High performed liquid chromatography (HPLC) or High pressure liquid chromatography

detail and complete study on the topic of high performance liquid chromatography done by the student fr the student by the student

1. High performance liquid chromatography ( HPLC) Ravish Yadav

2. High Performance Liquid chromatography (HPLC)

3. Instrumentation Mobile phase reservoir Pumps (reciprocating, displacement, pneumatic) (Self study-30 min 0.5 hr ) Sample injection systems ( Rheodyne injector and autosampler ) Column types (analytical, guard and preparative columns) and column packing ( porous, pellicular and monolithic), Detectors (Concept of solute and bulk property detector - Refractive index ,UV-Vis, Phototodiode array, fluorescence, , Electrochemical, Evaporative Light Scattering ) Difference between UPLC and HPLC (Self study-0.5 hr ) Applications, Advantages and Limitations of HPLC (Self study-0.5 hr )

4. Components of HPLC Instrument Mobile Phase Reservoir Pump Precolumn Injector Column Temperature controller/thermostat Detector

5. Mobile phase reservoir Reservoir – Holds single solvent or mixture of solvents used as mobile phase When elution carried out using single mobile phase of constant composition – isocratic elution When elution carried out using two or more than two mobile phases – gradient elution Therefore modern HPLC have two or more reservoirs from which solvent can be introduced into a mixing chamber at a rate to adjust polarity

6. Pump Injector Column Oven Detector Mobile Phase Simple system with one pump and one solvent reservoir. If more than one solvent is used, solvents should be premixed.

7. Data processor pump pump pump A B C Injector Column Oven Detector Mixer

8. Solvent Reservoir Inert to water, methanol, ACN etc. Usually glass / stainless steel Usually 500 to 1000 ml capacity Solvents used are HPLC grade

9. Reservoirs are often equipped with means to remove Filters - Particulate matter, dust etc. Degassers - Dissolves gases Filters Usually 1 – 5 µm microfilters

10. Degassing can be carried out by 1. By filtration under vacuum Removes all dissolved air or oxygen Millipore filters are used 2. By distillation of the mobile phase 3. By ultrasonication 4. By sparging an inert gas of low solubility Gas flushing system, involves bubbling inert gas of low solubility through the mobile phase

11. Pumps The solvents or mobile phase must be passed through a column at high pressures at up to 6000 psi( lb /in²) or 414 bar. As the particle size of stationary phase is smaller (5 to 10µ) the resistance to the flow of solvent will be high. That is, smaller the particle size of the stationary phase the greater is the resistance to the flow of solvents. Hence high pressure is recommended.

12. Requirements for pumps: Generation of pressure of about 5000 psi. Pulse free output & all materials in the pump should be chemically resistant to solvents. Flow rates ranging from 0.1 to 10 mL/min Pumps should be capable of taking the solvent from a single reservoir or more than one reservoir containing different solvents simultaneously.

13. Reciprocating Pumps Consists of Piston provided in syringe type chamber Syringe type Chamber is connected to two ball check valves on two ends Two chambers – Chamber 1 – collects mobile phase from reservoir, regulated by ball check valve 1 Chamber 2 – directs mobile phase from syringe type chamber into the column, regulated by ball check valve 2 The check valves open and close alternately

14. Working Piston is drawn back, ball checks 1 closes the entry into syringe type chamber Ball check 2 close the entry into chamber 2 Therefore mobile phase flows into chamber 1 but not into chamber 2 When piston direction is reversed, Ball check 1 closes entry from reservoir Ball check 2 closes entry into column Therefore mobile phase from chamber 1 enters chamber 2

15. When piston is withdrawn again Fresh mobile phase enters chamber 1 But mobile phase from chamber 2 cannot enter column, because no force to push it When piston direction is reversed again Mobile phase from chamber 1 enters chamber 2 and pushes the already existing mobile phase in chamber 2 into the column

16. 20 Plunger Reciprocating Pump motor and cam plunger plunger seal check valve pump head 5 - 50 µ L out in Mobile phase check valve

17. 21 21 Plunger Reciprocating Pump motor and cam plunger plunger seal check valve pump head 5 - 50 µ L out in Mobile phase check valve

18. Therefore When mobile phase enters chamber 1, no M.P goes into column When it goes into column no MP enters Chamber 1 Therefore we get pulsed flow in reciprocating column

19. Disadvantage can be overcome Reducing time of filling Use of dual piston pump Dual – Piston Reciprocating Pumps Produces pulse free flow One is filling and other is pumping

20. Advantages Internal volume is less – changeover from one MP to another is fast High output pressure Constant flow rate of mobile phase

