High performance liquid chromatograph HPLC

An introduction for HPLC

1. 1 High performance liquid chromatograph HPLC Introduction

2. Content Chromatography HPLC Chromatography Instrument Solvent Rack, Pump, Injector “ Autosampler”, Separation column, Detector. Applications 2

3. Chromatography 3

4. Chromatography Chromatography Is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. ( IUPAC) 4

5. Chromatography classifications 1. Classification according to the shape of the chromatographic bed 2. Classification according to the physical state of the mobile phase 3. Classification according to the mechanism of separation 5

6. 1. Classification according to the shape of the bed Column Chromatography A separation technique in which the stationary bed is within a tube . The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (Packed Column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (Open-Tubular Column). Planar Chromatography A separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (Paper Chromatography, PC) or a layer of solid particles spread on a support, e.g., a glass plate (Thin Layer Chromatography, TLC). Sometimes planar chromatography is also termed Open-Bed Chromatography. 6

7. 2 . Classification according to the physical state of the mobile phase Gas-liquid chromatography (GLC) Gas-solid chromatography (GSC) Liquid-liquid chromatography (LLC) Liquid-solid chromatography (LSC) Gas Chromatography (GC) A separation technique in which the mobile phase is a gas. Liquid Chromatography (LC) A separation technique in which the mobile phase is a liquid. 7

8. 3. Classification according to the mechanism of separation Adsorption Chromatography Separation is based mainly on differences between the adsorption affinities of the sample components for the surface of an active solid. Partition Chromatography Separation is based mainly on differences between the solubility's of the sample components in the stationary phase (gas chromatography), or on differences between the solubility's of the components in the mobile and stationary phases (liquid chromatography). Ion-Exchange Chromatography Separation is based mainly on differences in the ion exchange affinities of the sample components. Exclusion chromatography Separation is based mainly on exclusion effects, such as differences in molecular size and/or shape or in charge. Affinity Chromatography This expression characterizes the particular variant of chromatography in which the unique biological specificity of the analyte and legend interaction is utilized for the separation. 8

9. Specials techniques Reversed-Phase Chromatography Normal-Phase Chromatography Isocratic Analysis Gradient Elution Stepwise Elution Two-Dimensional Chromatography Isothermal Chromatography Programmed-Temperature Chromatography Programmed-Flow Chromatography Programmed-pressure Chromatography Pyrolysis-Gas Chromatography Reaction Chromatography Post-Column Derivatization 9

10. HPLC Chromatography 10

11. Liquid chromatography Schematic of HPLC A reservoir holds mobile phase, A high-pressure pump is used to generate and meter a specified flow rate of mobile phase, An injector introduce [inject] the sample into the continuously flowing mobile phase stream that carries the sample into the HPLC column. The column contains the chromatographic packing material needed to effect the separation. A detector is needed to  see  the separated compound bands as they elute from the column . 11

12. HPLC Separation Modes Separation depends on the triangle relationships among the sample, filler “ stationary phase”, and eluent! Polarity Electrical Charge “ion exchange” Molecular Size “size exclusion” 12

13. 1. Polarity Separation Modes A molecule’s structure, activity, and physicochemical characteristics are determined by the arrangement of its constituent atoms and the bonds between them. chromatographic separations based on polarity depend upon the stronger attraction between likes and the weaker attraction between opposites.   “ Like attracts like” and  opposites  may be repelled .   compounds in the sample that are similar in polarity to the stationary phase [column packing material] will be delayed because they are more strongly attracted to the particles.  Compounds whose polarity is similar to that of the mobile phase will be preferentially attracted to it and move faster. 13

14. Polarity phases Normal-Phase HPLC An elution procedure in which the stationary phase is more polar than the mobile phase. This term is used in liquid chromatography to emphasize the contrast to reversed-phase chromatography.. Hydrophilic-Interaction Chromatography [HILIC] Reversed-Phase HPLC The term reversed-phase describes the chromatography mode that is just the opposite of normal phase, namely the use of a polar mobile phase and a non-polar [hydrophobic] stationary phase. Hydrophobic-Interaction Chromatography [HIC] 14

