Chromatography Tutorial Notes

What is chromatography? The physical separation of analytes based on their differential migration in a mobile phase as they move along a stationary phase What is the mobile phase? Where the molecules can move. This is always a liquid, a gas or a supercritical fluid The mobile phase is also known as a eluent, and it is a solvent of mixture of solvents Examples are ethanol, acetone, and water What is the stationary phase? Where the molecules can't move. It is a solid or a really thick liquid, and is immiscible to the mobile phase. It is fixed in place in a column or on a solid surface and the sample absorbs onto it The particles are usually about 5-10 micrometres in average diameter (this is average since they are often irregularly shaped) Examples are silica and alumina What is elution? The process of extracting one material from another by washing with a solvent • Liquid chromatography Ò planar chromatography - thin layer chromatography (TLC) - paper chromatography Ò column chromatography - high performance liquid chromatograWhat are the types of chromatography? phy (HPLC) - adsorption chromatography - partition chromatography - ion exchange chromatography - size exclusion chromatography- gel filtration chromatography and gel permeation chromatography 1 / 21 - affinity chromatography Ò supercritical fluid chromatography • Gas chromatography Ò gas-liquid chromatography Ò gas-solid chromatography What is planar chromatography? The stationary phase is supported on a flat surface and the mobile phase moves through the stationary phase by capillary action or by gravity There are two forms: paper chromatography and thin layer chromatography What is thin layer chromatography? • It is a fast, simple and inexpensive analytical technique • It is used to determine or monitor - the number of components in a mixture - the identity of two substances - the effectiveness of a purification - the progress of a reaction - column chromatography effectiveness, and the appropriate conditions for a column chromatographic separation How does TLC work? • It involves spotting a dilute solution (1%) of a sample on one end of a small sheet that has been coated with silica or alumina- this is the stationary adsorbent phase. Silica is used for separation of more polar compounds while alumina is used in the separation of non polar compounds • The sheet is placed upright inside a jar in a small pool of solvent- this is the mobile phase. As the solvent rises up the sheet by capillary action, the components travel at different rates based on competing interactions with the mobile and adsorbent phases. • A polar solvent will carry a polar com2 / 21 pound farther while a non-polar solvent will carry a non polar compound farther What is the elutropic series? • A list of solvents in order of their power to carry a given compound through the stationary phase in TLC • The order of solvents runs in the order of their polarity • It is useful when selecting a solvent for a particular separation where more polar compounds will normally require a more polar solvent • For a new separation, you would start with choosing a medium polarity solvent eg dichloromethane or acetone, to see if this gives a good separation What is an Rf value? Retention factor The ratio of the distance the spot travels from the origin to the distance the solvent travels Rf = distance travelled by component/ distance travelled by solvent front What is column chromatography? • The stationary phase is held in a narrow tube through which the mobile phase is forced under pressure or by gravity • The mobile phase is either a liquid or gas, and the stationary phase is usually a viscous liquid bound to the inside of a tube or onto the surface of solid particles • Make sure to keep adding new solvent to never let the column go dry • The sample will slowly separate and the different fractions can be collected at the bottom of the column 3 / 21 What is the partition coefficient? • The ratio of a drug's concentrations in two different solutions • K = Cs / Cm where Cs is the concentration in the stationary phase and Cm is the concentration in the mobile phase • The larger the K, the more a molecule interacts with the column and the longer it is retained and the longer it takes to elute from the column • However K cannot be easily measured directly so instead we measure retention time tR which can be directly related to K What is retention time (tR)? • The time it takes a component in a mixture to pass through the column during gas chromatography • Whereas tM is the time taken for an unretained molecule to reach the detector (dead time) • The more polar the mobile phase, the less retention of the polar sample onto the stationary phase, the shorter the retention time What is high performance liquid chromatography? • Also called high pressure liquid chromatography • It is characterised by the use of high pressure to push a mobile phase solution through a column containing a stationary phase, allowing separation of complex mixtures with high resolution (more separation between peaks) • It is the most widely practiced form of quantitative analytical chromatography due to the wide range of molecule types and sizes which can be separated using HPLC • Solvent reservoirs • Pump system • Injection system 4 / 21 What are the components of a HPLC system? • Chromatographic