: analytical and preparative. Now we will see the difference between them. Analytical Type High Performance Liquid Chromatography (HPLC) provides analytical data as to what compounds were present in a sample, and their concentration. Preparative type HPLC can also supply a purified quantity of each compound that is collected in a “ Fraction ” of the flow output from the Detector. The instrument component that performs this function is called a “Fraction Collector”. This process is called “Preparative Chromatography”.
The scientist may need to obtain a certain target amount of purified compound. Depending on how much is required, a much bigger sample size may need to be processed. In general, as the sample size goes up, the size of the HPLC Column will become larger, and the pump will need the capacity to flow at faster flow rates.
Detectors measure the concentration of components present in sample. Selection of Detector depends upon the property of the compounds to be separated. Different detectors available for HPLC are: 1. uv/visible in which there is fixed wavelength and variable wavelength detectors including PDA, refractive index detector, fluorescence detector, electrochemical detector and others like conductivity, mass spectrometer, ELSD. UV detector detects absorbance of UV light by chromophores in the analyte compound. A refractive index detector will sense variation in refractive index of mobile phase stream passing through flow-cell as the sample/analyte mixed M.P enters the detector. Similarly Fluorescence Detectors check for Florescence. Electrochemical detectors rely on oxidation-reduction of sample.
Following criteria should be taken into consideration while choosing detectors. High sensitivity Negligible baseline noise Large linear dynamic range Response independent of variations in operating parameters (pressures, temperature, flow-rate…etc.) Response independent of mobile phase Low dead volume Non-destructive Stable over long periods of operations. Selective
A chromatogram is a representation of the separation that has chemically (chromatographically) occurred in the HPLC system. A series of “peaks” is drawn on a time axis. Each peak represents a different compound. The chromatogram is created by the Detector and Computer Data Station. Detectors detect various compounds as they elute out from column. The detector gives response in terms of a milivolt signal that is then processed by the computer (integrator) to give you a chromatogram.
First we will see the UV/Visible detector. Selective detection minimizing effects from other components. High sensitivity detection at maximum absorption wavelength. Most frequently used detector in HPLC analysis Compounds must contain a UV absorbing chromophore Must work in the linear range of Beer’s Law. It can be operated at fixed wavelength and variable wavelength.
Basically detector consists of a flow-cell through which the mobile phase and resolved sample moves. optics shine through the detector cell and variation in optical properties are detected.
The detector measures the concentration of sample bands as they leave the column and pass through the detector flow cell. When no band is passing through the detector, a constant signal is recorded -- called the Baseline of the chromatogram or detector. When a sample band reaches the detector, the detector responds to the difference in the mobile phase properties caused by the presence of the sample compound, giving rise to a change in detector signal, seen as a Peak.
This is deuterium lamp which gives maximum light intensity for 2000 hours.
This is basic diagram of UV/Visible instrument.
Choosing right wavelength is also very important for the separation of compounds. As shown in this diagram, when berberine is detected at 260 nm, impurity is detected but when it is detected at 340 nm, impurity does not give separate peak.
As a general rule, the wavelength is set to the absorbance maximum of the analyte. Using the wrong wavelength may result in decreased peak sizes, or even no peaks at all! Sometimes in sample, if two components are present and both absorb maximum light at different wavelength then wavelength programming is essential for which variable wavelength detector is used. When mixture of saccharin and sorbin acid is detected at fixed wavelength 265 nm, then very small peak of saccharin is observed because it absorbs light very less at this wavelength but when wavelength programming is done and saccharin is detected at 230 nm and sorbin acid is detected at 265 nm , then it improves sensitivity.
Photodiode array detectors are more versatile, because they allow simultaneous acquisition of both chromatographic and spectral information; they are frequently used in method development.
By using PDA, we can know the wavelength maxima of unknown compound and we can also know the peak purity.
First HPLC detector developed Typically referred to as Universal detectors. Detects all dissolved solutes- “non-specific”. RI response depends on the difference in RI between mobile phase and solute(s). Sensitivity reaches maximum when RI differences are greatest. Can detect g level but only for isocratic runs. Commonly used for: Sugars, Polymers and Fatty Acids
Refractive Index (RI) detectors monitor the index of refraction of the column effluent (the mobile phase leaving the column). Every liquid has a characteristic index of refraction, and this index changes when a solute (the analyte) is dissolved in the liquid. The change in refractive index thus indicates elution of analytes from the column. Because RI detection is based on a property of the mobile phase, it is universal. In this respect, it differs from UV detection which is sample-specific. RI is used primarily for analytes which do not absorb in the UV. It is sensitive to small changes in mobile phase flow or composition so in RI, gradient is not possible and can be very sensitive to small changes in ambient temperature (good temperature control is required for successful operation of RI detectors).
UV is selective detector while RI is universal detector. When sample is detected using UV detector then all components give sharp peak while if RI detector is used then it just detects the sample, sensitivity is not there.
Florescence detector Increased Selectivity and Sensitivity Sensitivity is in the pg region (up to 1000x UV) detector is very sensitive, but its response is only linear over a relatively limited concentration range. Mainly used in food industry.