15. Normal-Phase HPLC “NP-HPLC “ Adsorption strengths increase with increased analyte polarity. The interaction strength depends not only on the functional groups present in the structure of the analyte molecule, but also on steric factors. The use of more polar solvents in the mobile phase will decrease the retention time of analyte, whereas more hydrophobic solvents tend to induce slower elution (increased retention times). Very polar solvents such as traces of water in the mobile phase tend to adsorb to the solid surface of the stationary phase forming a stationary bound (water) layer which is considered to play an active role in retention. 15

16. Reversed-phase HPLC (RPC) RP-HPLC operates on the principle of hydrophobic interactions, which originates from the high symmetry in the dipolar water structure . The binding of the analyte to the stationary phase is proportional to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand on the stationary phase.   an analyte with a larger hydrophobic surface area (C–H, C–C, and generally non-polar atomic bonds, such as S-S and others) is retained longer because it is non-interacting with the water structure. On the other hand, analyte with higher polar surface area (conferred by the presence of polar groups, such as -OH, -NH 2 , COO –  or -NH 3 +  in their structure) are less retained as they are better integrated into water. 16

17. 2. Electric charge Separation Modes The rule is “ Likes may repel, while opposites are attracted to each other”. Stationary phases for ion-exchange separations are characterized by the nature and strength of the acidic or basic functions on their surfaces and the types of ions that they attract and retain. Cation  exchange is used to retain and separate positively charged ions on a  negative  surface. Conversely,  anion  exchange is used to retain and separate negatively charged ions on a positive  surface 17

18. 3. Molecular size Separation Modes Rule is “ Big ones come out first” The stationary phases have been synthesized with a pore-size distribution over a range that permits the analyte of interest to enter, or to be excluded from, more or less of the pore volume of the packing. The biggest molecules may be totally excluded from pores and pass only between the particles, eluting very quickly. the smaller molecules travel slower [because they move into and out of more of the pores] and elute later 18

19. Stationary phase particles size Smaller Particle Size Leads to Higher plate number Higher pressure Shorter run time (higher sample throughput) Lower detection limit 19

20. Instruments 20

21. Instrument 21

22. UltiMate 3000 System 22 The UltiMate 3000 systems are fully modular, allowing you to choose common system configurations or design the most suitable system for your application needs. RSLC nano Systems RSLC Systems Standard Systems

23. High pressure liquid chromatograph Instrumentation HPLC Is a form of liquid chromatography to separate compounds to identify and quantify each component that are dissolved in solution. HPLC instruments consist of: 1. Solvent rack, 2. Pump, 3. Injector, 4. Separation column, 5. Detector. 23

24. 1. Solvent Rack “SR” Instrumentation SR-3000 Solvent Rack without vacuum degasser typically for use with a LPG-3400, ISO-3100BM, or HPG-3200BX SRD-3200 Solvent Rack with analytical 2-channel vacuum degasser typically for use with the following pumps: - one HPG-3200 (SD or RS) - one ISO-3100SD 24

25. SRD “Solvent Rack Degasser” SRD-3400 Solvent Rack With analytical 4-channel vacuum degasser typically for use with the following pumps: - one HPG-3400 - two HPG-3200 (SD or RS) pumps in a two-stack system - one HPG-3200 (SD or RS) or ISO-3100SD if you want to degas the solvents and the wash solution of an UltiMate 3000 series autosampler SRD-3600 Solvent Rack With analytical 6-channel vacuum degasser typically for use with the following pumps: - one DGP-3600 - two HPG-3200 (SD or RS) pumps in a two-stack system - one HPG-3200 (SD or RS) and one HPG-3400 in a two-stack system - one HPG-3400 if you want to degas the solvents and the wash solution of an UltiMate 3000 series autosampler 25

26. Effect of Eluent composition in separation 26

27. Mobil phase 27 A fluid which percolates through or along the stationary bed, in a definite direction . It may be a liquid (Liquid Chromatography) or a gas (Gas Chromatography) or a supercritical fluid (Supercritical-Fluid Chromatography). In gas chromatography the expression Carrier Gas may be used for the mobile phase. In elution chromatography the expression Eluent is also used for the mobile phase.

