1. FACULTY OF PHARMACY &MEDICAL SCIENCE Petra University Dr. Wael Abu Dayyih 2012 1 ANALYSIS 501722

2. Dr. WAEL ABU DAYYIH 2011 2 An Introduction to Pharmaceutical Instrumental Methods

3. wabudayyih@uop.edu.jo wabudayyih@uop.edu.jo Office Hours: Sun.Tue, The. (11-12.00) And also by individual student appointments. ( Office hours are subject to change with any changes being announced in class) 3  1) Overview Pharmaceutical analysis  2) Analysis and pharmaceutical quality control  3) Evaluation of data in pharmaceutical analysis  4) Control of the quality of analytical methods  5) Physical and chemical properties of drug molecule  6) Spectral signals/Uv-Spectrometer  7) Spectral signals/IR-Spectrometer  8) Separation signal/Chromatography- HPLC  9) Electrical signals/ Potentiometry and ISE  10) Thermal signal /DSC  Course Text:  Principles of Instrumental Analysis (6th Edition) by Skoog, Holler and Crouch, published by Thomson Brooks/Cole.  Pharmaceutical Analysis 2ed edition by David Watson published by Elsevier Churchill Livingstone  Note: Supplementary course material,  Along with class handouts, will be provided in class or on the web and will be announced in-class. Evaluation: 15 marks first exam. 15 marks second exam  20 marks Lab. 10marks activity 40 marks final Scale 50-54 D 55-59 D+ 60-64 C- 65-69 C 70-74 C+ 75-79 B- 80-84 B 85-89 B+ 90-94 A- 95-100 A First exam( ) Second exam ( )

4. Introduction 4 Analytical Chemistry deals with methods for determining the chemical composition of samples of matter. A qualitative method yields information about the identity of atomic or molecular species or the functional groups in the sample; a quantitative method, in contrast, provides numerical information as to the relative amount of one or more of these components.

5. Classification of Analytical Methods 5 Analytical methods are often classified as being either classical or instrumental. This classification is largely historical with classical methods, sometimes called wet-chemical methods, preceding instrumental methods by a century or more. In the early years of chemistry, most analyses were carried out by separating components of interest in a sample by precipitation, extraction, or distillation.

6.  For qualititative analyses, the separated components were then treated with reagents that yielded products that could be recognized by their colors, boiling points or melting points, their solubility in a series of solvents, their odors, their optical activities, or their refractive indexes.  For quantitative analyses, the amount of analyte was determined by gravimetric or by titrimetric measurement. In the former, the mass of the analyte or some compound produced from the analyte was determined.  In Titrimetric procedures: the volume or mass of a standard reagent required to react completely with the analyte was measured. These classical methods for separating and determining analyte still fund use in many laboratories. The extent of their general application is, however, decreasing with the passage of time. 6

7. Instrumental Methods 7 In the mid-1930s, or somewhat before, chemists began to exploit phenomena other than those described in the previous section for solving analytical properties of analytes – such as conductivity, electrode potential, light absorption or emission, mass-to-charge ratio, and fluorescence – began to be employed for quantitative analysis of a variety of ignorance, organic, and biochemical analytes.

8. Instrumental Methods 8 Furthermore, highly efficient chromatographic separation techniques began to supplant distillation, extraction, and precipitation for the separation of components of complex mixtures prior to their qualitative or quantitative determination. These newer methods for separating and determining chemical species are known collectively as instrumental methods of analysis. Many of the phenomena upon which instrumental methods are based have been known for a century or more. Their application by most chemists, however, was delayed by a lack of reliable and simple instrumentation. In fact, the growth of the modern instrumental methods of analysis has paralleled the development of the electronic and computer industries

9. Types of Instrumental Methods 9 For this discussion, it is convenient to describe physical properties that are useful for qualitative or quantitative analysis as analytical signals. Table 1-1 lists most of the analytical signals that are currently used for instrumental analysis.

10. analytical signals. 10 Note that the first six involve electromagnetic radiation. The first, the radiant signal is produced by the analyte; the next five signals involve changes in a beam of radiation brought about by its passage into the sample. Four electrical signals then follow, finally, for miscellaneous signals are grouped together. These include mass-to-charge ratio, reaction rate, thermal signals, and radioactivity. The second column in Table 1.1 lists the names of instrumental methods that are based upon the various analytical signals. It should be understood that beyond their chronology, few features distinguish instrumental methods from their classical counterparts. Some instrumental techniques are more sensitive than classical techniques, but others are not. With certain combinations of elements or compounds, an instrumental method may be more selective; with others, a gravimetric or volumetric approach may suffer less from interference