This is basic diagram of fluorescence detector.
Conductivity Detector Generally used for Ion Chromatography Detects the ability of analyte to carry a charge solutes are ionic (i.e. acid and bases) Inorganic anions and cations
Electrochemical Detector “Destructive” detection mode Sample is either oxidized or reduced in the cell. High sensitivity with picogram (10-12 grams) to high femtogram (10-15 grams) range. Proper selection of buffer, electrode and voltage is critical to success as well as conditioning the mobile phase.
Electrochemical detectors measure compounds that undergo oxidation or reduction reactions. Usually accomplished by measuring gain or loss of electrons from migrating samples as they pass between electrodes at a given difference in electrical potential. Has sensitivity of 10-12 to 10-13 gm/ml
Table describing the summary of detectors.
Most widely used detector is UV/visible detector which is used in 70% of total literature published but in pharma industry it is used 95%. RI is used 5% mostly in preparative scale where sensitivity is not required. Fluorescence is used 10 %, mostly in food industry. Electrochemical detector is used 5% and others including Mass Spectrometry, Evaporative Light Scattering Detector are used 10%.
Detectors detects the sample and transfers the signals to the data system which are then processed. Data system process the detector output and integrate it to form a meaningful chromatogram. Modem Integration system do more than just that. They do processing of the chromatogram, calculations, statistical analysis, data back-up and storage. Data Systems also control various parameters of the system. Thanks to these advanced systems that we no longer have to cut chromatogram peaks and weigh them on analytical balances for interpretation!
Determined by length of column & particle size in the column. Longer Column Length = more run time, more separation but more solvent consumption & higher back pressure. Shorter column lengths = reduce run time and back pressure at the expense of reducing the mechanical separation power.
For a given particle chemistry and mobile phase, a column of the same length, but with a smaller particle size will provide more mechanical separation power. However, the back pressure will increase.
• The principle of separation is adsorption. Separation of
components takes place because of the difference in affinity
of compounds towards stationary phase. This principle is
seen in normal phase as well as reversed phase mode,
where adsorption takes place.
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Ion exchange chromatography
• The principle of separation is ion exchange, which is
reversible exchange of functional groups. In ion exchange
chromatography, an ion exchange resin is used to separate a
mixture of similar charged ions. For cations, a cation
exchange resin is used. For anions, an anion exchange resin
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• Affinity chromatography uses the affinity of the sample with
specific stationary phases. This technique is mostly used in
the field of biotechnology, microbiology, biochemistry etc.
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Size exclusion chromatography
• In this type of chromatography, a mixture of components with
different molecular sizes are separated by using gels. The gel used
acts as molecular sieve and hence a mixture of substances with
different molecular sizes are separated. Soft gels like dextran,
agarose or polyacrylamide are used. Semi rigid gels like
polystyrene, alkyl dextran in non aqueous medium are also used.
The mechanism of separation is by steric and diffusion effects.
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A modern HPLC apparatus is equipped with one or more
glass or stainless steel reservoirs, each of which contain 500
ml or more of solvent.
Mobile phase preparation and use:
• Sample must be soluble in mobile phase
• Solvent filtration.
• Solvent degassing.
Sample must be soluble in mobile phase. If it is not soluble
then sample may clot in the column and damage it.
Different grades are available. AR/ LR/ GR/ HPLC/
GRADIENT/ SPECTROSCOPIC etc. Clean, high purity HPLC
grade solvents and reagents should be used while preparing
• Solvents should be filtered through a 0.45 micron filter. This is
especially critical with salts and buffers. Also ensure that the
filter material is compatible with the solvent used. The benefit
of solvent filtering includes:
1. Improved pump performance (including seals, check valves
2. Increase in injector life
• MMoobbiillee After filtering, PPhhaassee
store the solvent in a covered reservoir to
prevent dust and debris from entering the solvent.
Mainly used for premixed mobile phase.
If the mobile phase reservoir is placed in an ultrasonic bath,
the sound waves remove small bubbles which can escape
not recommended for on-line mixing systems
often used in conjunction with vacuum degassing.
A stream of helium bubbles will sweep dissolved air out of
Helium sparging is very effective; it can reduce the dissolved
This makes helium sparging especially suitable for use with
on-line mixing systems.
This is the most convenient approach to degassing.
There is in built facility for degassing.
This only degas the solvent, not filter.
Improper solvent preparation leads to:
• Out gassing in pump head
• Air bubbles or particles trapped in the detector flow
• Mobile phase contamination
• Damage to pump, check valves and seals.
• Plugged in-line filters, frits, check valves or
• Poor injection precision.
• High system back pressure.
• Flow related base line noise.
• Shifting retention times.
• Abnormal peak shapes.
• In correct qualitative/ quantitative results.
Pumps are used to pass mobile phase through the column at
high pressure and at controlled flow rate.
• Generation of pressures up to 6000 psi
• Flow rate ranging from 0.1 to 10 ml/min
• Flow control and flow reproducibility of ± 0.5%
• It should be composition resistant and give a pulse free
• Mobile phase change should be easy.