28. Reservoir of mobile phase A water-based solvent, organic solvent, or a mixture of the two is mainly used as the mobile phase for HPLC. The way that they are mixed can cause large differences in analysis results. 28

29. Common HPLC Solvents 29 THF “ Tetrahydrofuran ” ( not compatible with PEEK) IPA “ Isopropyl Alcohol “

30. 2. Pump Instrumentation A device designed to deliver the mobile phase at a controlled flow-rate to the separation system. It is necessary to pump the eluent at a constant flow rate and pressure. isocratic   analysis in which the eluent composition remains unchanged during the analysis. solvent must be pre-mixed A  gradient analysis  allows the composition of the eluent to be changed during the analysis. Binary gradient pump –delivers two solvents Quaternary gradient pump –four solvents 30

31. Isocratic analysis In this mode, the mobile phase, either a pure solvent or a mixture,  remains the same throughout the run. solvent must be pre-mixed   A typical system is outlined  31

32. Gradient analysis Low-pressure mixing method: (LPG Pump) One pump is used for mixing. The eluent to be absorbed is switched via electromagnetic valves. Up to four eluents can be mixed. High-pressure mixing method:  ( HPG Pump) Two pumps are used. The eluents are mixed after pumping. The response of the gradient is superior because of the small volume from the mixing unit to the column. 32

33. UltiMate 3000 pump Operating Principle The pump is a zero-pulsation, serial dual-piston pump with electronic compressibility. The two pump heads are connected in series. Continuous delivery is achieved as follows: The working head delivers at the appropriate flow rate while simultaneously filling the serially connected equilibration head. The latter serves as a reservoir and delivers while the working head carries out the suction stroke. Pulsation during the pre-compression phase is reduced to a minimum by velocity modulation of the drive. The flow rate is always kept constant in relation to the atmospheric pressure. 33

34. Pump configurations Pump Descriptions Options 1 ISO -3100 A Isocratic pump ( analytical ; 1 solvent) 5035.0010 2 LPG-3400A Low-pressure gradient pump (analytical; 4 solvents) with integrated vacuum degasser and mixing chamber Mixing Chamber Extension Kit Micro Flow Kit 3 LPG -3400 M Low-pressure gradient pump optimized for micro flows (4 solvents) with integrated vacuum degasser. The pump has no mixing chamber. 4 LPG-3400A B Same as LPG-3400A, but biocompatible Version Mixing Chamber Micro Flow Kit 5 LPG-3400MB Same as LPG-3400M, but biocompatible 6 DGP -3600A Dual low-pressure gradient pump version(analytical): Two separate pumps with integrated mixing chambers in one enclosure (2x3 solvents) Mixing Chamber Extension Kit Micro Flow Kit 7 DGP-3600M Dual low-pressure gradient pump optimized for micro flows: Two separate pumps are installed in one enclosure (2x3 solvents). The pumps have no mixing chambers. 34

35. Pump Descriptions Options 8 DGP-3600AB Same as DGP-3600A, but biocompatible version Mixing Chamber Extension Kit 9 DGP-3600MB Same as DGP-3600M, but biocompatible version 10 HPG -3200A High-pressure gradient pump (analytical; 2 solvents) with integrated mixing chamber Mixing Chamber Extension Kit 11 HPG-3200M High-pressure gradient pump optimized for micro flows (2 solvents). The pump has no mixing chamber. 12 HPG-3200 P High-pressure gradient pump (semi preparative ; 2 solvents) with integrated mixing chamber and mixing chamber extension Mixing Chamber Extension Kit 13 HPG-3400A High-pressure gradient pump (analytical) with integrated mixing chamber and "2 from 4" solvent selectors Mixing Chamber Extension Kit 14 HPG-3400M High-pressure gradient pump optimized for micro flows with "2 from 4" solvent selectors. The pump has no mixing chamber. Pump configurations 35

36. Precautions When switching to another solvent, ensure that the new solvent is miscible with the one contained in the pump. If the solvents are not miscible, the pump can be damaged, for example, by flocculation. Never run the pump dry. Damage to the pistons or the piston seals could result. Before you start operating the pump, check the seal wash reservoir level and refill as needed. After turning on the pump, wait until the wash solution has passed all pump heads. If a leak occurs, turn off the pump and remedy the situation immediately. Some components are made of PEEK™. This polymer has superb chemical resistance to most organic solvents. However, it tends to swell when in contact with trichlormethane (CHCl3), dimethyl sulfoxide (DMSO), or Tetrahydrofuran (THF). In addition, it is attacked by concentrated acids, such as, sulfuric acid and nitric acid or a mixture of hexane, ethyl acetate, and methanol. In both cases, capillaries may start leaking or they can burst. Swelling or attack by concentrated acids is not a problem with brief flushing procedures. 36