column • Detector • Computer for control and display What is the pump system? • Mobile phase pressures of up to 6000psi are necessary to achieve reasonable column elution times (minutes) • Typical flow rates are 0.1 to 10ml/minute What is the injection system? • This is used to introduce small samples (0.1 to 500 microlitres) into the carrier stream under high pressure • The packed columns have a large volume • Most errors due to poor precision in HPLC occur during injection, because of the capillary action and the small dimensions/ cavities inside the injector • The injectors can be manual or automatic • The internal volume of the injection loop determines the injection volume What are the reservoirs? • Multiple solvents are necessary for performing gradient elutions (to change the polarity of the mobile phase during a run) • They often have a de-gassing unit built to prevent air bubbles entering the system • It is usually constructed of metals • It must operate at high pressure • It's typical dimensions are 10-30cm long and 1-3cm interior diameter • It contains packing material which holds What is the chromatographic column? the stationary phase. There are many types of packing material and the typical size of them are 5-10micrometers in diameter • A guard column is used to extend the life of the main column 5 / 21 What is the detector? • There are numerous types, some obscure • Original HPLC detectors were common lab instruments eg spectrophotometers • They must be solvent-compatible, stable etc • They can be universal or analyte specific. Universal ones respond to all analytes whereas analyte specific ones respond to specific properties of analytes • Most HPLC detectors are non-destructive but some can be destructive ie in mass spectrometry • Correct choice of column, so the equilibrium between analyte mobile phase and analyte stationary phase has meaning • Correct choice of mobile phase. • Decision on the type of mobile phase composition How do you optimise separations of an- constant composition = isocratic alytes in HPLC? - varying composition = gradient elution • Determining if flow rate should be constant (usually it is) • Decision on heating the column. Heating HPLC columns can influence the equilibrium between analyte mobile phase and analyte stationary phase What do the results show in HPLC? • A graph is obtained, with peaks for each substance detected in HPLC • The retention time of each peak can be matched to standards to determine what substances were present in the sample • The area under each peak increases as concentration increases. The area under each peak can be compared to standard areas of known compounds to estimate the concentration of each substance in 6 / 21 the sample • The separation of each peak is determined by the retention time. A greater difference in retention time results in increased separation. What is adsorption chromatography? Known as liquid-solid chromatography and is classic form of LC (very similar to normal phase) There is no additional phase on the stationary phase particles (silica, alumina) What is partition chromatography? Most widely used type in which the stationary phase is a second liquid that is immiscible with the liquid mobile phase, and the solute is partitioned between two liquid phases The stationary phase is coated/ bonded onto a solid support eg silica, alumina, divinylbenzene resin, and the solute dissolved in the liquid phase is bonded to the surface of the column The stationary phase particles are porous so small molecules get inside the pores and take a long time to move through the column whereas larger molecules remain outside and easily pass What is size exclusion chromatography? down the column. Separation occurs based on the molecular weights and therefore the size of molecules This can be gel filtration chromatography or gel permeation chromatography The stationary phase has an ionic charge. If the molecules in the sample have a positive charge, the stationary What is ion exchange chromatography? phase is kept negative and vice versa. Separation occurs based on the molecular electric charge 7 / 21 What is flash chromatography? A technique that uses air pressure to improve and speed up the separation It is also known as medium pressure column chromatography • Normal phase HPLC. The stationary phase is polar and the mobile phase is non-polar - the stationary phase can be silica or alumina - polar molecules are retained on the stationary phase - cyano, amino or diol terminations on the bonded phase What are the types of HPLC? • Reverse phase HPLC. The stationary phase is non-polar and the mobile phase is polar - non-polar molecules are retained on the stationary phase - C18 to about C8 terminations on the bonded phase - phenyl and cyano terminations on the bonded phase What is isocratic elution in HPLC? • Has a constant mobile phase composition • It is used when a sample contains components which have similar polarities, and where high reproducibility is required • Can often use one pump What are advantages of isocratic elution? • Allows mixing of solvents together ahead of time • Simpler, as no mixing chamber is required • It is good for a small number of samples 8 / 21 • We have little control as to how the peaks are eluted (how the samples come off the column). We have to rely on the interactions between the column phase