21. Syringe Pumps Constant flow rate pump Non-pulsating flow Low flow rates (1 to 100 mL/min) Isocratic flow only Refill required when reservoir (~50mL) expended

22. Syringe type/Displacement type pumps syringe-like chambers activated by screw-driven mechanism powered by a stepper motor advantages: output is pulse free disadvantage: limited solvent capacity (~20 mL) and inconvenience when solvents need to be changed

23. 27

24. Pneumatic Pumps Consists of collapsible bag containing MP, housed in metallic cylinder Compressed gas is passed into metallic cylinder This squeezes MP through the outlet Advantage – Simple, cheap, pulse free flow Disadvantages – Flow depends on viscosity and back pressure in column Not used for gradient elution

25. Precolumn/Guard column Placed between pump and injection system Also called guard column Broader than analytical columns Chemically identical to stationary phase in analytical column Particle size is bigger Only MP is passed short length of 2 to 10 cm

26. Rationale Impurities get adsorbed, so do not contaminate analytical column, hence do not interfere with separation MP becomes saturated with stationary phase, so no stripping of analytical column

27. Sample Injection system the most useful and widely used sampling device for modern LC is the microsampling injector valve samples can be introduced reproducibly into pressurized columns without significant interruption of flow, even at elevated temperatures. Rheodyne injector consists of six-port Rheodyne valve

28. Load position sample fills an external loop

29. Inject A clockwise rotation of the valve rotor places the sample-filled loop into the mobile-phase stream, with subsequent injection of the sample onto the top of the column through a low-volume, cleanly swept channel.

30. Automatic injectors Most of the autosamplers have a piston metering syringe type pump to suck the preestablished sample volume into a line and than transfer it to the relatively large loop (~100 ml) in a standard six-port valve.

31. Column types (analytical, guard and preparative columns) and column packing ( porous, pellicular and monolithic)

32. CLASSIFACTION OF CLOUMN column Main column Guard column Analytical column Preparative column Standard column Narrow bore Short fast column Micro preparative Preparative column Macro preparative 39 A) BASED ON APPLICATION

33. Columns HPLC column is made up of glass or stainless steel Glass columns can bear pressure upto 1000 psi Stainless steel coulmn can bear pressure from 2000 to 6000 psi Length ranges from 15 – 150 cm

34. Analytical columns Usual length – 10 to 30 cm Straight Occasionally coiled – results in loss in efficiency Inside diameter – 4 to 10 mm Particle size of packing – 5 to 10 µm Most commonly used column Length – 25 cm Inside diameter 4.6 mm Particle size 5 µm 40000 – 60000 plates/meter

35. Preparative Column Aim – to isolate compounds Length – 15 – 50 cm Diameter – 10 – 40 mm Packing size 5 to 60 µm Flow rate – 5 to 100 ml/min Load – 10 to 1000 mg

36. ANALYTICAL COLUMN STANDARD COLUMN Internal diameter 4 – 5 mm and length 10 – 30 cm. Size of stationary phase is 3 – 5 µm in diameter. Used for the estimation of drugs, metabolites, pharmaceutical preparation and body fluids like plasma. NARROW BORE COLUMN Internal diameter is 2 – 4 mm. Require high pressure to propel mobile phase. Used for the high resolution analytical work of compounds with very high Rt. 44

37. SHORT FAST COLUMN Length of column is 3 – 6 cm. Used for the substances which have good affinity towards the stationary phase. Analysis time is also less (1- 4 min for gradient elution & 15 – 120 sec for isocratic elution). PREPARATIVE COLUMN Used for analytical separation i.e. to isolate or purify sample in the range of 10-100 mg from complex mixture. Length – 25- 100 cm Internal diameter – 6 mm or more. 45

38. TYPES OF PREPARATIVE COLUMN Micro preparative or semi preparative column Modified version of analytical column Uses same packaging and meant for purifying sample less then 100 mg. Preparative column Inner diameter – 25 mm . Stationary phase diameter – 15- 100 µm Macro Preparative Column Column length – 20 – 30 cm Inner diameter – 600 mm 46

39. Scale up factor = Dp

40. Three types of column packing material Porous support Pellicular support Bonded phases Types of packing

41. Types of packing Porous support Porous, silica-based packings - Diffusive pores dominate a typical porous packing porous microparticulate packings - 5 to 10 µm major surface area of the particle is contained within these pores. In a porous particle, solutes transfer from the moving mobile phase outside of the particles into the stagnant mobile phase within the pores in order to interact with the stationary phase. solute molecule must diffuse out of the particle and continue its journey down the column. Such a mass transfer occurs many times as the differential separation process proceeds and the solute is eluted from the column. While the solute spends its time in the diffusive pores, the mobile phase in which it was located originally moves down the column ahead of the solute. This slow rate of mass transfer into and out of the porous particle is a major source of band broadening in HPLC

42. use of smaller particles shortens the pathlength of this diffusion process, improves mass transfer, and provides better efficiency. Manufacturers can now produce small diameter particles with fairly narrow particle size distributions down to 1.5-mm average diameter, although 3–3.5 mm and 5-mm particles are still the norm. with the improvement in efficiency, was the decrease in column permeability; that is, an increase in column back pressure.

