Lecture 5 - Chromatography Revision Session PDF

12/02/2024 CH3F2 - Chromatography Revision Session Use the app or go to: vevox.app Meeting number: 126-545-180 1 CH3F2 exam - Chromatography Two chromatography questions in 2023/24 (new!) – ONE compulsory question [20%] Similar style to the questions in the last 5 years, focussing on application of...

12/02/2024 CH3F2 - Chromatography Revision Session Use the app or go to: vevox.app Meeting number: 126-545-180 1 CH3F2 exam - Chromatography Two chromatography questions in 2023/24 (new!) – ONE compulsory question [20%] Similar style to the questions in the last 5 years, focussing on application of chromatographic techniques to an unseen previously problem. For revision please use: lecture slides and quizzes, revision session, past papers, DryLab session – ONE optional question [20%] Similar style to the workshop #1, focussing on chromatography calculations For revision please use: Workshop #1 problems, Workshop #1 Moodle quiz (for answers) and Chromatography FAQs. 2 1 12/02/2024 Join: vevox.app ID: 126-545-180 Enter Text and Press Send Type a word related to chromatography 45 3 Types of Chromatography Chromatography is divided into categories on the basis of the mechanism of interaction of the solute with the stationary phase Solute adsorbed on surface of stationary phase Adsorption Chromatography Cross section of open tubular column Solute dissolved in liquid phase bonded to the surface of the column Partition Chromatography 4 2 12/02/2024 Types of Chromatography Chromatography is divided into categories on the basis of the mechanism of interaction of the solute with the stationary phase Large molecules are excluded Mobile ions held near cations that are covalently attached to stationary phase Small molecules penetrate pores of particles Anion-exchange resin; only anions can be attracted to it Ion-exchange Chromatography Size-exclusion Chromatography 5 Join: vevox.app ID: 126-545-180 POLL OPEN Which method would you use to separate the molecules below? 1. Adsorption - because they have different polar groups 11.11% 2. Partition - because they have different solubilities/partition coeff. 25.93% 3. Size exclusion - because they are different size 3.7% 4. Gas chromatography - because they have different boiling points 59.26% 45 6 3 12/02/2024 1 Separation based on physical properties of the analytes Molecular characteristic Polarity Physical property Separation technique volatility Gas chromatography solubility Partition chromatography adsorptivity Adsorption chromatography charge Ion Exchange Chrom. Electrophoresis diffusion Size Exclusion Chrom. Dialysis sedimentation Ultracentrifugation ligand binding Affinity Chrom. Ionic Size (mass) Shape 7 Join: vevox.app ID: 126-545-180 POLL OPEN Which method will be best to use to separate a mixture of similar proteins? 1. Adsorption Chromatography 7.14% 2. Partition Chromatography 21.43% 3. Gas Chromatography 3.57% 4. Ion Exchange Chromatography 35.71% 5. Size Exclusion Chromatography 32.14% 45 8 4 12/02/2024 2 Optimising separation efficiency N: is the average column plate number for the two bands of interest k: is the average capacity factor α: separation factor of the two peaks k and α are in practice controlled by changing the column packing and by varying the composition of the mobile phase solvent (solvent parameters) N is controlled by changing the mobile phase flow rate, packing particle size and column dimensions 9 Join: vevox.app ID: 126-545-180 POLL OPEN A chromatogram shows poor separation for the critical pair, how would you optimise this separation without increasing the run time? Vote for up to 4 choices 1. Increase the column length 26.67% 2. Decrease the column length 13.33% 3. Increase the particle size 20% 4. Decrease the particle size 33.33% 5. Introduce solvent gradient 