UV-Visible Spectroscopy - BIOC3570_F24_8 PDF

Optical (UV-visible) absorbance spectroscopy 1 Absorbance spectroscopy Kimber, 2020 In absorbance spectroscopy, we measure the attenuation of light intensity after it has passed through a sample This atten...

Optical (UV-visible) absorbance spectroscopy 1 Absorbance spectroscopy Kimber, 2020 In absorbance spectroscopy, we measure the attenuation of light intensity after it has passed through a sample This attenuation is wavelength dependent The resulting UV-visible spectrum reveals information about chemical solutes present, their chemical state, and their concentration 2  456 electromagnetic spectrum Wikipedia≈ In UV Visible spectroscopy we are primarily interested in absorbance by visible and near UV light Other types of spectroscopy are also occasionally used – e.g. IR spectroscopy 3  463-466 Absorption of UV-visible radiation c = l n (wave equation) E = hn (Planck) The UV-visible spectral range is about 200-750 nm. This range corresponds to 600-150 kJ mol-1. Typical covalent bond energies are 150-900 kJ mol-1. UV-vis. light photons can excite valence electrons in molecules Molecules are raised to electronically excited quantum states Light energy is converted into vibration/rotation /translation (heat) energy Chemical changes (photochemistry) may also occur The net effect is that less light exits the sample than entered 4  Optical absorbance and visible colour If green light is absorbed from white light, the transmitted light appears as the complementary colour - red. complementary colours orange yellow red green violet blue Colours can be difficult to predict, because compounds/ mixtures often have multiple absorption bands, and the spectrum of “white” light varies with the source. Colour perception is a complex mix of physics, physiology and psychology. Analysing spectra is much more objective. 5 Optical absorbance and visible colour Rhodamine 6B Wikipedia≈ 6  Representative spectra of some coloured compounds 580 620 1.2 1.0 Absorbance 0.8 700 0.6 560 400 0.4 0.2 2,4-dinitrophenol 0.0 490 440 240 260 280 300 320 340 360 l (nm) Absorbance Absorbance Indocyanine Coomassie Green Brilliant Blue l (nm) l (nm) 7 UV/visible absorbance spectra originate in electronic transitions The energy of a photon is only absorbed if it corresponds to an allowed transition in a molecule For UV/visible light, electronic transitions involve an appropriate amount of energy Additional energy can be channeled into excited vibrational states This splits the electronic absorbance line into vibrionic bands Solvent collisions further spread the energies of a given quantum state into a smooth and continuous absorption peak 456-457 8 Solvents collisions broaden absorbance peaks UV absorption spectra for 1,2,4,5-tetrazine. a) In gas phase: many lines, due to electronic, vibrational, and rotational transitions. b) In a nonpolar solvent: distinct electronic transitions can be observed, but the vibrational and rotational structure has been lost. c) In a polar solvent: strong intermolecular forces cause the electronic peaks to broaden, giving a single smooth absorption band. Conclusion: while analytes have many sharp peaks, analytes in aqueous Skoog, Holler, and Crouch, Principles of Instrumental Analysis, 6th ed., p. 368 buffers will give few broad peaks 9 Spectrophotometers 466-469 10  Spectrophotometers 466-469 11 Thermo Scientific  Single-beam spectrophotometer: Schematic Monochromator Detector Sample Exit Slit Dispersion Device Entrance Slit Source Light source: a light emission device that produces a suitable spectrum Monochromator: selects out a single wavelength of light from the spectrum Detector: quantifies incoming light intensity 12  Light source 1: W-Halogen Lamp 1 low output in UV irradiance.1.01.001 200 400 600 800 l (nm.) 1 W-halogen lamps produce a black-body spectrum at 2900K Light has very low noise Lamps have a long lifetime Disadvantage: light output in the