Pharmaceutical Instrumental Analysis PHC 427 Dr. haya Al-johar

1. Pharmaceutical Instrumental Analysis PHC 427 Dr. haya Al-johar Chief of Research and Seized Department Saudi Food & Drug Authority E-mail : hijohar2@hotmail.com

2. LECTURES’ OUTLINE • High performance liquid chromatography (HPLC) • Analytical features of HPLC • External and internal standard methods • Stability-indicating methods of assay. • Chiral separation of pharmaceutical compounds • Chiral separation of pharmaceutical compound • Separation and quantification of related Substances • Gas Chromatographic • The thermodynamic of gas chromatography. • Instrumentation of gas chromatography. • Application of gas chromatography.

3. Capillary electrophoresis • Principles and instrumentation • Choice of optimum conditions for resolution. • Modes of electrophoretic separation • Applications of capillary electrophoresis • Atomic absorption and emission spectrophotometry • Instrumentation of atomic absorption • Quantitative analysis by of atomic absorption • Principles of atomic emission • Instrumentation of atomic emission. • Applications of atomic emission

4. High Performance Liquid Chromatography (HPLC) Identification: It is apart of chromatography techniques (physical separation) which is a general term applied to a wide variety of separation methods. based on the sample partitioning between a moving (mobile) phase, which can be a gas, liquid, or supercritical fluid, and a stationary phase, which may be either a liquid or a solid.

5. History: The discovery of chromatography is generally credited to Tswett, who in 1906 described his work on using a chalk column to separate pigments in green leaves. 1940 paper chromatography . 1950 gas and thin-layer chromatography. 1960 various gel or size-exclusion methods

6. Classification of chromatographic methods Gas column Gas Solid Chrom. (SSC or GC) Adsorption Chromatography Competition between a solid adsorbent and the mobile phase St. solid mo. liquid column Liquid Column Chrom. (LC) High Performance Liquid Chromatography (HPLC) Liquid Planer layer Thin Layer Chrom. (TLC) Paper Chrom. (PC) Gas column Gas Solid Chrom. (GLC) Partition Chromatography Competition between a liquid stationary phase and the mobile phase St. liquid Mo. liquid Liquid column Liquid Column Chrom. (LC) High Performance Liquid Chrmatography (HPLC) Ion Exchange Chromatography Competition between an ion exchange resin stationary phase and liquid mobile phase column Ion Exchange Chrom. (IEC) Liquid Permeation Chromatography Competition between a polymer matrix and liquid mobile phase Gel Permeation Chrom. (GPC) Liquid column

7. applications of hplc: • high-performance liquid chromatography (HPLC) is an analytical technique that is widely used for: • separation, • Identification (Qualitative analysis) • Determination (Quantitative analysis) • of the chemical component in a mixture and • Preparation of interest components.

8. It is the major and integral analytical tool applied in all stages of drug discovery, development, and production of (API).

9. HPLC is required when: • a mixture is too complex for direct analytical method e.g. spectroscopy. • the materials to be analyzed are very similar e.g. isomer. • it is necessary to prepare highly purified material. • a measurement of the amount of a particular material is needed.

10. In chromatographic process, there are 3 steps: • Injection. • Separation. • Elution. Injection Separation Elution

11. B B C C A A time Separation Mechanism Mobile phase (solvent) ↓ ↓ ↓ ↓ Column C > B > A Packing material

12. Separation is determined by column (packing material) and mobile phase (solvent) • The migration of the sample component through the stationary phase is a result of two forces: • movement driven by the mobile phase. • retardation resulting from the stationary phase. • Thus, the sample molecules are held by the stationary phase and transported by the mobile phase.

13. Results obtained by HPLC C A B Chromatogram containing three peaks Qualitative analysis (identification) and Quantitative analysis (determination) Can be performed using the information contained in the chromatogram

14. Identification C What is component A? A B Sample Caffeine Component (A ) elutes the same time as a caffeine peak. Component (A) is identified as caffeine.

15. C A B Caffeine (1mg/ml) 5ul injection (5ug) Determination What is the concentration of component A? Peak area (or height) is proportional to the concentration (or amount) of the component. The concentration of component A (caffeine) is determined by comparing the peak area with that of the standard caffeine peak.