and the analyte for separation What are disadvantages of isocratic elu- • It may not efficiently separate complex tion? mixtures, and a it requires longer analysis time for samples with a wide range of component polarities • Limited flexibility, so it is not used much in research, mostly for industrial chemistry or routine analysis What is gradient elution in HPLC? • Has a varying mobile phase composition • It is used for complex mixtures eg natural products, pharmaceutical and biochemical samples, where components vary widely in their polarities, as changing the mobile phase components changes the polarity index, which allows us to separate complex mixtures where each component has different polarities • It can also be used to subsequently elute compounds that were previously, intentionally stuck on the column • Uses multiple pumps, often 2-4 pumps (binary or quaternary systems), whose output is mixed together What are advantages of gradient elution? • It is more efficient for complex samples and allows for shorter run times for complex samples • We have control over how the peaks are eluted, by varying the composition of the mobile phase • Some additional wear on the stationary phase • The column has to re-equilibrate to original conditions after each run, which 9 / 21 takes additional time What are disadvantages of gradient elu- • Higher cost due to more advanced tion? equipment and potentially more expensive solvents • UPLC which is ultra performance liquid chromatography. It involves increasing resolution by decreasing particle size in the stationary phase, from around 5 micrometers in HPLC to around 1.7 micrometers in UPLC • As particle size decreases, the optimum flow rate to reach maximum column efficiency increases • However as back pressure is proportional to flow rate, the smaller particle size in UPLC requires much higher operating pressures than HPLC. UPLC reWhat are current advancements in chroquires higher pressures up to 15,000psi matography? to work whereas HPLC would work with pressures up to 6000psi, therefore new instrument is needed to deal with the massive back pressure increase • UPLC has a thinner stationary phase • It has better separation efficiency and higher resolution due to smaller particle size, and also shorter run times • It has increased sensitivity so is used when you are dealing with biological systems with low levels eg protein levels • It is commonly used in practice and industry • Chemical • Pharmaceuticals • Bioscience • Environmental • Consumer products • Clinical What are applications of HPLC? 10 / 21 • Drug analysis, during the process of its discovery, development and manufacturing. It can be either the active pharmaceutical ingredient or the drug in a formulation. • Compound identification (it is estimated that over 60% of existing molecules have been uniquely identified by HPLC testing) How is HPLC used in pharmaceuticals? • To control drug stability • To determine the quantity of drug from pharmaceutical dosage forms eg paracetamol in panadol tablets • To determine the quantity of drug from biological fluids eg blood glucose level • Therapeutic drug monitoring • Purity checking • Toxicology eg paracetamol poisoning • HPLC analysis of the novel antipsychotic drug quetiapine in human plasma - it was carried out on a C8 reversed-phase column - mixture of acetonitrile,methanol phenoll What are examples for when HPLC was and pH 1.9 phosphate buffer as the moused for drug analysis? bile phase - triprolidine was used as the internal standard • HPLC analysis of raloxifene hydrochloride and it's application to drug quality control studies 11 / 21 • For the simultaneous estimation of paracetamol and aceclofenac from pharmaceutical dosage forms - it was carried out on a Hichrom C18 reversed-phase column - the mobile phase consisted of acetonitrile: 20 mM phosphate buffer (pH 5.0) (60:40 v/v) at a flow rate of 0.8 ml/min. - detection was carried out at 265 nm. - etoricoxib was used as an internal standard - the retention time of paracetamol, aceWhat are examples for when HPLC was clofenac and etoricoxib was 4.75, 6.44 used to determine the quantity of drugs and 8.83 min, respectively. from dosage forms? • For the estimation of risperidone in tablet dosage forms - the column was in isocratic mode - the mobile phase contained methanol: acetonitrile: 50 mM potassium dihydrogen orthophosphate (80:10:10 v/v) - the flow rate was 1.3 ml/min and effluents were monitored at 234 nm. - clozapine was used as an internal standard - the retention time of risperidone and clozapine were 2.5 min and 3.3 min, respectively. • The rapid determination of metformin in human plasma using ion-pair HPLC - separation was performed on an analytical 150/4.6 mm i.d. µbondapak C18 column. What are examples for when HPLC was - the wavelength was set at 235 nm. used to determine the quantity of drugs - the mobile phase was 40% acetonitrile, from biological fluids? 