UV range is low  Light source 2: Deuterium (or H) Arc Lamp wikipedia Deuterium arc lamps use a high volt electric arc to excite deuterium; electronic relaxation emits light H2 ® H2* ® H + H + h< Produces intense light across the UV continuum (180 nm – 370 nm) Light produced has low noise Disadvantage: limited lifetime (~2000 - 5000 hrs) 14  Monochromator https://www.shimadzu.com Monochromators use a prism or a diffraction grating to split incoming light A slit then blocks all light except the desired wavelength Any light other than this wavelength that exits is termed “stray light” and adds noise to the signal, especially at high absorbance (since stray light may be outside the absorbance peak) Passing the light through 2 successive monochromators (i.e. a double monochrometer) reduces this stray light 15  Diffraction gratings Diffraction gratings are formed from any periodic repeating pattern of holes, ridges etc. A CD is an example Monochromator diffraction gratings are generally built as a series of ridges with set spacing and a set angle Light is diffracted only when the distance light reflecting from successive ridges travels is an exact whole number of wavelengths different Since this depends on the wavelength, only a specific wavelength diffracted at a given angle 16  Detectors: Photomultiplier tube hn dynodes e- photoemissive anode cathode Each successive dynode is » 200V higher Each e- impact yields » 4 secondary e- Typically ~10 stages ® » 410 » 106-fold amplification Photomultipliers have very high sensitivity (they can detect one photon) However, they are: expensive mechanically fragile dependent on a very stable high voltage power supply 17 Detectors: Photodiodes Photodiodes are light-sensitive semiconductor device – essentially a mini solar panel Light striking the p-layer creates metal contact electron-hole pair hn Travel of electron to n layer allows current to flow Small, light weight, and cheap V silica Very reproducible + - However, they have much lower sensitivity than PMT p layer Au block n layer 18 Double-beam spectrophotometers Achieve much higher sensitivity than single-beam instruments, minimizing drift (lamp and detector) by measuring the sample and reference cuvettes simultaneously. Monochromator Reference Exit Slit Source Dispersion Chopper Device Entrance Slit Detector Sample A “chopper” disc is a rotating gapped mirror that directs the beam alternately along the sample and reference light paths. The “chopping” strategy helps a lot, because the signal is modulated at the chopping frequency, but the noise is not. 20 Diode-array spectrophotometers Fastest spectral acquisition: measure all wavelengths simultaneously. Agilent 8453 diode-array spec.: optical diagram Shutter Lens Sample W lamp Lens D lamp Slit Grating Diode Array (1024 elements) note that there is no monochromator before the sample Light is split between the diodes by the diffraction grating after it has passed through the sample. 21 Diode-array detector: full spectral information 254 nm Contour graph 3D perspective graph Spectral view of HPLC chromatograms obtained from a 100 µM standard mixture. 1, hypoxanthine; 2, xanthine; 3, GMP;4, IMP; 5, inosine; 6, adenosine; 7, AMP; 8, GDP; 9, CTP; 10, GTP; 11, ADP; 12, ATP. Photodiode arrays are commonly used as readouts for chromatography systems This enables the full spectrum to be collected as the column runs Zur Nedden et al., Anal. Biochem. 388: 108-114, 2009. 