16. Column oven Data processor Auto sampler Pump Pump Injector Column Detector Fraction collector Mobile phase reservoir Reagent pump HPLC Instrumentation waste

17. HPLC Apparatus Mobile phase reservoirs A modern HPLC apparatus is equipped with one or more glass reservoirs each of which contain 500 ml or more of the mobile phase. Often the reservoirs contain a filtration system for filtering dust and particulate matters from the solvent to prevent these particles from damaging the pumps or injection valves or blocking the column.

18. HPLC Apparatus The mobile phase usually produce bubbles in the column and thereby can cause “band spreading” and interfere with the performance of the detector. Therefore, the solvent used must be “degassed” to overcome the dissolved gases. Degassers may consist of a vacuum system, ultrasonic vibration, a system of heating and stirring with reflux condenser, helium or system of sparging.

19. An elution of single solvent system (e.g. 50% water + 50 methanol) of constant composition is termed “isocratic”. In “gradient elution” two or more solvent system that differ in polarity are employed, e.g. methanl (10%, 20% and 30%), and water (90%, 80% and 70%). o

20. Isocratic versus Gradient Elution • Isocratic elution has a constant mobile phase composition • Gradient elution has a varying mobile phase composition

21. HPLC Apparatus • Pumping System • The function of the pump in HPLC is to pass the mobile phase through the stationary phase at high pressure and at a controlled flow rate. A pump capable of pumping solvent up to 4000 psi and at flows of up to 10 ml/min the Action of the Pump is most critical, since it must not mixed up the sample being analyzed with the solvent causing loss of resolution. The pump must be made of material, such as stainless steel or teflon; that resist the chemical reaction with the mobile phase.

22. HPLC Apparatus • Injector System • The Function of the injector is place the sample into the high pressure flow as narrow volume so the sample inters the column as homogenous. • There are two main types of injectors: • 1) Fixed loop injector. • 2) Variable volume injector. • The fixed loop has the advantage of high precision of injector volume. But the loop must be overfilled by several times its volume, that is, a 10 l loop requires 30 – 50 l to ensure that it is filled completely with the sample.

23. HPLC Apparatus The variable volume injector, as its name implies, can accommodate volumes from 1 l to 2ml. this has an advantage in that for limited sample sizes and for large preparative work, the same injector may be used without mechanical changing of the loops.

24. Columns Liquid chromatographic columns are constructed from smooth-bore stainless tubing or heavy walled glass tubing. the majority of columns range in length from 10 cm—30 cm, the inside diameter is often from 4 mm to 10 mm; the common particle size of packing are 3, 5, and 10 m the common column in use is one that is 25 cm in length, 4.6 mm in inside diameter and packed with 5 m particles.

25. HPLC Apparatus • Detectors • The characteristics of an ideal detector for HPLC include the followings: • a) should have a high sensitivity. • b) should respond universally with all solutes. • c) should have a linear response over several order of concentrations. • d) should be insensitive to temp changes & the mobile phase velocity change. • should be reliable and convenient to use. • Unfortunately, no single detector satisfies all these criteria.

26. HPLC Apparatus • The detector used in HPLC depends on the nature of the sample. • The most widely used detectors in HPLC are: UV detector

27. HPLC Apparatus Diode Array Detector (DAD)

28. HPLC Apparatus Evaporative Light Scattering Detector (ELSD)

29. HPLC Apparatus Electrochemical detector

30. HPLC Apparatus Refractive Index detector

31. HPLC Apparatus Flourescence detector

32. HPLC Apparatus HPLC recorder A data capture system, which may be a computing integrator or a PC with software suitable for processing chromatographic data

33. modes in HPLC LC mode Packing materials Mobile phase Interaction Normal phase Silica gel n-Hexane/IPE Adsorption Reversed phase a Silica-C18(ODS) MeOH/Water Hydrophobic Size exclusion Porous polymer THF Gelpermeation Ion exchange Ion exchange gel Buffer sol. Ion exchange Affinity Packings with Buffer sol. Affinity ligand

34. HPLC Modes • 1- Partition Chromatography (PC) • the stationary phase is a liquid coated or linked to a solid support • retention is due to the partitioning of the solute between the two liquid phases (relative solubility) • separation is based mainly on differences between the solubilities of the components in the mobile and stationary phases (liquid liquid chromatography) Cm Cs

35. HPLC Modes Two types of partition chromatography are encountered, namely, normal phase partition chromatography (NPPC) and reversed phase partitioning chromatography (RPPC). In normal phase the stationary phase is normal “polar” (such as triethylglycol or water) & the mobile phase is non polar (such as hexan or diethylether).