86.67% 6. Optmise the flow rate 73.33% (% = Percentage of Voters) 60 11 5 12/02/2024 LC/HPLC Modes of Separation Normal Phase: Classic form of liquid chromatography using polar stationary phases and non-polar mobile phases The analyte is retained by the interaction of its polar functional groups with the polar groups on the surface of the packing Analytes elute from the column starting with the least polar compound followed by other compounds in order of their increasing polarity Reversed Phase: The stationary phase is non-polar and the mobile phase is polar The analytes are attracted to the surface by their non-polar functional groups The most polar analyte elutes from the RP column first followed by other analytes in order of decreasing polarity Ion-exchange Size exclusion chromatography 13 Join: vevox.app ID: 126-545-180 POLL OPEN Which LC method is best suitable for separation of the following molecules? 1. Normal Phase LC/HPLC 26.92% ✓ 2. Reverse Phase LC/HPLC 61.54% 3. Size Exclusion Chromatography 0% 4. Ion Exchange Chromatography 11.54% 45 14 6 12/02/2024 Van Deemter model H = A + B/u + u [CM +CS] A: random movement through the stationary phase B: diffusion in the mobile phase C: interaction with the stationary phase H: plate height u: average linear velocity u = L/ tM 15 Join: vevox.app ID: 126-545-180 POLL OPEN Why does gradient elution (in HPLC) and temperature programming (in GC) limit band broadening? 1. Temperature and polarity both affect the plate height 23.33% ✓ 2. Solute spends more time in the SP where diffusion is less than in the MP 36.67% 3. Changing the solvent and temperature changes the visosity of the MP limiting diffusion 36.67% 4. Mass transfer is sped up by increasing the temperature 3.33% 60 16 7 12/02/2024 PAST EXAM QUESTIONS 17 Join: vevox.app ID: 126-545-180 POLL OPEN Which method would be most suitable to separate the molecules below: 1. Isocratic normal phase HPLC 6.45% 2. Gradient reverse phase HPLC 70.97% 3. Gas chromatography 19.35% 4. TLC 3.23% 5. Size Exclusion chromatography 0% 60 18 8 12/02/2024 Join: vevox.app ID: 126-545-180 POLL OPEN Which LC technique and mode would you use to separate the following mixtures of molecules? 1. Normal phase LC 7.14% 2. Reverse phase HPLC 57.14% 3. Ion Exchange 7.14% 4. Size Exclusion Chromatography 0% 5. Gas Chromatography 28.57% 60 19 Join: vevox.app ID: 126-545-180 POLL OPEN What detector might be suitable for HPLC separation of the following molecules? Vote for up to 3 choices 1. UV-Vis detector 60% ✓ 2. Refractive Index detector 80% ✓ 3. Electrochemical detector 20% ✓ 4. Mass spectrometry 40% 5. Fluorescence detector 20% (% = Percentage of Voters) 45 20 9 12/02/2024 Join: vevox.app ID: 126-545-180 POLL OPEN Which of the following mixtures are most suitable for separation using GC? W X Y Z Bovine Serum Albumin ✓ 1. Lysozyme Sex Hormone-Binding Globulin Vote for up to 2 choices W 91.67% 2. X 3. Y 4. Z 8.33% 4.17% ✓ 79.17% 60 (% = Percentage of Voters) 21 Join: vevox.app ID: 126-545-180 POLL OPEN What TWO methods in tandem could be used to purify protein X from the other proteins in the mixture, to obtain a high purity product? Protein Concentration (mg/ml) Ovalbumin Ovotransferrin Ovomucoid Ovomucin Protein X G2 globulin G3 globulin Ovoinhibitor Cystatin Isoelectric point (pI) 54 12 11 3.5 3.4 4 4 1.5 0.1 4.5 6.1 4.1 4.5-5.0 10.7 5.5 4.8 5.1 5.1 Molecular weight (kDa) 45 76 28 5500-8300 14 30-45 49 13 Denaturation temperature Td (°C) 84 61 77 75 93 - Vote for up to 2 choices 1. Normal phase TLC 2. Normal phase LC/HPLC 3. Reverse phase HPLC 4. Size Exclusion Chromatography 5. Gas Chromatography 6. Ion Exchange 0% 11.11% 27.78% 72.22% 0% 60 77.78% 22 10 12/02/2024 Join: vevox.app ID: 126-545-180 Pick one chromatographic method most suitable for separation of the compounds below Enter Text and Press Send 23 11