3/2/2020 Modern Instrumentation Methods and Technique 1. Separation Methods (GC, HPLC) 2. Spectroscopy 3. 4. 5. 6. 7. 8. Ultraviolet/ Visible Spectroscopy (UV-Vis) Infrared (FTIR) Spectroscopy Introduction to Nuclear Magnetic Resonance (NMR) Atomic Absorption (AAS) and Flame Emission Spectroscopy (FES) Elemental Analysis Statistical methods 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. Liquid Gas Column Flat sheet Major types: --- gas chromatography (GC) --- liquid chromatography (LC) 1 3/2/2020 Equilibration based on stationary phase 1. Adsorption 2. Partition 3. Ion exchange 4. Size dependent pore penetration mobile phase stationary phase mobile phase inert solid stationary phase Basic chromatographic process Sample: mixture of components a, b, and c SP= stationary phase MP= mobile phase 2 3/2/2020 Basic chromatographic process Sample: mixture of components a, b, and c MP= mobile phase SP= stationary phase Detector Elution is a process in which solutes are washed through a stationary phase by the movement of a mobile phase. Eluate is the mobile phase that exits the column. An eluent is a solvent used to carry the components of a mixture through a stationary phase. a b c 3 3/2/2020 Chromatogram is a plot of some function of solute concentration versus elution time. tR= retention time time required for the analyte to reach the detector tM= dead time (void time) time it takes for an unretained species to pass through a chromatographic column. tS (or t’R): adjusted retention time; time the analyte spends in the stationary phase Retention factor, kA: related to the rate at which A migrates through a column Selectivity factor, a, for solutes A and B is defined as the ratio of the distribution constant of the more strongly retained solute (B) to the distribution constant for the less strongly held solute (A). Skoog and West: Fundamentals of Analytical Chemistry 9E Column efficiency Number of theoretical plates: N Height equivalent to a theoretical plate: HETP or H L= length of the column Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed. 4 3/2/2020 Column efficiency Column resolution (Rs) Quantitative measure of the ability of the column to separate two analytes Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. Column resolution (Rs) If WA = WB Number of theoretical plates: N Retention factor, k Selectivity factor, a, Christian, Dasgupta, Schug - Analytical Chemistry 7th ed. 5 3/2/2020 Effect of column length on the resolution. Chemical Analysis - Modern Instrumentation Methods and Techniques 2nd ed - F. Rouessac, A. Rouessac (Wiley, 2007) Column efficiency Optimal flow rate Indicator ----- band broadening Variables That Influence Column Efficiency --- Velocity of mobile phase --- Diffusion coefficient in mobile phase --- Diffusion coefficient in stationary phase --- Retention factor --- Diameter and uniformity of packing particles --- Thickness of liquid coating on stationary phase velocity of the mobile phase components mix faster than they migrate Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. 6 3/2/2020 Optimization of chromatographic analysis --- choice of column --- length of column --- diameter of the column --- stationary phase composition --- mobile phase composition --- flow rate --- temperature ------------------- always required !!!!!!! The resolution and the elution time are the two most important dependent variables to consider. the resolution varies with the square root of the column length Effect of solvent on resolution and elution time water/acetonitrile 50/50% 55/45 water/acetonitrile 60/40% 65/35 How could you separate these two components? Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed. 7 3/2/2020 Retention factor, kA: related to the rate at which A migrates through a column k is optimal for 1 and 2 k is optimal for 5 and 6 k is optimal first for 3 and 4, Gradient elution then for 5 and 6 Gradient elution: steady changes of the mobile phase composition (HPLC) or temperature (GC) during the chromatographic run. Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. Solve the following problem Chromatographic response of an artificial mixture containing 6 essential oil components and methyl benzoate as an internal standard. Using the cinnamaldehyde peak 1. Determine the number of theoretical plates for the column. 2. Determine the height equivalent to a theoretical plate, if a fused-silica column of 0.25 mm x 30 cm with 0.25 μm film was used. Cinnamaldehyde Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. 8 3/2/2020 1. Determine the number of theoretical plates for the column. 