Cromatografia Líquida de Alta Eficiência Farmacologia Clínica

1. Cromatografia Líquida de Alta EficiênciaFarmacologia Clínica Universidade Mogi das Cruzes Março 2004

2. HPLC - state of the art High Performance Liquid Chromatography (HPLC) is one mode of chromatography, the most widely used analytical technique. Chromatographic processes can be defined as separation techniques involving mass- transfer between stationaryand mobile phases.

3. HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components (or analytes) are first dissolved in a solvent, and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture is resolved into its components. The amount of resolution is important, and is dependent upon the extent of interaction between the solute components and the stationary phase. The stationary phase is defined as the immobile packing material in the column.

4. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.

6. Fast and high-efficient separation of some aromatics. Hypersil-C8 (100x2) 3 mm, 60% MeOH in Water, 1.5 ml/min., 1 - Benzamide, 2 - Benzil Alcohol, 3 - Acetophenone, 4 - Methyl Benzoate, 5 - Phenetole, 6 - Naphthalene, 7 - Benzophenone 8 - Biphenyl.

8. Types of HPLC There are many ways to classify liquid column chromatography. If this classification is based on the nature of the stationary phase and the separation process, two modes can be specified. In adsorption chromatography the stationary phase is an adsorbent (like silica gel or any other silica based packings) and the separation is based on repeated adsorption- desorption steps.

9. In ion-exchange chromatography the stationary bed has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusi- vely with ionic or ionizable samples. • In size exclusion chromatography the column is filled with material having precisely controlled pore sizes, and the sample is simply screened or filtered according to its solvated molecular size.

10. Concerning the first type, two modes are defined depending on the relative polarity of the two phases: • Normal phase chromatography, the stationary bed is strongly polar in nature (e.g., silica gel), and the mo- bile phase is nonpolar (such as n-hexane or tetrahydro- furan). • Reversed-phase chromatography is the inverse of this. The stationary bed is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile.

11. Eluent polarity plays the highest role in all types of HPLC. There are two elution types: • Isocratic: the constant eluent composition is pumped through the column during the whole analysis. • Gradient: the constant eluent composition (and strength) is steadily changed during the run.

12. Overlay of the four components trace analysis chromatograms. (A) is the isocratic elution, (B) is the gradient elution, shadow line is the gradient profile from 30% acetonitrile in water to 65% acetonitrile.

13. Instrumentation - HPLC Systems

14. Functional description of the instrument • Mobile phase • Pumps • Columns • Detectors • Injectors • Data systems

17. Mobile phases Mobile phase is the most important parameter in reversed-phase HPLC. Type of mobile phase used may have a big effect on the retention. It can promote or suppress an ionization of the analyte molecules, and it also can shield an accessible residual silanol or any other active adsorption centers on the adsorbent surface.

18. Proper selection of the mobile phase is the second most important step in the development of the separation method (the first one is the selection of the adsorbent type). The main requirement for the mobile phase is that it has to dissolve the analytes up to the concentration suitable for the detection. In HPLC type and composition of the mobile phase (eluent) is one of the variable influencing the separation. Variation of the eluent composition provides the great flexibility of HPLC separations. Despite of the large variety of solvents used in HPLC, there are several common properties:

19. Purity • Detector compatibility • Solubility of the sample • Low viscosity • Chemical inertness • Reasonable price

20. Each mode of HPLC has its own requirements for:. • normal phase mode solvents are mainly nonpolar; • reversed-phase eluents are usually a mixture of water with some polar organic solvent such as acetonitrile.

21. Table shows the most important parameters of some common solvents often used as eluent components in HPLC.

22. PUMPS The HPLC pump is considered to be one of the most important components in a liquid chromatography system which has to provide a continuous constant flow of the eluent through the HPLC injector, column, and detector. The two basic classifications are:

23. constant-pressure pump: is used only for column packing and we will describe it only briefly. • constant-flow pump: is the most widely used in all common HPLC applications

25. Schematic of the single piston pump. CAM is pushing a sapphire piston back and force. When the piston is moving backwards it sucks the eluent through the inlet check valve (on the bottom). The sapphire ball is lifted and opens the path for the eluent. When the piston moves forward, the liquid pushes the inlet ball down and closes the path, but the outlet ball is lifted and opens the outlet valve (upper).

26. Schematic of a dual-head pumps.

27. The schematic of this dual piston pumps which have three or even two check valves. The first piston, called low pressure, is sucks the liquid from the reservoir while the second (high pressure piston) is supplying the eluent to the system. Then the first piston refills the second piston very fast, during 1/100 of the whole pump cycle. This scheme allows the use of only 3 check valve, one of which is working under low pressure. There are no cavitation effects. Because the piston volumes are small (~100 µl), pressure pulastions are small and sharp and easy to damp.

28. Another type of dual piston pump uses only two check valves, but piston volumes are different. While the smaller piston dispenses an eluent in the HPLC system, the bigger piston is sucking an eluent. When pistons change their direction, the bigger piston simultaneously refill the smaller chamber and dispenses eluent into the system. This set-up allows only two check valves for the dual piston pump.

29. Standard HPLC pump requirements • Flow rate range: from 0.01 to 10 ml/min • Pressure range: from 1 to 5,000 psi • Pressure pulsation: less than 1% for normal and reversed phase mode less than 0.2% for size exclusion mode.