37. SD and SDN pumps ISO-3100SD, LPG-3400SD(N), DGP-3600SD(N), HPG-3200 SD, HPG-3400SD SD “ Standard” 37 No Description 1 Peristaltic pump 2 Detector of the rear seal wash system 3 Capillary guides 4 Pump head with working cylinder and equilibration cylinder 5 Leak sensor 6 Inline filter 7 Pump unit without purge valve and pressure transducer for the system pressure 8 Pump lights 9 Pump block status LED

38. ISO 3400 SD Operating Principle 38 No. Description 1 pump head with Working cylinder ( no. 1a) and equilibrium cylinder (no. 1B) 2 Purge unit with Purge valve knob ( no.2a) and outlet nozzle (no. 2b) 3 Inline filter 4 Pump outlet

39. RS pumps LPG-3400RS, DGP-3600RS, HPG-3200RS, HPG-3400RS RS “ Rapid Separation” 39 No Description 1 Peristaltic pump 2 Detector of the rear seal wash system 3 Capillary guides 4 Pump head with working cylinder and equilibration cylinder 5 Leak sensor 6 LPG-3400SD and RS: Capillary mixer LPG-3400BM: Capillary from purge unit to inline filter 7 4-channel vacuum degasser 8 Pump lights 9 LPG-3400SD and RS: Static mixer LPG-3400BM: Inline filter 10 Purge unit with purge valve and pressure transducer for the system pressure 11 Pump block status LED 12 4-channel proportioning valve

40. LPG 3400 RS operating principle 40 No. Description 1 Inbuilt vacuum degasser 2 Proportioning valve 3 Pump head with Working cylinder ( no. 3a)and outlet nozzle ( no.4) 4 Purge unit with Purge valve knob (no. 4a) and outlet nozzle ( no. 4b) 5 LPG – 3400 SD and LPG – 3400 RS Two steps mixing system with capillary mixer( no.5) and static mixer (no. 6) LPG 3400 BM Capillary from purge unit to inline filter( no. 5) and filter ( no.6) 6 7 Pump outlet

41. BM pumps ISO-3100BM, LPG-3400BM, DGP-3600BM 41 No Description 1 Peristaltic pump 2 Detector of the rear seal wash system 3 Capillary guides 4 Pump head with working cylinder and equilibration cylinder 5 Leak sensor 6 Pulse damper 7 Inline filter 8 Purge unit with purge valve and pressure transducer for the system pressure 9 Pump light 10 Pump block status LED

42. ISO 3100 BM Operating Principle 42 No. Description 1 pump head with Working cylinder ( no. 1a) and equilibrium cylinder (no. 1b) 2 Purge unit with Purge valve knob ( no.2a) and outlet nozzle (no. 2b) 3 Pulse damper 4 Inline filter 5 Pump outlet

43. BX pump HPG-3200BX 43 No Description 1 Peristaltic pump 2 Detector of the rear seal wash system 3 Capillary guides 4 Pump head with working cylinder and equilibration cylinder 5 Leak sensor 6 Purge unit with purge valve and pressure transducer for the system pressure 7 Pump lights 8 Pump block status LED 9 Capillary mixer (HPG – 3200 SD and RS) or capillary from purge unit to static mixer (HPG -3200 BX) 10 Static mixer

44. HPG 3200 BX Operating Principle 44 No Element NO Element 1 1a 1b Left pump head with working cylinder Equilibrium cylinder 4+5 HPG-3200SD & HPG 3200 RS Two- step mixing system with Capillary mixer (no.4) and Static mixer (no. 5) 2 2a 2b Right pump head with working cylinder Equilibrium cylinder HPG 3200 BX Capillary from purge unit to static mixer ( no. 5) 3 3a 3b Purge unit with Purge valve knob Outlet nozzle 6 Pump outlet

45. Visual Inspection 45 When a problem occurs, it is advisable to perform a quick visual check of the instrument and column. This will pick up leaks, loose or disconnected tubing, changes in instrument settings etc. These problems are easy to rectify and will save time.