2. Determine the height equivalent to a theoretical plate, if a fused-silica column of 0.25 mm x 30 cm with 0.25 μm film was used. 19.0 Cinnamaldehyde = 28523 H = 10.5 μm 0.45 Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. In what order wolud the following compounds be eluted from a column having an apolar stationary phase using a protic polar solvent? Benzene, acetone, and benzoic acid? Apolar molecule Organic acid Polar molecule Fastest: benzoic acid Second: acetone Last: benzene 9 3/2/2020 Consider two compounds for which tM= 1 min, t1= 11.30 min and t2=12 min. The peak widths at half-height are 10 s and 12 s, respectively. Calculate the time the analytes spend in the stationary phase. Calculate the column resolution. tS= tR – tM= Compound 1: 11.30 – 1 = 10.30 min Compound 2: 12 – 1 = 11 min = (12 – 11.30)∙60 = 18 s = 0.82 10 + 12 = 22 s Gas chromatography (GC) The stationary phase --- gas – solid (adsorption) chromatography --- gas – liquid (partition) chromatography porous solid immobilized liquid Analyte in the vapor state distributes between the stationary phase and the carrier gas. Gas-phase equilibria are rapid, so resolution (and the number of plates) can be high. Application: --- all gases --- volatile and thermostable liquids and solids (e.g. nonionized organic molecules upto 25 carbon atoms, organometallic compounds) --- non-volatile compounds can be derivatized https://www.agilent.com/en/products/gas-chromatography/gc-systems/7820a-gc-system 10 3/2/2020 Analyte in the vapor state distributes between the stationary phase and the carrier gas. The most important factor in gas chromatography is the selection of the proper column (stationary phase) for the particular separation to be attempted. The nature of the liquid or solid phase will determine the exchange equilibrium with the sample components; and this will depend on --- the solubility or adsorbability of the analytes, --- the polarity of the stationary phase and sample molecules, --- the degree of hydrogen bonding, and specific chemical interactions. Components of GC instruments: 1. 2. 3. 4. Sample injector Column Detector Data system Christian, Dasgupta, Schug - Analytical Chemistry 7th ed. 11 3/2/2020 Components of GC instruments: 1. 2. 3. 4. Sample injector Column Detector Data system The sample injection port, column, and detector are heated to temperatures at which the sample has a vapor pressure of at least 10 torr, usually about 50 °C above the boiling point of the highest boiling solute. The injection port and detector are usually kept somewhat warmer than the column to promote rapid vaporization of the injected sample and prevent sample condensation in the detector. Christian, Dasgupta, Schug - Analytical Chemistry 7th ed. Carrier gas free of all traces of hydrocarbons, water vapour and oxygen The carrier gas is a chemically inert gas available in pure form such as argon, helium, or nitrogen, or hydrogen. It has no significant influence on partition coefficient. A highly dense gas gives best efficiency since diffusivity is lower, but a low-density gas gives faster speed. The viscosity of the carrier gas and its flow rate have an effect on the analytes’ dispersion in the stationary phase and on their diffusion in the mobile phase. The choice of gas is often dictated by the type of detector. Flow rate is regulated by controlling the gas inlet. --- enables reproducibility of the retention times Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed. 12 3/2/2020 Carrier gas free of all traces of hydrocarbons, water vapour and oxygen The carrier gas is a chemically inert gas available in pure form such as argon, helium, or nitrogen, or hydrogen. It has no significant influence on partition coefficient. A highly dense gas gives best efficiency since diffusivity is lower, but a low-density gas gives faster speed. The viscosity of the carrier gas and its flow rate have an effect on the analytes’ dispersion in the stationary phase and on their diffusion in the mobile phase. The choice of gas is often dictated by the type of detector. Flow rate is regulated by controlling the gas inlet. --- enables reproducibility of the retention times The nature of the carrier gas has no significant influence upon the values of the partition coefficients K of the compounds between the stationary and mobile phases, owing to an absence of interaction between the gas and solutes. Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed. Injector Suitably sized sample inserted as a „plug” of vapour. --- packed columns: 0.1 – 20 μl liquid --- direct vaporization --- capillary columns: 2 order of magnitude smaller ----- split/splitless Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed. 13 3/2/2020 Colums Packed columns Capillary columns Colums Packed columns Capillary columns steel, glass 1-3 m (3-6 mm), spherical support (0.2 mm) high purity fused silica 12-100 m (0.1-0.5 mm/0.5 mm) --- routine applications --- increased capacity --- easy manifacturing, --- wide variety of stationary phases --- higher resolution --- higher speed --- trace analysis 14 3/2/2020 Stationary phases „like dissolve like” Very polar Non polar Highly polar Effect of temperature 45 °C optimum resolution vs. elution time low temp. higher temp. 145 °C Programmed 30 – 180 °C Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. 