43. totally porous silica particles give considerable improvements in column efficiency, sample capacity, and speed of analysis.

44. Pellicular Packing Thin layer of stationary phase coated on glass beads also referred to as superficially porous packings or porous layered beads, good efficiency relative to the large porous particles (with diameters of 100 mm or so), poor sample capacity - due to their small specific surface areas transition from large porous particles and pellicular materials to small porous particles occurred in the early 1970s when microparticulate silica gel ( dp , 10 mm)

45. Monoliths Monoliths are columns that are cast as continuous homogeneous phases rather than packed as individual particles. Monolithic columns have great potential in offering a stable, easily replaced column for both analytical and preparative separations. Both silica-based and polymer-based monoliths have been studied extensively. These columns are solid rods of silica monolith Rods are prepared by a polymerization process either in situ

46. There are two important characteristics for current silica monolith columns: they have the efficiency equivalent to about a 3–5 mm silica particle pressure drop is about 40–50% lower than a 5-mm silica particle. Thus, columns can be coupled in a serial manner thereby generating higher plate counts for more difficult separations. The polymeric monolith columns also have made their mark on separation science. These columns consist of a continuous crosslinked, porous monolithic polymer usually polymethacrylates or methyacrylate copolymerizates .

47. show higher permeability and lower flow resistance than conventional liquid chromatography columns

48. Bonded-Phase Supports : molecules, comprising the stationary phase, i.e. , the surfaces of the silica particles, are covalently bonded to a silica-based support particle. most popular bonded-phase, siloxanes , are formed by heating the silica particles-in dilute acid for a day or two so as to generate the reactive silonal group : which is subsequently treated with an organochlorosilane :

49. These bonded phases are fairly stable between the pH range 2 to 9 and upto temperatures of about 80 °C. nature of the R group of the silane solely determines the surface polarity of the bonded phase. common bonded phase is made with a linear C18 hydrocarbon, also known as ODS ( octadecyl silane ) Column are called as bondapack columns When R = C 18 H 37 – C 18 column R = C 8 H 17 – C8 column

50. The procedure chosen for column packing depends chiefly on the Mechanical strength of the packing particle size. Particles of diameter >20 pm can usually be dry packed particles with diameters < 20 pm slurry packing techniques are used in which the particles are suspended in a suitable solvent and the suspension (or slurry) driven into the column under pressure.

51. Detectors Liquid chromatographic detectors are of two basic types. Bulk property detectors respond to a mobile-phase bulk property, measure the difference in some physical property of the solute in the mobile phase compared to the mobile phase alone Example - refractive index, dielectric constant, or density. Solute property detectors respond to some property of solutes, respond to a particular physical or chemical property of the solute, being ideally independent of the mobile phase Example - UV absorbance, fluorescence, or diffusion current Detectors to be studied in detail (Refractive index ,UV-Vis, Phototodiode array, fluorescence, , Electrochemical, Evaporative Light Scattering)

52. Refractive index detectors bulk property detector are based on the change of refractive index of the eluent from the column with respect to pure mobile phase. disadvantages – lack of high sensitivity, lack of suitability for gradient elution, need for strict temperature control to operate at their highest sensitivity. A pulseless pump, or a reciprocating pump equipped with a pulse dampener limitations may be overcome by the use of differential systems in which the column eluent is compared with a reference flow of pure mobile phase.

