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Lecture 1: Analytical Instrumentation 1: Units of concentration and quantification (not quantitation)    

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ppm (grams per million grams) / ppb (grams per billion grams) prefixes: milli, micro, nano, pico, femto, atto, zepto, yocto parts per notation External standard calibration:  Sample => spectrophotometry => Straight line (calibration curve) => concentration range  (+) simple, less time consuming, useful for simple matrices  (-) variability in sample preparation / matrix effects  dont use word methodology in report, use something means as developing the method Internal standard calibration:  Interanal standard is added to Standard solutions & samples  Samples vs ratio of responses of component to internal standard  Different from above method: RATIO Standard addition calibration:  More accurate method  Standard is added directly to sample  Absolute value of x-inter => determine concentration  Common way, advantage method for complex matrices (most accurate way), compensates for sample matrices Quantification tutorial (example of question/answer): refer to tutorial data What method is the best out of 3? Not enough information it should be analysed more to

Lecture 2: GAS CHROMATOGRAPHY -

Produce a stable flow and pressure for the mobile phase Stable flow requires a tank of compressed gas a pressure regulator and a value

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Example of THE CHROMATOGRAM

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Gas Chromatography:  provides both qualitative and quantitative information about individual components in a sample. Compounds that have volatility below 350 to 400 degrees are suitable for analysis GC. Compounds must be stable at high temp, rapidly transformed into the vapour state without reaction with other compounds.  General rules: - most HYDROCARBONS are suitable - inorganic compounds (metals, salts) do not posses the required volatility => not suitable - many biomolecules and pharmaceuticals are sensitive => not suitable - the greater the molecular weight/the more polar a compound, the lower the volatility => overall it’s estimated that only 10% of all compounds can be analysed by GC

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Factors Affecting Selectivity: there are 3 main factors  The nature of the compound  The stationary phase  The column temp

 Others: column dimension and carrier gas do not have a direct affect upon selectivity 

Stationary phases:  WCOT: wall-coated open tubular consist of a capillary tube whose walls are coated with liquid stationary phase.  PLOT: porous layer open tubular columns in which a thin layer of adsorbent is affixed to the inner walls of the capillary.  SCOT: support coated open tubular consist of capillary lined with a thin layer of support material, onto which stationary phase has been adsorbed.

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Column comparisons table

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Column Dimensions  Internal Diameter: the smaller the diameter, the greater the efficiency => better resolution. Reduce the diameter by half and the column efficiency doubles.  Film Thickness: - Sample loading: thick film column is recommended to use for samples with a variation in solute concentration. This will reduce the possibility of broad overloaded peaks co-eluting with other compounds of interest. If the separation of 2 solutes is sufficient, then a thinner film can be used. - Volatility of solute: the greater the film thickness, the greater the retention of a solute => the higher the elution temperature. - Separation of a series of compounds on capillary columns with the same internal diameter, but different film thicknesses, show that the retention times of components increase with thicker film layers. - A greater film thickness allows for a greater fraction of time spent in the stationary phase

 Phase ratio: - As long as the  (phase ratio) values are the same, the columns will give similar elution times and patterns.

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Column Selection  Column length: select the shortest column length that will provide the required resolution for the application (12-30 meters). Resolution is proportional to the square root of the column efficiency. => double the column length will only increase the resolving power of the column by approx. 40%

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Chromatographic Process  Compound molecules dissolve in the stationary phase  For a given compound, it may dissolve in 1 stationary phase more than another => result in longer retention.

 Different molecules dissolve in the stationary phase at different rates according to their polarity and the polarity of the stationary phase  At higher temp, there are fewer molecules in the stationary phase => Results in shorter retention times  At higher temp, the differences between the distribution constants for different compounds, become less. Smaller distribution differences results in less separation. 

Column Bleed  Is the elution of degradation products of stationary phase, causing a background signal in GC detector

 All columns produce bleed products, the most common degradation shown opposite

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Van Deemter Equation

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Linear Velocity vs Flow rate

 Linear velocity: speed (cm/sec) at which the carrier gas travels thru the column  Flow rate: volume of carrier gas passing thru the column per unit time (ml/min)  Linear velocity is more meaningful for capillary column as it affects efficiency & retention time  Linear velocity is not uniform throughout the column => average linear velocity is used for measurement purposes 

Optimum Average Linear Velocity  There is an optimal average linear velocity for each carrier gas where maximum efficiency is obtained  The optimal average velocity is when the curve is at its lowest point (highest N)  Using OPGV (optimal practical gas velocity) is recommended as a higher average linear velocity, this results in shorter retention time, compensates for lower linear velocities when temp is increased

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Carrier Gases  Hydrogen, helium, nitrogen and argon are often used as carrier gases  Helium and hydrogen are best for capillary columns  Nitrogen is suitable carrier gas, but not recommend except for special cases  Argon/methane should not be used for capillary columns Nitrogen  Provides the best efficiency when compared to helium and hydrogen  Optimum average linear velocity is very low resulting in long retention times  Van deemter curve is steep => efficiency drops sharply with any deviation from the optimum  Poor carrier gas for compounds that elute over a wide temp range 

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Lecture 4: HPLC - High Performance Liquid Chromatography Learning outcomes: -types of chromatography -basic chromatography theory:  Van Deemter equation  HETP  Plate count  Resolution  Capacity factors  Selectivity factors