22 UV-visible spectrometers design trade-offs Visible-only is simpler than UV-visible UV range demands higher energy light sources, and UV transparent materials Single-wavelength (e.g. 280 only) vs scanning vs diode-array designs have cost/efficiency/sensitivity trade-offs. Photomultiplier has high sensitivity but is expensive; photodiode is cheap but much less sensitive. Single-beam has a simpler construction, but double-beam is much more sensitive 23 Sample cuvettes Cuvettes are typically available with different path lengths, but 1 cm is most common Narrow and shallow sample holder designs fit standard holders but allow measurements of a few microliters Measuring UV absorption spectra requires quartz cuvettes – other materials do not transmit UV light 24 Eppendorf UVette disposable UV/Vis cuvette a light transmission of 220 nm to 1,600 nm using crystal clear plastic $2.50 per Uvette 25  Absorbance: quantitation l I0 I monochromator sample detector I T = x 100% I0 Transmittance is the primary quantity measured by the instrument. 26 For calculating concentrations, the derived quantity A (absorbance) is more useful than T. 3 I0 100% A = log = log I T 2 Abs. 1 Note: at Abs 1, T = 10%, so only 1/10th of the light is transmitted At Abs 3, 0.1 % of the light was transmitted 0 0 25 50 75 100 % Transmittance 27 Beer-Lambert law I0 I Beer-Lambert Law: A=ecl Beer: Absorbance is proportional to the number of molecules in path. Lambert: Each successive “layer” (path-length element) absorbs the same fraction of the light incident upon it A is unitless e (molar extinction coefficient) units mol-1 L cm-1 e is a molecular property (and so a characteristic of a specific molecular species) 28 Practicalities emax: 103 average 104 strong 105 very strong For a standard cuvette (l = 1 cm) and a typical chromophore (say, e = 2,000), a good “starting point” concentration for quantitative analysis is about 0.1 mM. A = 2,000 × (0.1 × 10-3) × 1 = 0.2 Generally molecules absorb at UV-Vis when they have several conjugated double bonds Peptide bonds absorb at 190 nm and (more weakly) at 210- 220 nm 29  Cut-off wavelength Below a certain l, any solvent will UV cutoffs (nm) absorb UV light so strongly that (approximate) almost no light is transmitted This is the “cut-off” l, beyond which Acetonitrile Methanol 190 you can no longer measure the Water absorbance of solutes 190 The cut-off effectively defines the Methanol shortest wavelength measurable in 205 Ethanol UV-Vis spectroscopy 210 Water and CH3CN have the lowest Chloroform cut-offs 245 Ethyl Acetate Note that some organic solvents 255 absorb significantly at 260 – 280 DMSO nm, and can interfere with DNA or 270 protein quantitation Toluene 285 Acetone 30 330  Optimal reading ranges The instrument will always give a reading … but it is not necessarily meaningful! Photometric accuracy: Most accurate range is A ~ 0.1 - 1. A > 2.0: I0 is too small; insufficient light (< 1 %) is reaching detector; you should dilute the sample. relative error A < 0.01: difference between I and I0 is too small; you have reached the limit of sensitivity. 0 1 2 3 4 5 Absorbance 31 Characteristics of a good optical spectrum “black” at short λ smooth drop to A = 0 at long λ smooth, broad peaks ~Gaussian peak shapes wavelength (nm) Flint et al., J. Biol. Chem. 271:16053-16067, 1996. 32  Scattering also attenuates incident light Kimber, 2020 Scattering attenuates incident light by redirecting it In absorbance spectroscopy, total attenuation of the incident beam is the sum of the scattering and absorbance by the sample The steep rise of scattering at short wavelengths is often obscured by peaks (e.g. protein, DNA) Be careful of samples where peaks do not return to baseline Any visible cloudiness in a sample makes it unusable for measuring absorbance 33  Common artefacts in UV-vis spectrophotometry Particulates or colloids in the sample (Tyndall scattering): sharp increase in “absorbance” at long wavelengths Air bubbles in the cuvette, more commonly in FPLC flow cells Incompletely-filled cuvettes – the beam has to pass through the sample! Interfering substances in buffers e.g. acetone, disulfides, oxidized DTT Incomplete mixing, esp. in enzyme assays conducted in cuvettes Dirty cuvettes/ residual liquid in the cuvette before addition of the sample Always record full spectra (say, 250-350 nm) of samples Single-l reads should only be used for monitoring chromatography column eluates. 