11. Signals Employed in Instrumental Methods 11  (X-ray, UV, visible, electron, Auger); fluorescence, phosphorescence, and luminescence (X-ray, UV, and visible)  Emission of radiation  Spectrophotometry and photometry (X-ray, UV, visible, IR); photoacoustic spectroscopy; nuclear magnetic resonance and electron spin resonance spectroscopy  Absorption of radiation  Turbidimetry; nephelometry; Raman spectroscopy  Scattering of radiation  Refractometry; interferometry  Refraction of radiation  X-Ray and electron diffraction methods  Diffraction of radiation  Polarimetry; optical rotary dispersion; circular dichroism  Rotation of radiation o Potentiometry; chronopotentiometry  Electrical potential o Coulometry  Electrical charge o Polarography; amperometry  Electrical current o Conductometry  Electrical resistance o Mass spectrometry  Mass-to-charge ratio o Kinetic methods  Rate of reaction o Thermal conductivity and enthalpy  Thermal properties o Activation and isotope dilution methods  Radioactivity

12. N0TE…..!!! 12 Generalization on the basis of accuracy, convenience, or expenditure of time are equally difficult to draw. is it necessary true that instrumental procedures employ more sophisticated or more costly apparatus; indeed, the modern electronic analytical balance used for gravimetric determinations is a more complex and refined instrument than some of those used in the other methods listed in Table 1-1. As mentioned earlier, in addition to the numerous methods listed in the second column of Table 1-1, there exists a group of instrumental procedures that are employed for separation and resolution of closely related compounds, The majority of these procedures are based upon chromatography. One of the signals listed in Table 1-1 is ordinarily used to complete the analysis following chromatographic separations. Thermal conductivity, ultraviolet and infrared absorption, refractive index, and electrical conductance have been employed for this purpose. This text deals with the principles, the applications, and the performance characteristics of the instrumental methods listed in Table 1-1 and of chromatographic separation procedures as well. No space is devoted to the classical methods, the assumption being that the reader will have encountered these techniques in earlier studies

13. Instrument for Analysis 13 In the broadest sense, and instrument for chemical analysis converts an analytical signal that is usually not directly detectable and understandable by a human to a form that is. Thus, an analytical instrument can be viewed as a communication device between the systems under study and the scientist. An instrument for chemical analysis is general made up of just four basic components. As shown in Figure, these components include a signal generator, an input transducer (called a detector), a signal processor, and an output transducer or readout device. A general description of these components follows A signal generator produces a signal that reflects the presence and usually the concentration of the analyte. In many instances, the signal generator is simply a compound or iron generated from the analyte itself. For atomic emission analysis, the signal generator is excited atoms or ions of the analyte that emit photo of radiation. For a pH determination, the signal is the hydrogen ion activity of a solution containing the sample. In many other instruments, however, the signal generator for an instrument for an infrared absorption analysis includes, in addition to the sample, a sound of infrared radiation, a monochromator, a beam chopper and splitter, a radiation attenuator, and a sample holder.

14. Some Examples of Instrument Components TAB.1.2 14 INSTRUMENTSIGNAL GENERATO R ANALYTICAL SIGNAL INPUT TRANSDUCER TRANSDU CER SIGNAL SIGNAL PROCESSOR READOUT PHOTOMETERTUNGSTEN LAMP,GLASS FILTER, SAMPLE ATTENUTED LIGHT BEAM PHOTOCELL ELECTRICAL CURENT NONECURRENT METER ATOMIC EMISSION SPECT. FLAM MONO- CHROMATOR,SAMPLE UV OR VISIBLE RADIATION PHOTOMULTIPLIER TUBE ELECTRICAL POTENTIAL AMPLIFIER DEMODULATOR CHART RECORDER COULOMETERDC SOURCE SAMPLE CELL CURRENT ELECTRODES ELECTRICAL AMPLIFIER CURRENT CHART RECORDER pH METERSOLUTION SAMPLE HYDROGEN ION ACTIVITY GLASS CALOMEL ELECTRODES ELECTRICAL POTENTIAL AMPLIFIER DIGITIZER DIGITAL UNIT X-RAY POWDER DIFFRACTOMETER X-RAY TUBE SAMPLE DIFFRACTED RADIATION PHOTOGRAPHIC FILM LATENT IMAGE CHEMICAL DEVELOPER BLACK IMAGES ON FILM COLOR COMPARATOR SUNLIGHT SAMPLE COLOREYEOPTIC NERVE SIGNAL BRAINVISUALCOLO R RESPONSE