0.01 M sodium dodecyl sulphate, 0.01 M sodium dihydrogen phosphate, and distilled water to 100%, adjusted to pH 5.1 at a flow rate of 1.5 ml/min. - phenytoin is the internal standard 12 / 21 What is gas chromatography? • Used to separate volatile compounds, which easily get vaporised at room temperature • A sample is vaporised by injection into a heated system, carried and eluted through a column by an inert gaseous mobile phase and then it is detected • The stationary phase is packed on the inside wall of the column. It can be silicone grease or wax, which can withstand a high temperature • The mobile phase is a volatile gas, which is the carrier gas (there are no solvents). Carrier gases must be chemically inert eg helium, argon and nitrogen. Often, the detector dictates the choice of carrier gas • The less volatile molecules in the sample interact more with the stationary phase and move slowly through the column. • If the compound is non volatile, then derivatization technique must be used • It starts with a cylinder containing the mobile phase • This is connected to the septum of the injector by a trap, scrubber or cartridge of molecular sieves. This purifies the mobile phase gas by removing unwanted hydrocarbons, water vapour and oxygen in the gas. • Then there is the injector oven, with an injector port and a silicone rubber septum. The mobile phase/ carrier gas enters here to get into the column oven/ chamber. The injector is a microsyringe injecting 1 to 20 microlitres rapidly into the column. The injector is usually 50°C 13 / 21 hotter than the boiling point of the sample, and it is also hotter than the column, to allow for rapid volatilisation of the sample. The injector port can be a septum of synthetic rubber which is punctured by a syringe. Some instruments have an autosampler that does the injection for you. How is a gas chromatogram designed? • There is a long column inside a column oven. The column is normally thin, metal, coiled and heated in the oven, and a uniform temperature is maintained. • The end of the column is in the detector oven, with a detector that is connected to a computer. The carrier gas exits from the detector oven • Gas-solid chromatography (not used much) What are the different types of gas chro- • Gas-liquid chromatography (most commatography? mon) • Gas-bonded phase chromatography (relatively new) What is gas-liquid chromatography? • Separation technique that uses an inert gas as the mobile phase and liquid as the stationary phase. • The liquid stationary phase can be coated on the inside of an open-tubular capillary column or on particles inside a packed column (if the packed-column GC uses non-coated solid stationary phases, it is gas-solid chromatography) • The analyte is in the gas phase in the GC and partitions between the mobile phase (carrier gas) and the liquid stationary phase • Volatile molecules have lower boiling point so spend more time in the gas 14 / 21 phase and move faster while higher BP molecules spend more time in stationary phase and move slower What are the different types of columns used in gas chromatography? • Packed columns • Capillary columns What are packed columns? • They consist of either - fine particles of solid support coated with liquid stationary phase - fine particles of solid support that are the stationary phase • Packed columns have greater sample capacity than capillary columns • However, they give broader peaks, longer retention times and less resolution (peaks closer together) • Packed columns are made of stainless steel or glass, and are 3-6mm in diameter and 1-5m in length What are capillary columns? • The column frame is constructed of fused silica tubing. A polyamide coating on the outside gives it strength. It can be mounted on a wire cage to make them easier to handle • The liquid stationary phases are coated or bonded to the inside of the tubing. The stationary phase is usually 0.10 to 5 micrometers in thickness • The column is around 0.1-0.53mm internal diameter and 5-100 metres in length. The smaller the diameter/ the more narrow the column, the higher the resolution (separation between peaks). • Increasing stationary phase thickness increases retention time and increases resolution of early peaks but can lead to column bleed. • Resolution also increases in proportion to the square root of column length 15 / 21 • They are are better than packed columns as they give better separation and shorter analysis time • pg/ml (ppt) to microgram/ml (ppb) concentration range for volatile liquid analytes • WCOT wall-coated open tubular column, which has a stationary liquid phase surrounding the whole column wall • SCOT support-coated open tubular column, which has solid support that is coated with the stationary liquid phase surrounding the whole column wall • PLOT porous-layer open tubular column, which has stationary solid-phase particles surrounding the whole column What are the types of capillary columns? wall • WCOT column gas chromatography is the primary type of GC used in qualitative analysis, because - it has a higher resolution so greater ability to discriminate between components - even though the column has a smaller capacity, it is not important as long as there is sufficient analyte available for detection 16 / 21 • The simplest way to alter the separation in GC is to alter the temperature program in the oven. • Isothermal has a constant temperature whereas gradient has a varied temperature where temperature is used to elute the sample, so the fractions come out of the column as increasing boiling point • By altering the temperature, you vary the rate of the equilibrium reaction for any analyte. They spend more or less time in the stationary phase. The greater the difference in the times between anaHow can separation be improved on gas