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Chromatography: separation method. The components are separated bewtten 2 phases: stationary phase (solid), mobile phase (liquid or gas)  Column chromatography  Absorption chromatography  Ion-exchange chromatography: separation based on eletrostatic attraction of solute ions of opposite charge ions present on stationary phase  Size-exclusion chromatography: no attraction between solid phase and solutes; separation based on size; known as molecular exclusion chromatography and gel permeation chromatography  Other types – thin layer chromatography/gas chromatography/electrophoresis

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High Performance Liquid Chromatography: liquid mobile phases is pumped through a column packed with a stationary phase. Separation occurs due to attraction of the solute the liquid and stationary phase The chromatogram: refer to the graph Reversed Phase:  Non polar stationary phase  Polar phase  Polar compounds elute first, neutral compounds elute last Reversed Phase – Isocratic: HPLC C18 Height equivalent to a theoretical plate: measure of the efficiency of a column (example refer to photo)

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Van Deemter equation: A = multiple paths B = longitudinal diffusion C = mass transfer U = linear velocity of mobile phase

 Multiple paths (A): molecules pass thru stationary phase that differ in path and length  Longitudinal diffusion (B): solute spreads out due to a concentration gradient since the solute spends time in the column  Mass transfer (C):  About van deemter plot: 

Plate count, N

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Capacity factor: measure of retention relative to an unretained solute K’ should be between 1-10

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Selectivity factor: describes the separation of 2 peaks relative to each other; defined as the ration of the capacity factor. The high the selectivity factor, the better the separation

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Resolution: degree of separation of 1 component from another

 resolution equation shows the dependence of resolution on selectivity, capacity factor and plate number

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Mixing solvents: low pressure gradient / high pressure gradient

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TUTORIAL QUESTIONS:

Lecture 5: METHOD VALIDATION 

Types of Analytical Procedures to be Validated:  Identification tests  Quantitative tests (impurities’ content)  Limit tests (control of impurities)  Quantitative tests of the active moiety in samples

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Identification tests: to ensure the identity of an analyte in a sample, achieved by comparison of a sample compared to a reference standard (spectrum, chemical reactivity, etc) Impurity testing: either a quantitative test or limit test, reflect the purity characteristics of the sample Assay procedures: to measure the analyte present in a given sample.  assay represents a quantitative measurement of the major component in drug substance  dissolution:

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Validation characteristics:  Accuracy: how close to the result  Precision: repeatability –, Intermediate precision –, reproducibility- method has been changed to be tested in a various ?, last step, harmonize method  Specificity: How specific the sample, absolutely sure  Detection limit: smallest amount of the amount that can be detected without being quantify as an exact value  Quantification limit: smallest amount can be measured with being determined with suitable precision and accuracy  Linearity: how linear the detection is, are directly proportional to the concentration of analyte in sample  Range: interval between the upper and lower concentration in sample

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Analytical Procedure: the way to form a task, performing the analysis, this should be described in detail with steps necessary perform in each test eg. The sample, the reference standard, reagents preparations, use of apparatus, calibration curve, formula for calculation

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Specificity: ability to assess the analyte, this includes impurities, degradants, matrix,..  Identification: ensure the identity of an analyte

 Purity tests:  Assay: give an exact result that allows an accurate statement on content/potency of the analyte in sample 

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Accuracy: express the closeness of agreement between the value (which is accepted/reference) and the value found, accuracy maybe inferred once precision, linearity and specificity have been established Precision: express the closeness of agreement between a series of measurement obtained from multiple sampling in same homogeneous sample.  Considered at 3 levels (repeatability, intermediate precision and reproducibility)  Precision procedure: expressed as the variance, standard deviation or coefficient of variation

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Robustness: measure of its capacity to remain unaffected by small, deliberate variations, indicate its reliability during normal usage Considerations: main objective of validation of analytical procedure is to demonstrate that the procedure is suitable for its purpose Detection limit:

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System suitability:

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Tutorial question:

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Lecture 8: MICROWAVE PLASMA-ATOMIC EMISSION SPECTROSCOPY (MP-AES) 

Atomic Spectroscopy  Emission from a thermally populated excited state  Absorption of sharp lines from a hollow cathode lamp  Fluorescence following the absorption of laser radiation

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Atomic Absorption Spectroscopy  Samples are vaporized at 2000-6000K, decomposed into atoms  Concentration are measured by absorption of characteristic wavelengths of radiation  Analytes are measured between ppm(ug/g) and ppt (pg/g) levels  Each element analysis requires a specific, different lamp cathode

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Atomic Absorption (AAS)

 Liquid sample is aspirated into a flame with temperature 2000-3000K  Liquid evaporated, the remaining solid is atomised in the flame  The pathlength of the flame is 10cm, increase total absorbance

 The cathode is bombarded with energetic Ne+ or Ar+ ions  Excited Fe atoms vaporise, emit light at the same wavenlengths absorbed by Fe atoms in the flame  A detector measures the amount of light passing through the flame  Different lamp is required for each element In AAS, the element being analysed is measured by the light it absorbs. Isotopes of an element absorb at the same wavelenghts.  AAS does not distinguish bewteern isotopes.

Development of atomic absorption (pg2), Tetron’s entry into atomic absorption (pg3), Atomic absorption milestones at varian (pg3), Agilent 4100 MP-AES (pg3) => refer to lecture slide 

Excitation sources  Air/Acetylene : 2200C  Nitrous oxide/ Acetylene: 2900C  N2 plasma: 3500-5500K  Ar plasma: 6000-10000K  Plasma is not a Flame

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Absorption vs Emission

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Effect of temperature on Emission

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Atomic Absorption overview

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Microwave plasma Emission overview...