15. Detectors (Input Transducers) 15 A transducer is a device that converts one kind of energy (or signal) to another. Examples include a themocouple, which converts a radiant heat signal into an electric voltage; a photocell, which converts light into an electric current; or the beam of a balance, which converts a mass imbalance into a displacement of the beam of a balance from the horizontal. Transducers that act on a chemical signal are called detectors. Most detectors convert analytical signals to an electric voltage or current that is readily amplified or modified to drive a readout device. Note, however, that the last two detectors listed in Table 1-2 produce non-electric signals

16. Signal Processors 16 The signal processor modifies that transduced signal from the detector in such a way as to make it more convenient for operation of the readout device. Perhaps the most common modification is amplification – a process in which the signal is multiplied by a constant greater than unity. In a two-pan balance, the signal processor is a pointer on a scale whose displacement is considerably greater than the displacement of the beam itself.

17. AMPLIFIER 17 Amplification by a photographic film is enormous; here, a single photon may produce as many as 10 12 silver atoms. Electric signals, of course, are readily amplified by a factor of 10 12 or more. A variety of other modifications are also commonly carried out on electric signals. In addition to amplification, signals are often filtered to reduce noise,

18. Readout Devices 18 A readout device is a transducer that converts a processed signal that is understandable by a human observer. Usually, the transducer signal takes the form of the position of a pointer on a meter scale, the output of a cathode-ray tube. In some instances, the readout device give an analyte concentration directly.

19. Circuits and Electrical Devices in Instruments 19 The detector in most modern analytical instruments converts the analytical signal to an electric one, which is processed in various ways and them displayed by a readout device.

20. Microprocessors and Computers in Instruments 20 Modern analytical instruments generally employ one or more sophisticated electronic devices – such as operational amplifiers, integrated circuits, analog-to-digital and digital-to-analog converters, counters, microprocessors, and computers.

21. SELECTING AND ANALYTICAL METHOD 21  DEFINING THE PROBLEM In order to select an analytical method intelligently, it is essential to define clearly the nature of the analytical problem. Such a definition requires answers to the following questions:  What accuracy and precision are required?  How much sample is available?  What is the concentration range of the analyte?  What components of the sample will cause interference?  What are the physical and chemical properties of the sample matrix?  How many samples are to be analyzed?

22. SELECTING AND ANALYTICAL METHOD 22 The answer to question 1 is to of vital importance because it determines how much time and care will be needed for the analysis. The answers to questions 2 and 3 determine how sensitive the method must be and how wide a range of concentrations must be accommodated. The answer to question 4 determines the selectivity required of the method. The answers to question 5 are important because some analytical methods in Table 1-1 are applicable to solutions (usually aqueous) of the analyte. Others are more easily applied to gaseous samples, whereas still others are suited to the direct analysis of solids.

23. SELECTING AND ANALYTICAL METHOD 23 The number of samples to be analyzed question 6 is also an important consideration from the economic standpoint. If this number is large, considerable time and money can be spent on instrumentation, method development, and calibration. Furthermore, if the number is to be large, a method should be chosen that requires the least operator time per sample. On the other hand, if only a few samples are to be analyzed, a simpler but more time-consuming method that requires little or no preliminary work is often the wiser choice.

24. Conclusion 24 With answers to the foregoing six questions, a method can then be chosen – provided the performance characteristics of the various instrumental methods shown in Table 1-1 are known.

25. factors that should be considered when choosing an available method 25 I. The type of available instrument II. The experience of the analysis is using the instrument III. The excepted concentration range within with the sample falls IV. The required precession and accuracy of assay V. The possible interferes f the assay VI. The # of the samples that must assayed with the instrument VII. The rate at which must the result be obtained VIII. The expense of each assay while using the instrument

26. Lists quantitative performance criteria of instruments, criteria that can be used to decide whether factors that should be considered in glossary. 26 Criteria 1. Precision------------------------- 2. Bias------------------------------- 3. Sensitivity----------------------- 4. Detection limit----------------- 5. Concentration range---------- 6. Selectivity------------------------ Figure of Merit 1.Absolute standard deviation, relative standard deviation, coefficient of variation, variance 2.Absolute systematic, error, relative systematic error 3.Calibration sensitivity, analytical sensitivity 4.Blank plus three times standard deviation of a blank 5.Concentration limit of quantitation (LOQ) to concentration limit of linearity (LOL) 6.Coefficient of selectivity

27. Characteristics to Be Considered in Method Choice 27  Speed  Ease and Convenience  Skill required of operator  Cost and availability of equipment  Per-sample cost

28. Analysis and Pharmaceutical Quality Control 28 Types of Analysis: 1.Qualitative Analysis: 2.Quantitative Analysis:

29. Quantitative Analysis 29 How much of a compound is there present? *Most Useful Methods: a.NMR: good at percent concentration levels not at trace levels. b.IR: needs high concentrations and mostly done for liquid samples. c.UV: used especially for unsaturated compounds in natural products, not for trace levels but for percent levels, it is subject to spectral overlap and interference from other compounds in the sample but can be overcome by certain techniques. Other: Thermal analysis, X-ray diffraction.