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Pumps are categorized into
1. Single head reciprocating piston pump (Dual Piston Pump)
2. Syringe pump
3. Diaphragm pump
Depending upon elution technique, they are categorized into
1. ISOCRATIC ELUTION
2. GRADIENT ELUTION
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Pressure gauge (Dampener)
• To reduce pulsations
• For suction of mobile phase from solvent reservoir and its
withdrawal to column
• Piston in co ordination with check valve maintain flow rate
• For purging of system. If there is any bubble in mobile phase
and it is passed into the system then there are chances of
damage of piston, check valves, column etc. so purging is
done in which some of the mobile phase is bypassed and
then it is allowed to pass into the system.
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Types of HPLC pumps based on elution techniques:
1. ISOCRATIC: A separation that employs a single solvent of
constant composition is termed as isocratic elution.
2. GRADIENT: A separation that employs two (or more) solvent
systems that differ significantly in polarity is termed as
• Frequently separation technique is greatly enhanced by
gradient elution. Here the proportion of the solvent is varied in
programmed way, sometimes continuously and sometimes in
a series of steps. Modern HPLC equipment is often equipped
with devices that introduce solvents from two or more
reservoirs into a mixing chamber at rates that vary
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• Samples are loaded by the analyst
• Loads and inject samples; limited pre-injection treatment of
samples (e.g., dilution or addition of aliquots of a standard)
• Integrates sample pre-treatment and injection with other
system parameters such as the selection of the column,
analytical conditions and processing method.
Protects the analytical column.
It is located between the pump and the sample injector.
Used when mobile phase ph is greater than 8.
Particle size is larger than analytical column.
It saturates the mobile phase so high PH mobile phase does
not dissolve the silica inside the analytical column.
It is a short column.
Located between sample injector and analytical column.
Used when dirty samples are to be injected like blood, urine,
soil samples etc.
Contain same particle size and packing material as that of
It is cheap and can be refilled.
• Used in bulk drugs.
• Mainly for synthesis purpose
• Preparatory column has a large column diameter to facilitate
large volume injections.
Micro bore Columns
• used for analytical and small volume assays.
• Diameter is 1-2 mm.
• Increase in sensitivity without loss in resolution.
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Normal Phase Chromatography
• Retention by interaction between polar stationary phase and
polar sample molecules using non polar mobile phase.
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Reversed Phase Chromatography
• Retention by interaction between non polar hydrocarbon
chain of stationary phase and non polar part of sample
molecules using polar mobile phase.
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Q : Find out polarity index of
Methanol : Water : ACN
60: 20: 20
Polarity index of methanol= 5.1
Polarity index of water= 9.0
Polarity index of ACN= 5.8
Polarity index of mobile phase= PaPb + PcPd + PePf
Pa= Polarity Index of methanol
Pb= Ratio Of Methanol
Pc= Polarity Index of water
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Pe= Polarity Index of ACN
Pf= Ratio Of ACN
Ans : Polarity Index Of Mobile Phase =
(5.1)(0.6) + (9.0)(0.2) + (5.8)(0.2)
= 3.06 + 1.8 + 1.16
Q : Prepare two mobile phases which polarity index is less
than 6.02 and more than 6.02.
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• High Performance Liquid Chromatography (HPLC)
provides analytical data as to what compounds were
present in a sample, and their concentration.
• HPLC can also supply a purified quantity of each
compound that is collected in a “ Fraction ” of the flow
output from the Detector.
• The instrument component that performs this function is
called a “Fraction Collector”.
• This process is called “Preparative Chromatography”.
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Selective detection minimizing effects from other
High sensitivity detection at maximum absorption wavelength.
Most frequently used detector in HPLC analysis
Compounds must contain a UV absorbing chromophore
Must work in the linear range of Beer’s Law.
It can be operated at fixed wavelength and variable
Common Column Problems and Remedies
Three most important problems in HPLC method
Variability in retention and resolution
Short column lifetime
Retention and resolution irreproducibility
• Sample retention should be repeatable from run to run
• It is important to check column retention during method
development at least daily, using a particular set of
• Values of K and a should not change by more than 2-3% over
Retention and Resolution Variations in HPLC
Variation in support, bonding
Disturbance in bed, loss of bonded
phase, dissolution of silica support,
buildup of non-eluted material
From system to system: large injn.
Volume, large tubing volume between.
injection valve and column
Poor control of
Changes in mobile phase composition,
flow rate and temp.
Column overload Too large a sample mass
Solutions to the Irreproducibility Problems
Initially select a good column of less-acidic highly
purified support (if silica based) and maintain other
column parameters same throughout the application
Eliminate chemical or silanol effects for silica-based
columns by using favourable mobile phase conditions.
Equilibrate the column with proper mobile phase
Use proper laboratory techniques that ensure stable
day-to- day operation.
Ensure continuing supply of the same column.
Causes of Tailing (Asymmetrical) Peaks
Bad column: An initial bad column (poorly packed).
Plugged frit or void.
Wrong solvent for sample
Extra column effects
Chemical or secondary retention (silanol) effects
Contaminating heavy metals