Column Chromatography (Week 1) PDF

BIOM2702 Column Chromatography (HPLC & GC) Dr. Mark D. Evans [email protected] Gas Chromatography - Learning Objectives Explain how the chromatography in a GC works Describe the types of sample that can be analysed by GC and the physicochemical characteristics of such...

BIOM2702 Column Chromatography (HPLC & GC) Dr. Mark D. Evans [email protected] Gas Chromatography - Learning Objectives Explain how the chromatography in a GC works Describe the types of sample that can be analysed by GC and the physicochemical characteristics of such samples Describe some biomedical examples of applications of GC  Chromatography Instruments paration of individual components in mplex mixture by chromatography Complex mixture Injection port Detector Colu mn Optional Column Oven Mobile Phase Detection, quantification & identification of individual components in complex mixtu  Gas Chromatography (GC) Injection port Detector Mobile phase = a gas Colu Stationary phase = liquid or liquid coated particles Carrier Gas mn Column Oven Helium, GC is most suited to Detector response nitrogen, analysing volatile argon, compounds. hydrogen Some compounds can be derivatised to make them volatile. Gas-Liquid Chromatography (GLC) Retention time  GC in HB 2.08 Photographs - Dr Antonio Peña- Fernández  Chromatography tography exploits different physicochemical properties of compounds: , size, charge, selective binding… cilitate separation Mobile phase (contains the compounds to be separated.) Stationary phase (has features enabling separation of the compounds by exploiting the physicochemical properties of those compounds.) Compounds separate based on their relative affinity for stationary and mobile phases There is nothing for compounds to interact with in the mobile phase in GC. Compounds separate based on a combination of their volatility and interaction with stationary phase.  Gas Chromatography Column Still a glass column, just longer, narrower, more Capillary GC: Most common form of flexible uses narrow bore capillary columns pending on the type of stationary phase various interactions tween compounds in mixture and stationary phase can occur  Van der Waals  Dipole-dipole  Hydrogen bonding  GC - Detectors FID Ions form in flame e- Collected by electrode Give a signal, more ions = more compou Mass Spectrometer. Gas chromatography – Mass Spectrometry (GC-MS) or GC-MS/MS (tandem mass spectrometry)  Biomedical Applications of GC b.: Specialist lab. within a clinical biochemistry (chemical pathology) lab. s of abuse eutic Drug Monitoring (TDM) GC-Mass Spectrometry (GC-MS) of a drug (or a metabolite) in blood or urine sample might TDM be indicated? therapeutic levels do not differ greatly from toxic levels (narrow therapeutic index), e.g. lithium, digoxi ents who have impaired clearance of a drug with a narrow therapeutic index, e.g. patients with renal fai sed clearance of digoxin, so higher risk of toxicity.  Sampling for GC Analysis Volatile Where might you find volatiles in biologically relevant samples? Volatile Organic Compounds (VOCs) 1. Inert gas Trap Trap with adsorptive packing 2. e.g. polydimethylsiloxane Heat 1. GC inlet Pump Analy sis Purge & Trap Gas Chromatography - Learning Objectives Explain how the chromatography in a GC works  Describe the types of sample that can be analysed by GC and the physicochemical characteristics of such samples  Describe some biomedical examples of applications of GC   HPLC - Learning Objectives Describe the different components of a HPLC instrument Describe how the chromatography in HPLC works Describe the types of detector commonly used in HPLC and their relative advantages and disadvantages Give examples of the use of HPLC in biomedical science  HPLC Instrument Injection port Detector Colu mn Column Oven Mobile Phase Pump HPLC can analyse non- volatile and (soluble) volatile compounds In HPLC, the mobile phase is a liquid  HPLC Instrument – HB2.08 Degasser Mobile Phase Injector Column Pump Detector Dr Antonio Peña-Fernández  HPLC Instrument – HB2.08 Degasser Mobile Phase Injector Pump Column Detector Dr Antonio Peña-Fernández  HPLC Stationary Phase 2 μm – 10 μm Porous, spherical microparticles of silica Very large surface area Analytical HPLC High pressures to force mobile phase through column Stationary phase is packed into a steel col  HPLC Column Accessories HPLC column oven HPLC Guard Column: Helps keep the more expensive analytical column ‘cl  Sample Injection Glass syringe or autosampler Sample Loop How a rheodyne valve works: https://www.youtube.com/watch?v=8-D-mM  Learning Objectives Describe the different components of a HPLC instrument  Describe how the chromatography in HPLC works Describe the types of detector commonly used in HPLC and their relative advantages and disadvantages Give examples of the use of HPLC in biomedical science  HPLC Stationary Phase lanol groups are often derivatised Compounds being separated partition between mobile and stationar End capping Stationary Mobile Octadecylsilane (ODS) – C18 TMS = trimethylsilane –Si(CH3)3  HPLC Mobile Phase Baseline Reverse phase HPLC mobile phase: Aqueous buffer solution plus some organic solvent, e.g. acetonitrile, m Unreactive ‘Degassing’ – stabilise UV/Vis absorbance detection (baseline stability)  Isocratic elution – unchanging composition of mobile phase during HPLC nt elution – mobile phase changes composition during HPLC analysis  Learning Objectives Describe the different components of a HPLC instrument  Describe how the chromatography in HPLC works  Describe the types of detector commonly used in HPLC and their relative advantages and disadvantages Give examples of the use of HPLC in biomedical science  Peak Resolution Ideally peaks should be sharp, symmetrical and well resolved Low resolution High resolution Tailing Baseline  HPLC Detectors Electrochemical Spectrophotometric Fluorescence Applicable to analytes that can be oxidised or red Sensitive Aldehydes Ketones Peroxides (R-OO-R’) Excite with laser & measure fluorescence Phenols Sensitive Aromatic amines Plasma irinotecan Plasma homocysteine, cysteine, cysteamine Mass spectrometric  HPLC Detectors Spectrophotometric (UV-visible) Single wavelength or Diode Array Detector (DAD) Sensitive to low concentrations of every analyte. Insensitive to changes in temperature & mobile phase compo Enables collection of entire spectrum for a peak Helps with confirmation of peak (compound) identificatio  Learning Objectives Describe the different components of a HPLC instrument  Describe how the chromatography in HPLC works  Describe the types of detector commonly used in HPLC and their relative advantages and disadvantages  Give examples of the use of HPLC in biomedical science  HPLC Applications Rapid Widely applicable Sensitive Amino acids Proteins Nucleotides Nucleic acids Drugs of abuse/therapeutic drugs Vitamins (fat and water soluble) Carbohydrates and oligosaccharides Ultra-High Performance Liquid Chromatography (UPLC) Key features of UPLC versus HPLC HPLC: Packing material size = 3-5 µm & typical operating pressures = 2000 - 4000 psi. UPLC: Packing material size = 1.7 µm & typical operating pressures = 6000 - 15000 psi. UPLC faster chromatography UPLC better peak resolution UPLC higher sensitivity UPLC lower solvent usage  HPLC - Learning Objectives Describe the different components of a HPLC instrument  Describe how the chromatography in HPLC works  Describe the types of detector commonly used in HPLC and their relative advantages and disadvantages  Give examples of the use of HPLC in biomedical science 