46. Pump Chromeleon page 46

47. 3. Injector Instrumentation A device by which a liquid, solid or gaseous sample is introduced into the mobile phase or the chromatographic bed. The injector serves to introduce the liquid sample into the flow stream of the mobile phase. The injector must also be able to withstand the high pressures of the liquid system. A sample is injected into the flow path for analysis. Each type is equipped with six-port valves, so that a sample can be injected into the flow path at continuous pressure. 47

48. Autosampler Excellent retention time and gradient precision. Pulled-loop injection principle (full- and partial-loop injections). Fast and stable column thermostatting between 5 °C above ambient and 50 °C. High detection sensitivity with low detector noise and drift. Robust, dependable performance at low cost of ownership. 48

49. Pressure troubleshooting 49 System pressure is affected by a number of variables including the viscosity of the solvent used, column variables, flow rate and temperature . Pressure problems fall into one of three categories: high, low or fluctuating pressure. They can occur suddenly or be a gradual process.

50. High Pressure troubleshooting 50 Has the ambient temperature changed ? Is the flow rate correct ? Is the eluent viscous ? Is the pressure transducer operating correctly ? Loosen detector waste outlet fitting. Loosen detector inlet fitting . Loosen column outlet fitting. Loosen column inlet fitting. Loosen fitting at guard or in-line filter. Loosen injector outlet fitting. Loosen pump outlet fittings.

51. Low Pressure troubleshooting 51 Is the pump fuse in working order ? Is the pump on ? Is there solvent flow ? Is there solvent in the reservoir ? Is the low pressure cut-off higher than the operating pressure ? Does solvent flow out of the purge valve when opened ? Was the pump primed ? Is air visible in the solvent lines ? Are the pump heads functioning correctly? Is the flow rate set correctly ? Is the column temperature constant ? Are there any leaks ? Is the correct solvent being used ? Was the purge valve closed after priming ? Is the auto injector in prime mode ? Is the flow rate delivered same as the rate entered ?

52. Fluctuating Pressure Reading 52 Is the pressure functioning correctly ? Was the pump primed properly ? Are you performing a gradient analysis ? Are the pump heads functioning correctly ? Are all the solvents degassed ? Are all solvents miscible ? Are the solvents volatile ?

53. Autosampler Chromeleon page 53

54. 4. Column Instrumentation Is The tube and the stationary phase contained within, through which the mobile phase passes. The heart of the chromatograph, the column’s stationary phase separates the sample components of interest using various physical and chemical parameters. The pump push hard to move the mobile phase through the column and this resistance causes a high pressure. A column is selected to suit both the sample and the purpose of separation. 54

55. Column compartment Freely-configurable and user-interchangeable high-pressure switching valves Accommodation of up to 12 columns Temperatures from 5 to 110 °C with the Rapid Separation Thermostatted Column Compartment Short equilibration times for temperature step gradients and fast application switching Low-dispersion eluent preconditioned for better peak shapes at elevated column temperature Homogeneous temperature distribution via a fan-based, forced-air design Column identification system and comprehensive system wellness features 55

56. Separation power principals Efficiency Mechanical separation power, created by the column length, particle size, and packed-bed uniformity, Efficiency is a measure of mechanical separation power. 56

57. Separation power principals Selectivity Chemical separation power, created by the physicochemical competition for compounds between the packing material and the mobile phase. Selectivity is a measure of chemical separation power. 57

58. Column heating Reproducibility Retention in HPLC is temperature-dependent If temperature varies, then it is difficult to assign “peaks” to specific compounds in the chromatogram and the peak areas/heights may vary Solubility Certain chemical compounds may have low solubility in the HPLC mobile phase If they are injected into the flow stream they may precipitate or other difficulties may arise Stability Certain chemical compounds, especially biological compounds such as enzymes or proteins, may not be stable at room temperature or higher The temperature needs to be much lower down to 4°C 58

59. Home page Chromeleon 59

60. 5. Detector Instrumentation A device that measures the change in the composition of the eluent by measuring physical or chemical properties. The desirable features of a detector are: Sensitivity towards solute over mobile phase Low cell volumes to minimize memory effects Low detector noise Low detection limits Large linear dynamic range 60