15 3/2/2020 Detectors universal vs. discriminating (selective) destructive vs. non-destructive single information vs. multiple information tR tR + structural info. Detectors Gas chromatography always uses flow-through detectors that automatically detect the analytes as they elute from the column; the majority of GC detectors are destructive. Flame ionization detector (FID) General Destructive Mass-sensitive Insensitive toward non-combustable gases (H2O, CO2, SO2 NOx) Not-sensitive to functional groups containing electronegative elements. (carbonyl, halogen, alcohol, amine) Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. 16 3/2/2020 Detectors Termal conductivity detectors Simple, general use Non-destructive Relatively low sensitivity Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. Detectors Electron Capture detectors High sensitivity for halogen cont. comp. Not-destructive Radioactive source: β-emitter Ni-63 Sensitive to molecules such as halogens, nitro compounds, peroxides Insensitive toward hydrocarbons, alcohols, amines Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. 17 3/2/2020 Detectors Mass spectrometry detectors (GC/MS) Typical gas chromatogram of unleaded gasoline, a complex mixture, using a capillary column. Christian, Dasgupta, Schug - Analytical Chemistry 7th ed. 18 3/2/2020 Application of GC Applicable to relatively volatile and thermally stable molecules. --- qualitative analysis (purity) --- quantitative analysis (calibration, internal standards) Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. Christian, Dasgupta, Schug - Analytical Chemistry 7th ed. https://www.legalhelplawyers.com/blood-alcohol-testing-drunk-driving-cases 19 3/2/2020 449 377 Accidently only 8 μL injected 20 3/2/2020 High Performance Liquid Chromatography (HPLC) --- adsorption chromatography --- partition chromatography --- normal phase chromatography polar stationary phase and nonpolar or inremediate polarity solvents silica hexan, THF, etc.... --- reverse phase chromatography nonpolar stationary phase and polar solvents octadecyl silica water – acetonitrile, methanol – water mixture --- ion exchange chromatorgraphy --- size exclusion chromatography Application: --- nonvolatile compounds --- small molecules --- macromolecules --- ionic species Chromatography In everyday’s life - Forensic Testing CSI and Law and Order? Investigating criminal caseases Crime scene testing: blood or cloth sample Arson verification: chemical residues after fire or explosions (GC) - Quality testing in food and pharmaceutical Industry - Is it really beefmeet in the burger? In 2013, In Europe more than 300 tonnes of horsemeat was sold as beefmeat. - Performance Enhancing Drug Testing Doping test from blood (HPLC) http://lasvegas.csiexhibit.com/ https://en.wikipedia.org/wiki/Hamburger http://post.jagran.com/iitroorkee-engineers-develop-sensor-to-give-dope-test-results-in-5-minutes-1312087343 21 3/2/2020 Basic components of a HPLC system Christian, Dasgupta, Schug - Analytical Chemistry 7th ed. Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed. Guard columns --- usually contain the same packing as the analytical Analytical columns Stainless steel tubing 4.6 mm inner diameter 5 – 250 mm lengths Christian, Dasgupta, Schug - Analytical Chemistry 7th ed. 22 3/2/2020 Stationary phases in HPLC Spherical microporous silica particles, 10 μm CH3 Christian, Dasgupta, Schug – Analytical Chemistry 7th ed. Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed. 23 3/2/2020 The effect of column temperature upon compound separation. (a) 25°C, (b) 35°C, (c) 45°C Chemical Analysis - Modern Instrumentation Methods and Techniques 2nd ed - F. Rouessac, A. Rouessac (Wiley, 2007) Detectors UV detectors Fluorescence flow cell Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed. 24 3/2/2020 Chromatograms of a sample containing two compounds A and B, for which the UV spectra are different. Schematics of a differential refractive index detector. Chemical Analysis - Modern Instrumentation Methods and Techniques 2nd ed - F. Rouessac, A. Rouessac (Wiley, 2007) 25 3/2/2020 Comparison of HPLC and GC Both methods: --- efficient, highly selective, widely applicable --- only small amounts of sample required --- readily adapted to quantitative analysis HPLC --- can accommodate nonvolatile and thermally unstable compounds --- generally applicable to inorganic ions GC: --- simple and inexpensive equipment --- rapid --- easy to interface with mass spectrometry --- not for macromolecules or salts Thin-Layer Chromatography (TLC) Planar form of chromy. Stationary phase: --- finely divided sorbent on glass, metal (Al), plastic sheet --- multiple samples can be simultaneously analysed Three stages: 1. Sample spotting (0.5 – 5 μl) 2. Plate