34 Typical applications of UV-visible absorbance spectroscopy Directly measuring the concentration of pure proteins, nucleic acids, and small molecules Indirectly measuring the concentrations of substances via chromogenic assays Monitoring chemical changes, e.g., protonation equilibria, redox state changes in metals or co-factors Monitoring eluate from chromatography column Characterizing protein prosthetic groups, e.g., heme, flavins Diverse enzyme assays that exploit absorbance changes in the co-factor, substrate, or a coupled reaction (e.g., NADH depletion) 35 UV-visible spectroscopy of biomolecules: overview Nucleic acids: excellent Proteins: generally good, but only accurate quantitation if you know the composition Prosthetic groups: excellent if they absorb Carbohydrates: usually poor Lipids: unsaturated or conjugated only A wide variety of groups can, however, be reliably measured using suitable coupling to chromogenic substances Many of these can substances can be detected using chromogenic assays 36  UV spectra of protein and DNA DNA: A260/A280 = 1.8 RNA: A260/A280 = 2.0 protein: A260/A280 < 1 DNA protein 37 Unless they contain prosthetic groups, most proteins have similar UV spectra. Arginase Ornithine transcarbamoylase Abs drops to ~0 ~300 nm Green et al., Eisenstein et al., J. Biol. Chem. 265: 1601-1607, 1990. J. Biol. Chem. 259: 5139-5145, 1984. 38 For most polypeptides, the UV spectrum is similar to that of tryptophan. Protein (GST Theta) Trp Abs. l (nm) Note: the peptide backbone absorbs at 190 – 220 nm, and more weakly at 210:220 This can be useful for monitoring proteins with little Tyr or Trp, but be aware that most molecules with double bonds also absorb in this region. 39  488 Protein UV absorbance depends on Trp, Tyr and Cystine e280 (M-1 cm-1) = (#Trp ´ 5,500) + (#Tyr ´ 1,490) + (#Cystine ´ 125) e280 contribution for 1 Trp Trp Tyr Cystine Note – not cysteine! Trp has the strongest UV absorbance, but can be rare (1.3 % of a.a. in proteins) Tyr is ~1/3rd as strong, but is ~2.5 x more common, so about as important Cystine is only pertinent to some proteins, and contributes only slightly (88 Cys residues = 1 Trp!) Pace, C.N., et al., How to measure and predict the molar absorption coefficient of a protein 40 Protein Sci. 4: 2411-2423, 1995. Sample calculation of protein absorbance e280 (M-1 cm-1) = (#Trp ´ 5,500) + (#Tyr ´ 1,490) + (#Cystine ´ 125) 1 μM bovine serum albumin (BSA) solution BSA: 607 aa; MW = 69 kDa; contains 3 W, 21 Y, and 35C (17 cystines) \ e280 = 49,900 (approx. 50,000) 1 cm cuvette: A = e 1 c = 50 x 103 x 10-6 = 50 x 10-3 = 0.050 1 μM = 10-6 x 69 x 103 g/L = 69 mg/L = 0.069 mg/mL very rough rule: protein concentration in mg/mL ~A280 but: very dependent on tryptophan content IMPORTANT: theoretical e280 values assume an unfolded protein; always use urea or GdnCl to denature/unfold your protein before measurement 41 UV spectroscopy can detect protein prosthetic groups adrenodoxin reductase (flavoprotein) Absorbance due to flavin adenine dinucleotide (FAD) cofactor Abs due to protein Sagara et al., Biochim. Biophys. Acta 1434: 284-295, 1999. 42 Changes in some active sites can be monitored by changes in the absorbance spectrum Cytochrome c is a small electron carrying protein It has a heme containing prosthetic group Changes to the redox state of the heme can be measured in the UV-Vis spectrum Oxygen binding to heme also changes its absorbtion spectrum (so arterial blood is redder than veinous blood) Nelson & Cox Fig. 19-4 43 Isosbestic points AA = AB Abs. l If a reaction follows the simple stoichiometry A ® B, then one (or more) isosbestic (equal absorbance) points will be observed during the progress of the reaction. not true for: A ® B + C 44 Isosbestic points are often seen in redox changes, titrations, ligand binding, and other stoichiometrically simple reactions. HN O H N N C6F5 C6F5 prodigiosin derivative Spectral changes during titration of a prodigiosin derivative (10 mM) with Zn2+ (0-1.2 eq.). Hong et al., Development of a prodigiosin derivative as a fluorescent Zn(II) probe, RSC Adv. 4: 6133-6140, 2014. 