30. Isocratic Pump

31. Isocratic Pump • Flow Precision: <0.3 % RSD (typically <0.15 % RSD) Range Set points: from 0.001 to 10.0 ml/min, in 0.001 ml/min increments • Pressure Operating range: 0 - 40 MPa (0 - 400 bar, 0 - 5880 psi) up to 5 ml/min 0 - 20 MPa (0 - 200 bar, 0 - 2950 psi) up to 10 ml/min Pulsation: <2 % amplitude (typically <1 %) at 1 ml/min isopropanol at all pressures >1 MPa (> 10 bar, >147 psi)

32. Compressibility compensation: User selectable, based on mobile phase compressibility • Recommended pH range 1.0 - 12.5Solvents with pH <2.3 should not contain acids which attack steel • GLP features Electronic records of maintenance and errors .Early maintenance feedback (EMF) for continuous tracking of instrument usage, display of feedback messages if preset limits are exceeded.

33. Binary Pump

34. Binary Pump • Flow Precision: <0.3 % RSD (typically <0.15 % RSD) Range Set points: from 0.001 to 5.0 ml/min, in 0.001 ml/min increments • Pressure Operating range: 0 - 40 MPa (0 - 400 bar, 0 - 5880 psi) Pulsation: <2 % amplitude (typically <1 %) at 1 ml/min isopropanol at all pressures >1 MPa (> 10 bar, >147 psi) Compressibility compensation: User selectable, based on mobile phase compressibility.

35. Gradient Delay volume: 180 - 480 µl (600-900 µl with mixer) dependent on back pressure. Composition precision: <0.20 % SD, at 0.1 and 1 ml/min • Recommended pH range: 1.0 - 12.5 Solvents with pH <2.3 should not contain acids which attack steel • GLP features Electronic records of maintenance and errors .Early maintenance feedback (EMF) for continuous tracking of instrument usage, display of feedback messages if preset limits are exceeded.

36. Quaternary Pump

37. Quaternary Pump • Flow Precision <0.3 % RSD (typically <0.15 % RSD) Range Setpoints from 0.001 to 10.0 ml/min, in 0.001 ml/min increments. • Pressure Operating range: 0 - 40 MPa (0 - 400 bar, 0 - 5880 psi) up to 5 ml/min 0 - 20 MPa (0 - 200 bar, 0 - 2950 psi) up to 10 ml/min Pulsation: <2 % amplitude (typically <1 %) at 1 ml/min isopropanol at all pressures >1 MPa (> 10 bar, >147 psi)

38. Compressibility compensation: User selectable, based on mobile phase compressibility • Gradient Delay volume: 800 - 1100 µl dependent on back pressure Composition precision: <0.20 % SD, at 0.2 and 1 ml/min • pH range: 1.0 - 12.5 Solvents with • Recommended pH <2.3 should not contain acids which attack steel • GLP features Electronic records of maintenance and errors.Early maintenance feedback (EMF) for continuous tracking of instrument usage, display of feedback messages if preset limits are exceeded.

39. Columns

40. Typical LC columns are 10, 15 and 25 cm in length and are fitted with extremely small diameter (3, 5 or 10 mm) particles. The internal diameter of the columns is usually 4 or 4.6 mm; this is considered the best compro- mise among sample capacity, mobile phase consumption, speed and resolution. However, if pure substances are to be collected (preparative scale), larger diameter columns may be needed. Packing of the column tubing with the small diameter particles requires high skill and speciali- zed equipment.

41. For this reason, it is generally recommended that all but the most experienced chromatographers purchase prepacked columns, since it is difficult to match the high performance of professionally packed LC columns without a large investment in time and equipment.

44. Column Parameter • System Suitability • Length, Internal Diameter • Packing Material: • Type (Silica, Zirconia, Polymer- based) • Size & Shape (particle diameter,spherical irregular) • Porous, non- porous • Porous size,pore volume, surface area • Bonded Phase Type (C18,C8,CN,NH2,Diol etc.) • Bonded Phase Density (Carbon Load)

45. Performance • Efficiency • Selectivity • Specificity Interaction • pH Stability • Life Time

46. System Suitability Available HPLC system set margins for column selection • 20 µL detector flow-cell incompatible with < 3mm I.D columns • 10 µl sample loop incompatible with < 1 mm I.D columns • 0.2 µl micro injector is useless for conventional columns Suitability Rules 1.Injection Volume < Volume Cell 2. Column Dead > Vcell  N Column Dead Volume ~ 0.7 of the empty column volume

47. Column Length • Column length is compromise between the efficiency • and backpressure • Column efficiency is proportional to the column length • Specific efficiency (# of particles per H) decreases • with lengthincrease

49. Column Diameter Solvent consumption and the sample loading are depending on the column internal diameter • 3 - 5 cm I.D, conventional columns (over 80%) • over 5 mm I.D, semi- preparative • 0.5 - 3 mm I.D, midi-bore columns (old LC-MS) • below 0.5 mm I.D, micro-bore columns (only research)

50. Packing Material Type • Silica • Rigid porous (or non-porous) particles • wide variety of particles and porous sizes • soluble in water at pH >8 • Zirconia • Rigid porous particles • average pore diameter ~ 250 Å • better stability at high pH