53. The two chief types of RI detector are as follows. 1. Deflection refractometer measures the deflection of a beam of monochromatic light by a double prism in which the reference and sample cells are separated by a diagonal glass divide. When both cells contain solvent of the same composition, no deflection of the light beam occurs; if, however, the composition of the column mobile phase is changed because of the presence of a solute, then the altered refractive index causes the beam to be deflected. The magnitude of this deflection is dependent on the concentration of the solute in the mobile phase. Refractive index detectors

54. 2. Fresnel refractometer measures the change in the fractions of reflected and transmitted light at a glass-liquid interface as the refractive index of the liquid changes. In this detector both the column mobile phase and a reference flow of solvent are passed through small cells on the back surface of a prism. When the two liquids are identical there is no difference between the two beams reaching the photocell, but when the mobile phase containing solute passes through the cell there is a change in the amount of light transmitted to the photocell, and a signal is produced. The smaller cell volume (about 3 µl) in this detector makes it more suitable for high-efficiency columns for sensitive operation, the cell windows must be kept scrupulously clean. Refractive index detectors

55. Ultraviolet detectors solute property detector, most widely used in HPLC, based on the principle of absorption of UV visible light as the effluent from the column is passed through a small flow cell held in the radiation beam. It is characterized by high sensitivity (detection limit of about 1 x 10-'g mL-' for highly absorbing compounds) since it is a solute property detector, it is relatively insensitive to changes of temperature and flow rate. The detector is generally suitable for gradient The presence of air bubbles in the mobile phase can greatly impair the detector signal, this effect can be minimized by degassing the mobile phase Both single and double beam instruments are commercially available. single- or dual-wavelength instruments (254 and/or 280 nm), variable-wavelength detectors covering the range 210-800 nm

56. Diode array (multichannel) detector, polychromatic light is passed through the flow cell. The emerging radiation is diffracted by a grating and then falls on to an array of photodiodes, each photodiode receiving a different narrow wavelength band. A microprocessor scans the array of diodes many times a second and the spectrum so obtained may be displayed An important feature of the multichannel detector is that it can be programmed to give changes in detection wavelength at specified points in the chromatogram; this facility can be used to 'clean up' a chromatogram e.g. by discriminating against interfering peaks due to compounds in the sample which are not of interest to the analyst. Ultraviolet detectors

57. Diode permits qualitative information to be obtained beyond simple identification by retention time. There are two major advantages of diode array detection. allows for the best wavelength(s) to be selected for actual analysis. This is particularly important when no information is available on molar absorptivities at different wavelengths. The second major advantage is related to the problem of peak purity. Often, the peak shape in itself does not reveal that it actually corresponds to two (or even more) components. In such a case, absorbance rationing at several wavelengths is particularly helpful in deciding whether the peak represents a single compound or, is in fact, a composite peak.

58. Fluorescence detectors enable fluorescent compounds (solutes) present in the mobile phase to be detected passing the column effluent through a cell irradiated with ultraviolet light and measuring any resultant fluorescent radiation. Radiation from a Xenon-radiation or a Deuterium-source is focused on the flow cell through a filter. The fluorescent radiation emitted by the sample is usually measured at 90° to the incident beam. The second filter picks up a suitable wavelength and avoids all scattered light to reach ultimately the photomultiplier detector.

59. Although only a small proportion of inorganic and organic compounds are naturally fluorescent, many biologically active compounds (e.g. drugs) and environmental contaminants (e.g. polycyclic aromatic hydrocarbons) are fluorescent Because both the excitation wavelength and the detected wavelength can be varied, the detector can be made selective. The application of fluorescence detectors has been extended by means of pre- and post-column derivatization of non-fluorescent or weakly fluorescing compounds Fluorescence detectors

60. Electrochemical detectors. refers to amperometric or coulometric detectors - measure the current associated with the oxidation or reduction of solutes. serious interference (large background current) caused by reduction of oxygen in the mobile phase. Complete removal of oxygen is difficult so that electrochemical detection is usually based on oxidation of the solute. Examples of compounds are phenols, aromatic amines, heterocyclic nitrogen compounds, ketones, and aldehydes. detectors are selective and selectivity may be further increased by adjusting the potential applied to the detector to discriminate between different electroactive species. an anode becomes a stronger oxidising agent as its electrode potential becomes more positive. requires conducting mobile phases, e.g. containing inorganic salts or mixtures of water with water-miscible organic solvents

61. Amperometric detector is widely used electrochemical detector, having the advantages of high sensitivity and very small internal cell volume. Three electrodes are used: 1. the working electrode - made of glassy carbon - the electrode at which the electroactive solute species is monitored; 2. the reference electrode, usually a silver-silver chloride electrode, gives a stable, reproducible voltage to which the potential of the working electrode is referred; 3. the auxiliary electrode is the current-carrying electrode and usually made of stainless steel. amperometric detectors have a more limited range of applications, used for trace analyses where the ultraviolet detector does not have sufficient sensitivity. Electrochemical detectors.

62. Evaporative Light Scattering Detector Column effluent is passed into nebulizer Converted to fine mist At controlled temperature evaporation of mobile phase takes place, leading to formation of particles of analyte Analyte particles then pass through laser beam The scattered light is detected at right angles by silicon photodiode