lytes, the better the separation chromatography columns? • However, if the temperature at a given time is too high, it can cause the peaks to co-elute. There needs to be a balance between poorer resolution and faster separation. If the temperature at a given time is too low, you can still get a good separation with adequate resolution but separation would take a long time. Thus, you need a compromise temperature or program • The pressure of the carrier gas can also be altered to alter the separation but this is less common • Adequate sensitivity for desired analysis (10^-8 to 10^-15g analyte/ sec) • Stable, where the background is constant with time • Reproducible and with good precision What are characteristics of an ideal de- • Linear response over several orders of tector in gas chromatography? magnitude • Temperature range of between room temp to 400°C • Rapid response time! • Independent of flow rate • Easy to use, even by inexperienced 17 / 21 operators • Either selective or universal response • Non-destructive There are dozens of detectors available but not one detector exhibits all of these characteristics • Flame ionisation detectors (FID) - FID detects only hydrocarbons at levels >10ppm What are examples of detectors used in • Flame photometric detectors (FPD) gas chromatography? • Thermal conductivity detectors (TCD) - TCD detects all classes of compounds but is not very sensitive. How does FID work? • FID has 3 inlets: one for the carrier gas which comes from the column, and the other two for hydrogen and oxygen. • The igniter ignites the hydrogen and oxygen to produce a flame. • The sample molecules reach the flame and get ionised to release electrons. • There are two electrodes on either side of the flame, which detect the electrons generated in the form of a current, which is amplified and detected by the computer. This gives peaks at different retention times. • Consider the goal of the analysis • Consider if any sample preparation is What are some method development strategies when choosing a gas chronecessary matography procedure to follow to solve • Choose a detector a particular problem? • Choose a column • Choose an injection method How do you consider the goal of the analysis? • What is required of the analysis? • Quantitative or qualitative analysis? • How important is the resolution? • Is a high precision required? • Will the sample be diluted or concen18 / 21 trated? • How much can the analysis cost? How do you consider if any sample preparation is necessary? • It may be necessary to clean up samples. If you do not clean your samples, chromatograms could contain a broad forest of unresolved peaks. • Non-volatile substances can ruin chromatographic columns • The analyte may be too dilute and require concentration before running, or the analyte may be too concentrated and require dilution before running How do you choose a detector? • Do you need info about everything in the sample? • Do you need to detect a particular compound or class of compounds? How do you choose a column? • Choose the type of stationary phase, the column diameter, the length, and the thickness of the stationary phase • However there are only a few combinations of these factors that are sensible • Is the column compatible with the analytes? Polar analytes require polar stationary phases so they will spend some time in the stationary phase, and non-polar analytes require non-polar stationary phases. There usually needs to be a compromise on the stationary phase to get a good column for your analytes (which are probably a mix of polar and non-polar) • DB-5, HP-5, EC-5, RTX-5 (95% dimethyl, 5% diphenyl polysiloxane) is most common general use column. • GCs are usually set up for either packed or capillary columns, which is decided based on sample capacity and resolution 19 / 21 • Thin-film narrow bore - advantages: high resolution, fast separations, low temperatures, elutes compounds with a high boiling point - disadvantages: low capacity, requires high sensitivity detector • Thick-film narrow bore - advantages: good capacity, good resolution, easy to use, retains volatile comHow do you choose the bore for a col- pounds, good for mass spectrometry umn? - disadvantages: moderate resolution, long retention time for compounds with a high boiling point • Thick-film wide bore - advantages: high capacity, good for thermal conductivity and infrared detectors, simple injection techniques - disadvantages: low resolution, long retention time for compounds with high boiling points How do you choose an injection method? • Split injections are used for concentrated samples and dirty samples with packed liner. They give high resolution but they can cause thermal decomposition • Splitless injections are used for dilute samples. They give high resolution but they require solvent trapping or cold trapping • On-column injections are best for quantitative analysis. They are good for thermally sensitive compounds but they give low resolution • Quality control and analysis of drug products like antibiotics (penicillin), antivirals (amantadine) and general anaesthetics • Assay of drugs. The purity of a com20 / 21 What are some applications of GC in drug analysis? pound can be determined for drugs like atropine sulphate, clove oil and stearic acid • Determine the levels of metabolites in body fluids like plasma, serum and urine 21 / 21