45 UV-visible spectroscopy: Summary Principle: Molecules absorb photons, exciting electrons in molecular orbitals; excited states decay by collisional processes, ultimately releasing energy as heat. Applications: Quantitative analysis of molecules with useful chromophores. Some structural information may be gleaned, e.g. protonation or redox equilibria. Strengths: Routine instrumentation. Can be used in high-throughput mode (microplate readers). Direct measurement of analyte concentration. Weaknesses: Applicable only to molecules with good chromophores. Very prone to interferences. Lower sensitivity than fluorescence spectroscopy. 46 Practice question Oxidoreductase enzyme kinetics often rely on measuring the NAD+ reduction to NADH Given the spectrum shown below for these two molecules, what UV wavelength would you recommend measuring to monitor an enzyme that reduces NAD+? A. 240 nm B. 260 nm C. 275 nm D. 340 nm E. 360 nm 47 Answer A. 240 nm B. 260 nm – strongest absorbance for both NAD and NADH, so little change during reaction C. 275 nm – isosbestic point, so no change to monitor D. 340 nm – largest change gives strongest difference signal E. 360 nm 48  471-475 Circular Dichroism Spectroscopy Recall that UV light ( 10 nm), scattering angle falls off at high angles in a way that depends on r Most protein complexes are too small to allow reliable measurements 55  ClpP SEC-MALS 164 kDa 338 kDa SEC-MALS can resolve the oligomerization equilibrium of ClpP protease Monica Goncalves, PhD Student Vahidi Lab @UoGuelph Static light scattering gives s reliable measure of MW only when a single particle type is present In SEC-MALS, size exclusion chromatography (SEC) is used to separate out species before multi-angled light scattering (MALS) measures the sample The instrument also simultaneously measures UV absorption and refractive index to allow the concentration of protein and other solutes to be calculated It can then calculate MW with ~5 % accuracy 56  526-529 Surface Plasmon Resonance Light shone at a surface between two media can propagate along the surface in a wave known as a surface plasmon This only occurs under specific circumstances, and the exact angle it occurs under is very sensitive to the index of refraction (IoR) of the media This sensitivity allows an SPR experiment to report very small changes in the IoR in a surface boundary layer that extends one wavelength from the surface E.g. by binding a macromolecule to a properly prepared surface, we can observe binding of protein or even small molecules as changes in the IoR 57  flow cell SPR Prepared surface Light strikes the surface at a range of angles Light at a certain angle is absorbed (surface plasmon); this angle is the signal The signal is the shift in the (infrared) plasmon angle relative to a reference cell through which the same buffer is flowing through plot the differences 58 wikipedia  kon L+A L A koff gives kon gives koff nicoya The ligand may be bound to the chemically modified chip surface through either chemical linkage, or using an affinity tag (e.g. His tag) Once the ligand is bound and the signal stable, a buffer with the analyte is flowed over the chip This increase in signal monitors binding during the association phase; this exponential curve yields the binding constant, kon (in M-1s-1) Once binding is complete, the system switches back to running buffer, and the analyte dissociates; this exponential decay curve gives the dissociation constant, koff (in s-1) kon/koff = KD : i.e. the dissociation constant 59 SPR gives binding constants measured data data fit to single ligand model Zhuoyang et al, JBC, 2016 Accurate estimates of KD require running the experiment over a range of ligand concentrations, and then fitting the data Note that the precision of the measurement is ~ 1% 60