development 3. Detection Christian, Dasgupta, Schug – Analytical Chemistry 7th ed. 26 3/2/2020 Stationary phase: Mobile phase: --- finely divided sorbent on glass, metal (Al), plastic sheet --- if silica or alumina used as stationary phase 60 Å pore size silica 10 -12 μm mean particle size 200 – 250 µm sorbent layer thickness 10 – 15 cm migration distance Developing eluting power of solvents increases in the order of their polarities (e.g. from hexane to acetone to alcohol to water) Detection of spots / Visualization --- UV lamp (265 nm or 365 nm) --- fluorescent analyte --- plate contains fluorescent zinc-salt --- iodine vapours --- colored species can be visually observed Analyte is characterized by the Rf (retardation factor) value: Resolution: Rs=2(x1–x2)/(w1+w2) Efficiency: N=16(x1/w)2 w= spot diameter Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed. 27 3/2/2020 Two-dimensional TLC --- two successive elution with two mobile phases Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed. Questions 1: GC response of an artificial mixture containing 6 essential Oil components and methyl benzoate as an internal standard. 1. Determine the number of theoretical plates for the column. 2. Determine the height equivalent to a theoretical plate, if a fused-silica column of 0.25 mm x 30 cm with 0.25 μm film was used. Cinnamaldehyde Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. 28 3/2/2020 1. Determine the number of theoretical plates for the column. 2. Determine the height equivalent to a theoretical plate, if a fused-silica column of 0.25 mm x 30 cm with 0.25 μm film was used. 19.0 = 28523 Cinnamaldehyde H = 10.5 μm 0.45 Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. Question 2: Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. 29 3/2/2020 The largest loss of ibuprofen as a percentage occurs between 1.0 and 1.5 hours. Question 3: How could you calculate the efficiency (N) of a TLC plate for a compound whose migration distance is x and spot diameter is w? How could you define the H value? Resolution: Rs=2(x1–x2)/(w1+w2) Efficiency: N=16(x1/w)2 w= spot diameter 30 3/2/2020 Question 4: In what order would the following compounds be eluted from an alumina column using n-hexane as the eluting solvent? CH3CH2OH CH3CHO CH3CO2H They would elute in increasing order of polarity: fatest: acetaldehyde second: ethanol last: acetic acid Christian, Dasgupta, Schug – Analytical Chemistry 6th ed. Question 5: Fastest: benzoic acid Second: acetone Last: benzene Christian, Dasgupta, Schug – Analytical Chemistry 7th ed. 31 3/2/2020 Question 6: What solvent would you choose to separate a group of hydrocarbons, CH3(CH2)xCH3, on an alumina column? A nonpolar solvent like hexane or heptane. Christian, Dasgupta, Schug – Analytical Chemistry 6th ed. Question 7: What is normal-phase chromatography? Reversed-phase chromatography? Stationary phase Mobile phase Normal phase chromatography: polar non-polar non-polar molecule move faster Reversed-phase chromatography: non-polar polar polar molecule move faster Christian, Dasgupta, Schug – Analytical Chemistry 6th ed. 32 3/2/2020 Question 8: Christian, Dasgupta, Schug – Analytical Chemistry 7th ed. Question 9: Christian, Dasgupta, Schug – Analytical Chemistry 7th ed. 33 3/2/2020 Question 10: Fastest: 1 Second: 3 Third: 2 Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed. Question 11: Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. 34 3/2/2020 tR= retention time time required for the analyte to reach the detector tM= dead time (void time) time it takes for an unretained species to pass through a chromatographic column. tS (or t’R): adjusted retention time; time the analyte spends in the stationary phase Retention factor, kA tR= 30 min Selectivity factor, a tR= 50 min tM= 4 min Skoog and West: Fundamentals of Analytical Chemistry 9E original column longer column 35 3/2/2020 Question 12: Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. Question 13: Substances A and B have retention times of 16.40 and 17.63 min, respectively, on a 30 cm column. An unretained species passes through the column in 1.30 min. The peak widths (at base) for A and B are 1.11 and 1.21 min, respectively. Calculate (a) The column resolution, (b) The average number of plates in the column, (c) The plate height, (d) The length of column required to achieve a resolution of 1.5, and (e) The time required to elute substance B on the column that gives an Rs value of 1.5. Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. 36 3/2/2020 a) b) c) Retention factor, kB d) Selectivity factor, a original column longer column 37 3/2/2020 e) Question 14: Retention factor, kA Void time is 1.05 min Selectivity factor, a 0.03735 0.90337 N= [Rs∙4/(0.03735∙0.90337)]2 = Rs2∙14053.52 kB= [(10.5+22/60)‒1.05]/1.05= 9.3492 kA= [10.5‒1.05]/1.05= 9.0 α= 9.3492/9= 1.0388 Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed. 38