61. UV/UV-VIS Detectors (variable λ detector) 61 Wavelength range of 190–900 nm with combined use of deuterium and tungsten lamp on one optical axis. Sensitive detection in the UV, visible, and near-infrared range. Up to four wavelengths can be recorded simultaneously, making the four-channel mode compatible with even fast chromatography. 61

62. VWD Principle A UV detector employs a deuterium discharge lamp (D2 lamp) as a light source, with the wavelength of its light ranging from 190 to 380 nm. If components are to be detected at wavelength longer than this, a UV-VIS detector is used, which employs an additional tungsten lamp (W lamp). By monitoring the reference light divided from the light in front of the flow cell, the difference in light intensity can be determined between the back and front of the flow cell, and this is output as absorbance. 62

63. UV Detector Chromeleon page 63

64. Diode array detector (DAD, PDA) Photodiode arrays (semiconductor devices) are used in the detection unit. Incorporation of large number of diodes which serve as detector elements makes possible simultaneous monitoring of more than one absorbing component at different wavelengths. This provides benefit of time saving and cost reduction on expensive solvents. Optical System, Single-beam, reverse optics design with concave holographic grating Wavelength Range 190 to 800 nm 64

65. PDA Principle 65 The tungsten and deuterium lamps emits light in UV and visible ranges (190 -800) nm. The polychromatic beam passes the flow cell. The grating splits up the polychromatic beam to different wavelengths, the intensities of which are measured by an array or photodiodes. A  photodiode  is a semiconductor device that converts light into current. The current is generated when photons are absorbed in the photodiode.

66. Fluorescence Detector Xenon lamp Light source for the UV to near-infrared wavelength range. PMT Photomultiplier tube (PMT) - Converts the emitted light to a measureable current signal. Wavelength range 200 – 900 nm (dual). Fluorescence detection offers greater sensitivity than a UV-VIS detector. However, the number of naturally fluorescent compounds is smaller in comparison to light absorbing compounds. This limitation is overcome by post column Derivatization 66

67. FL Principle FL is a phenomenon in which a substance absorbs light to reach a high-energy level and then emits light to return to its original level. Such a substance has specific wavelengths of light that it absorbs (excitation wavelengths) and emits (emission wavelengths). While a UV/UV-VIS detector detects light that has passed through the flow cell, an FL detector detects fluorescence emitted in the direction orthogonal to the exciting light. A UV/UV-VIS detector monitors the absorption of light with a specified wavelength. However, some substances absorb light at one wavelength, and then emit light called fluorescence at another wavelength. 67

68. FL Detector Chromeleon page 68

69. Refracted index Detector The response is dependent on changes in refractive index of eluting compounds in the mobile phase. The mobile phase itself should have refractive index different from the sample. Gradient programming is not possible due to resulting changes in refractive index of mobile phase. Temperature control is necessary as it has high temperature sensitivity. Any component in the eluate can be detected; thus, the RI detector is often called a “universal detector”. Typical applications are in Size Exclusion Chromatography. The detector is less sensitive than UV-VIS detector. 69

70. RI Principle RI detector detects components based on the refraction of light in solution. The reference-side cell is filled with eluate, and the column eluate is introduced into the sample-side cell through the changed flow channel. When components are eluted from the column, the chemical composition changes in the sample-side solution, which changes its photorefractive level. As a result, the amount of light received by the light-receiving section changes, showing a peak which can be detected. 70

71. Applications 71

72. Applications fields 72 Environmental applications Food applications Pharmaceutical applications Bioanalytical applications Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol) Salts like sodium chloride and potassium phosphate Proteins like egg white or blood protein Organic chemicals like polymers (e.g. polystyrene, polyethylene) Heavy hydrocarbons like asphalt or motor oil Many natural products such as ginseng, herbal medicines, plant extracts Thermally unstable compounds such as trinitrotoluene (TNT), enzymes

73. Preparation of Pure Compounds Preparative chromatography By collecting the chromatographic peaks at the exit of the detector, - and concentrating the compound (analyte) by removing/evaporating the solvent, - a pure substance can be prepared for later use (e.g. organic synthesis, clinical studies, toxicology studies 73

74. 74 Thanks [email protected]