Instrumental Analysis Lecture Notes (PDF)

Instrumental Analytics Harald Hundsberger, Krems Wintersemester 23/24 INSA Overview Centrifugation UV/Vis-Spectroscopy and applications Measurements: Kinetic/Endpoint Fluorescence Spectroscopy & Applications Fluorescence Intensity (RT-PCR, HTS, Sequencing, CE, Laser Scanning Microscopy, Lumin...

Instrumental Analytics Harald Hundsberger, Krems Wintersemester 23/24 INSA Overview Centrifugation UV/Vis-Spectroscopy and applications Measurements: Kinetic/Endpoint Fluorescence Spectroscopy & Applications Fluorescence Intensity (RT-PCR, HTS, Sequencing, CE, Laser Scanning Microscopy, Luminescence Chromatografy Principles IEX FPLC HPLC Electrophoresis/CE 2D Electrophoresis Mass Spectroscopy  Instrumental Analytics Lecture deals with instruments but main focus is the application ! Flow Cytometry https://oncohemakey.com/principles-of-flow- cytometry/ Part-I Centrifugation Centrifugation- Types of Centrifuges Centrifugation is a wide spread technique All labs is industry and academia do have a lot of different centrifuges for different applications Benchtop C. low volumes and low speed Centrifuges with higher volumes (several liters) -big laboratory centrifuges Ultracentrifuges - Vacuum is applied (100.000 rpm) high g forces to seperate subcellular components. Elutriation Centrifuges, for cell separation (size) Safety Considerations UC Safety Considerations using Ultra Centrifuges (and other centrifuges) Use specified rotors only (user manual) Use specified sample holders only Weight samples and avoid unbalance (sub milligrams !!!). Rotors and other equipment sometimes have specified lifetime !!! DANGER OF LIFE !! Classification on Rotor Type Fixed angle rotors Sample Swing out rotor separation Centrifugation-Basics V= sedimentation speed g = relative centrifugal accelaration d = size of particle ρ(p)= density of particle ρ(m)= density of medium η = viscosity of medium V= d2(ρp-ρm)g/18η Svedberg Equation If centrifugation occurs in media with lower density of particles and low viscosity, the size is the dominating factor for sedimentation !! Centrifugation Techniques Differential Centrifugation Cell disruption Pellet nuclei Pellet mitochondria Pellet ER and other membranes Zonal Centrifugation Medium consists of a density gradient for example sucrose: density and viscosity are increasing Centrifugation is done with high g-forces and over longer time periods Gradients slows down fast particles maximum density of medium should be higher than the lowest density of the separated particles Recommended for particles which differ in size ! C. is stopped before particles will pellet Fraction collection Media for density Gradients Cesium Chloride Percoll advantage is low viscosity, disadvantage good osmolarity capabilities for whole organelle high osmolarity, used for nucleic acid preparations separation Sucrose Adv.: non-inionic, no interaction between biological material, Disadv.: low resolution  Applications for Centrifugation Pellet Cells, Harvest Cells Yeast, Bacteria other cells from fermentation Protein Purification Fractions of cells Purification of membrane proteins Desalting/Concentration of protein solutions (Amicon tubes) Pellet Nucleic Acids Plasmid Prep RNA Prep Spin columns (Silica based) Subcellular Fractionation Cell separation Elutriation Part II - Spectroscopy Basics Optical Spectroscopy Advantages: Little requirements on sample preparations, sample purity and work load Well defined instrumentation (Laboratory standard equipment) Applications for Spectroscopy in Biochemical Disciplines: Structure (CD-Circular Dichroism) Concentration (UV/Vis, Fluorecence) Bound/non bound state (Fluorescence Polarization) Reaction/Kinetcis (Fluorescence, Absorbance) Analysis of substances (Absorbance, IR-Spectroscopy) What is Light ? Electric & magnetic wave = Light ! Electric & magnetic vector are oscillating sinusuidally Dualism of light: light can act as particle: corpuscular theory Polarized Light Electro Magnetic Spectrum E=h.v E……………….Energy The higher v the more h………………..Planck`s constant energy light has: v………………...frequency UV light causes sunburn  Interaction of Light and Material/Sample Light can interact with material: photons can bring electrons to higher energy levels Transitions are very fast (S0 to S1 10-14 sec !) Light can be absorbed without later radiation Light can be absorbed and can cause later radiation Excited state has finite life time Excited state is more unstable/reactive fast:fluorescence slower:phosphorescence Part III - Spectroscopy UV/Vis/NIR-Spectroscopy Basics of Photometry Photometry: Measurement of the specific absorbence of light (absorbance at specific wavelength) Coloured liquids absorb a certain part of the wavelength spectrum Yellow colour means: blue light is absorbed Beer-Lambert law: relationship between absorbence and concentration of the absorbing substance.  