30. Qualitative Analysis 30 WHAT ARE THE COMPONENTES PRESENTS ? *Most useful methods: a.NMR:identifies type of H, C in organic compounds and revels the position of functional groups relative to each other. b.IR: indicates for functional groups and hetero elements and distinguishes between aliphatic and aromatic systems c.MS: useful to identify very high molecular weight and identifies functional groups and structure of the compound. It also confirms the presence of hetero elements. d.UV: Determination of unsaturated compounds especially aromatics and other functional groups. Other: X-ray fluorescence and X-ray diffraction.

31. Classification of Analytical Methods 31 A.Classical Analysis: Titration  Quantitative While reacting with a reagent to give a product with a color, boiling point or melting point, solubility and refractive index.

32. Classification of Analytical Methods 32 B.Instrumental Analysis: I.based on measurement of a certain physical property (non- Seperational technique) Spectroscopy, Techniques: (Fluorescence, IR, NMR, UV, MS) Electrochemical: Potentiometry II.Seperational Techniques Chromatographic techniques: (HPLC, GC, CE, TLC) Crystalization Extraction Distillation

33. Areas of Applying Analysis 33 Fundamentals research Product development Product Quality control Monitoring and control of pollutants Assay Medical and chemical studies

34. Application in Pharmaceutical Industry 34 Design and testing of new drugs "Combinatorial chemistry” QC/QA of raw materials, intermediates and formulated products "Intermediate Analysis" Stability testing "Accelerated degradation" PK ( pharmacokinetic.)and metabolism studies

35. Sampling 35 The most important step in analysis: It should be representative, sufficient for all analysis to be carried out comfortably Pre-treatment: To decrease interference bring the analyte into a desired range of concentration by dissolution, fusion, separation, dilution concentration, derivatization Control of the chemical environment, controlling the atmosphere which the sample is exposed to and controlling different instrumental parameters Sampling different sample types:

36. sample types 36 Gas Samples: Single spot sample taken by balloon or syring after sample has been stirred well with a fan pr paddle Several composite samples of gas can be bleeding gas slowly in a suitable container over a period of time

37. sample types 37 Liquid Samples Stir before sampling Sample remote from sources of contamination River or sea samples should be taken at points where contamination is minimal, taken at several depths and distances from shore.

38. sample types 38 Solid Samples: Most difficult to handle, can't be mixed well  take portions from different parts and mix them and make a representative sample.

39. sample types 39 Metal Sample: When a molten metal solidifies the purist solidifies first The core contains most impurities so ground a representative cross section of the sample, Bulk Samples: as coal, soil,…

40. Storage of Sample 40 Container should be clean and air tight Liquid and gas can be stored in plastic containers Solid samples should be sealed All samples should be stored in room not too hot or too humid Avoid storage for long times (decomposition, evaporation) special avoid glass bottles Advised freeze sample

41. Steps in an Analysis 41 1.Definition of the Problem: This is the first stage in planning the analysis, and can be considered as the process of deciding what analytical information is needed. 2.Choice of the Technique: Having decided what information is required, you next decide in detail how are you going to obtain it. 3.Sampling: The process of selecting and removing a small representative part of a whole on which you will perform the analysis. 4.Sample pre-treatment and separation: Only in the simplest of situations can the sample be analyzed without some form of pre-treatment to convert it into a form suitable for analysis. This pre-treatment may or may not involve separation.

42. Steps in an Analysis 42 5.Qualitative Analysis: Test on the (pre-treated) sample. (Under controlled conditions) Tests on reference materials for comparison. (Under controlled conditions). Interpretation of the tests. 6.Quantitative Analysis: (Needs more controlled conditions) Calibration – obtaining raw analytical data from suitably prepared standards. Measurements – obtaining the raw analytical data from measurements on the (pre-treated) sample. Maybe recalibration due to not valid for a long time. Evaluation – calculating a meaningful analytical result from the raw data.7 7.Action: The result is used to make a decision as to what action to take in relation to the original problem.

43. NEXT CHAPTER 43  CONTROLE OF QUALITY OF ANALYTICAL METHODS  VALIDATION METHODS Good Luck Dr. WAEL ABU DAYYIH