36. HPLC Modes • In reversed phase chromatography, the stationary phase is non polar (such as C18 phase, C8 phase) and the mobile phase is relatively polar (such as water, methanol) • Reversed methods are the most commonly used to prepare bonded phase from silica involves the reaction of the silica with a substituent such as dimethylchlorosilane.

37. HPLC Modes R group may be hydrocarbon such as C18 or C8 or a hydrocarbon with a polar group such as CN or NH2. e.g. to prepare C18 phase.

38. In normal phase chromatography, the least polar elute first. In contrast in the reversed phase the most polar component elute first. “Like Attracts Like, and Opposites are Not Attracted” “Like Attracts Like, and Opposites are Not Attracted”

39. Reversed-Phase OH CH3 OH CH3 Non polar Highly polar OH CH3 OH CH3 Moder- ately polar Moder- ately polar Polar stationary phase Non-polar mobile phase No-Polar stationary phase Polar mobile phase OH CH3 OH CH3 Non polar Highly polar OH CH3 OH CH3 Normal-Phase

40. HPLC Modes • Adsorption Chromatography (AC) • In adsorption chromatography, the analyte species are adsorbed on either the surface of polar solid stationary phase such as silica, alumina, porous glass, when the mobile phase is relatively non polar such as hexane(NPAC) or the surface of non polar solid stationary phase such as polymer beads when the mobile phase is polar, such as water or acetonitrile (RPAC). • In adsorption chromatography, the only variable that affect the partition coefficient of analytes is the composition of the mobile phase, in contrast with partition chromatography when the polarity of the stationary phase can also be varied.

44. The effect of pH on the HPLC retention time An additional factor which can be used to control the solvent strength of the mobile phase is pH. Control of he rate of elution via the pH of the mobile phase is only applicable to compounds in which the degree of ionization dependent on pH but this covers a majority of commonly used drugs. The pH of the mobile phase can only be within the range of 2-8.5 pH units because of the tendency for extremes of pH to dissolve silica gel and break the bonds between silane-coating agents and the silica gel support.

45. The effect of pH on the HPLC retention time The effects of pH on retention time are as yet not fully understood. The greatest effects of alteration of pH in the mobile phase are observed within one pH unit either side of the pKa value of the drug, i.e: where the partition coefficient of the partially ionized drug varies between 90% and 10% of the partition coefficient of the un-ionized drug.

47. HPLC Modes • 3-Size exclusion chromatography (SEC) • the stationary phase is a porous material having controlled pore size • separation is based mainly on exclusion effects, such as differences in molecular size and/or shape • the terms Gel Filtration and Gel-Permeation Chromatography • (GPC) were used earlier to describe this process

48. HPLC Modes • 4-Ion exchange chromatography (IEC) • the stationary phase has ionically charged groups at the surface • the retention is due to the attractive interactions between ionic solutes and the opposite charged stationary phase • separation is based mainly on differences in the ion exchange affinities of the sample components • this technique is now often referred to as Ion Chromatography (IC)

49. Theoretical Consideration Retention Time (tR) The retention time (tR) is the time between injection of a sample and the appearance of a solute peak at the detection of a chromatographic column. The retention volume (VR) is the volume of mobile phase required to elute a component from the column. VR = F tR where F is the volume flow rate of the mobile phase.

50. Theoretical Consideration Capacity Factor (KA) The capacity factor is a measure of the position of a sample peak in the chromatogram. It is specific for a given substance. KAdepdnds on the stationary phase, the mobile phase, the temperature or where, t0 = the time taken for unretained molecule to pass through the void volume tR= the time taken for the analyte 1 to pass through the column vo = the void volume of the column vt= retention volume of the analyte. A large capacity factor favors good separation and leads to increase the elution time (2-10 min).