Photometric Measurements Concentration of a solution can be determined with spectrophorometry if: pathlength (usally 1cm) molar extinction coefficiant (if not known, a standard curve is done) Absorbance is known (measured) https://www.youtube.com/watch?reload=9& v=BST5GRsAnPk Photometric Measurements Measurement unit for absorbance is OD (Optical Density) Modern Photometers display absorbance values in OD (optical density) What is OD ? A = -log T (Transmission) T = I/I0 I.....Light intensity after passing through the sample I0....initial light intensity Relationship Transmission and OD T=100% OD=0 I=100, I0=100; T= I/I0 = 100/100 = 1; A= -log T = log 1 = 0 T=10% OD=1 I=10, I0=100; T= I/I0 = 10/100 = 0.1; A= -log T = log 10 = 1 T=1% OD=2 T=0.1% OD=3 Classification of Photometric Devices Lightbeam Single beam/dual beam Wavelength selection Filters/Monochromator Filters:limited wavelengths Monochromator:continious spectrum Detector Single Photodiode Diode array Specifications Important Spec ! NIST Standard Cuvettes: National Institutes of Standards and Technology Applications UV/Vis-Spectroscopy Protein Quantitation: Biuret 570nm Bradford at 595nm Lowry at 750nm, 650nm or 540nm BCA 562nm Applications UV/Vis-Spectroscopy Protein Quantitation: Bradford at 595nm Lowry at 750nm, 650nm or 540nm BCA 562nm 06.01.2025 Fußzeile | Titel der Präsentation 30  Applications UV/Vis- Spectroscopy Absorption at 280nm (semi-quantitative) Absorption due to tryptophan (tyrosine) residues (fast) - disturbance by other substances with absorption at 280nm - absorption different for different proteins, due to variation in Trp content Rule of thumb: A280 = 1 corresponds to 0.5 – 2 mg.ml-1 protein Absorption at 205nm (semi-quantitative) Peptide bonds do absorb at 205 nm not dependent on protein composition little dynamic range (1-100µg) Applications UV/Vis-Spectroscopy Enzyme Kinetics Many Spectrophotometers do have evaluation software avaulble for Enzyme Kinetics Many enzyme kinetic reactions are measured at 340nm (NADH cofactor) Proteases: colorimetric substrates available Applications UV/Vis-Spectroscopy Applications UV/Vis-Spectroscopy DNA Quantification Protein Quantification Main application for microplate based photometers: ELISA !! Enzyme Linked Immune Sorbent Assay Measurement Modes Single Wavelength Measurement Done with cuvette photometers Blank measurement is done and value is subtracted form sample (necessary because cell itself scatters light) Application example: Optical density of a bacterial culture Measurement Modes Dual Wavelength Measurement: Usally done with microplate readers Second measurement can compensate for unspecific signals Reference wavlength for ELISA mostly between 620-650nm Application Example: Substrates for AP : pNPP (para-nitrophenyl phosphate): 405 nm aminoantipyrene, phenyl phosphate: 492 nm Reference value at 620nm is subtracted from 405 or 492nm. Part V –Circular Dichroism Spectroscopy  Circular Dichroism Spectroscopy Polarisation of Light Linear Polarisation Circular Polarisation Circular Dichroism, often abbreviated to "CD", is displayed when an optically active substance absorbs left or right handed circularly polarized light preferentially Circular Dichroism is the difference between the absorption of left and right handed circularly- polarised light and is measured as a function of wavelength This absorbance method works only with chiral molecules (optical active molecules).  Circular Dichroism Spectroscopy CD is measured as a quantity called mean residue ellipticity, whose units are degrees-cm2/dmol. Chiral or asymmetric molecules produce a CD spectrum because they absorb left and right handed polarised light to different extents and thus are considered to be "optically active". Biological macromolecules such as proteins and DNA are composed of optically active elements and because they can adopt different types of three-dimensional structures, each type of molecule produces a distinct CD spectra.  Circular Dichroism Spectroscopy Secondary structure analysis of proteins with CD: Scan from 190nm to 240nm is performed Helices, Sheets , Turns and Coils give different CD spectra Biosimilar Analytics „Rituximab“ 06.01.2025 Fußzeile | Titel der Präsentation 42