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Chemical Analysis Facility
- 1. Chemical Analysis Facility ICMR, Feb 2010
- 2. The Facility • The facility is a multi-instrument laboratory organised and run by academic staff of the Departments of Chemistry and Pharmacy. • It is free for the use of staff, students, graduate students, and post-doctoral workers in the University. • The facility is a University investment of £4.5 million. • It includes instruments for structure determination, spectroscopy, mass- spectrometry, and calorimetry.
- 3. Academic leaders of CAF groups Andy Russell – project director Geoff Brown - nmr Becky Green – thermal analysis Adam Squires - molecular spectroscopyJohn McKendrick – mass spec Christine Cardin – X-ray diffraction
- 4. The plan • To create a facility run by academics for academics, and free at the point of use • Planning started about four years ago • University funding was approved about two and a half years ago • Building and conversion work started in Autumn 2008 • Major instruments installed summer 2009
- 5. Main CAF floor plan
- 6. X-ray lab floor plan
- 7. Technical support • Currently limited to • Peter Heath (nmr) • Martin Reeves ( mass spec) • So these are the only two real services, in general, facilities are available either as collaborations or as paid-for access.
- 8. NMR services Dr Geoff Brown (academic) Peter Heath (technical) Spectrometers: Bruker Nanobay 400 MHz Bruker DPX 400 MHz Bruker Avance III 500 MHz Bruker Avance III 700 MHz Two ways to run Use the Open Access NMR service (Bruker Nanobay 400 MHz and/or Bruker DPX 400 MHz) to perform NMR experiments on your sample yourself Submit your sample to the Internal NMR service (Bruker Avance III 500 MHz and/or Bruker Avance III 700 MHz instruments) Once an NMR spectrum has been acquired, users of the Open Access NMR and Internal NMR Service can process and view their data using one of two software packages available through CAF for Off-Line NMR Data Processing.
- 9. 700MHz Bruker Avance III 700 (with cryoprobe) for the highest resolution e.g. macromolecules
- 10. Bruker Avance III 500 (with solids capability) Bruker DPX400
- 11. Dr Geoff Brown
- 12. 400MHz Bruker Avance III 400 Nanobay (inverse probe) and Bruker DPx400 (normal probe) • The inverse probe has the H coils inside and is more sensitive for proton spectra, while the normal probe has the hetero-element coils inside and is more sensitive for „X“ nuclei, e.g. C. • In general the inverse probe is better for all 2D experiments, while the normal probe has higher sensitivity for C, and for DEPT experiments
- 13. 13-C Spectrum artemisenin 1 Recorded in 6 minutes (128 scans) on the Nano (less sensitive) instrument.
- 15. 13-C Spectrum artemisenin 3 With only 2 mg, we lose a quaternary C (172 ppm) entirely, and might conclude there were only 14 C atoms in the molecule.
- 16. 13-C DEPT Spectrum artemisenin All methyl, methylene, and methine groups identified from 64 scans recorded in 3.5 minutes.
- 17. Artemisinin DEPT spectrum on 2 mg. Spectrum is acceptable if not good; so could be collected for longer, or recorded using the normal probe. A 1 mg sample did not give an acceptable spectrum with these conditions.
- 18. Heternuclear 2D spectrum using 1 mg. Recorded on 1 mg in 5 minutes. All carbons are clearly visible. 0.5 mg gives information, but the noise is then evident with the real signals.
- 19. Mass spectrometry Dr John McKendrick (academic) Martin Reeves (technical)
- 20. Accurate Mass MS Thermo Fisher Orbitrap XL (with liquid chromatography input) These features allow protein mixtures of high complexity to be analysed comprehensively, and for protein abundances to be quantified from samples that have been isotopically labelled (SILAC).
- 21. Configuration of Orbitrap Mass- spectrometer
- 22. An example of resolution
- 23. Resolution and mass accuracy
- 24. Mass accuracy
- 25. Isotope Ratio MS Thermo Fisher Delta V (with gas bench) • Can be used for accurate isotope ratio measurements. • Works by conversion of substances into simple gases (e.g., CO2,) and operates over a mass range of 1-96 Dalton. • Typical uses are for stable isotope ratios for isotope fractionation in natural systems, or radiogenic isotope analysis for radiometric dating.
- 26. Sample isotope ratio data: 13C
- 27. Sample isotope ratio data: 15N
- 28. X-ray diffraction is in a separate laboratory on the lower ground floor
- 29. X-ray experiments powder diffraction single crystal diffraction small angle X-ray scattering (SAXS)
- 30. X-ray people Dr Ken Shankland - pharmacy (pharmaceutics) Dr Ann Chippindale and Dr Simon Hibble (inorganic solid state) Dr Clare Rawlinson – pharmacy practice Prof Ian Hamley and Dr Adam Squires (SAXS)
- 31. Powder X-ray diffraction Instrument 1 • High throughput Instrument 2 • Sample environment
- 32. #1: Bruker D8 Advance High throughput • Monochromatic (CuK 1) • LynxEye detector (~3.5 2 ) • Sample changing robot • Up to 90 samples • 1y operation in reflection mode but can also do transmission Main applications • Sample screening • Phase identification 32
- 33. #2: Bruker D8 Advance Sample environment • Monochromatic (CuK 1) • LynxEye detector (~3.5 2 ) • Reflection stage • Capillary transmission stage • Low T / Humidity – -193 C to 450 C • High T oven – Up to 1200 C Applications • Phase transformations as a function of T and RH • Rietveld refinement • Structure solution 33
- 34. XRPD: Sample data 5045403530252015105 1,050 1,000 950 900 850 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 L glutamic acid, few mg on a zero background plate. ca. 15 mins. data 5-55 FWHM 0.045 Zero point -0.003 4140.54039.53938.53837.53736.53635.53534.53433.53332.53231.53130.53029.5 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20
- 35. 35 Zopiclone Phase Transformation monoclinic dihydrate 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 298 K monoclinic anhydrous 325 K 350 K orthorhombic anhydrous Temperature
- 36. The Gemini-S-Ultra single crystal diffractometer • Designed for single crystal structure determination of both chemical and biological samples • Two wavelengths – copper – for large organic and biomolecules - molybdenum – for well diffracting and heavy atom- containing crystals • Typical data collection times 3 hours (a morning)- 60 hours (a weekend) • Instrument managed by Professor Christine Cardin.
- 39. Single crystals – small and large molecules CJC CH4I1 2007-8 Mount the crystal on a glass fibre or in a loop, mount on a goniometer head, place on the diffractometer, record an image
- 40. Guangzhou 2008 Sr2+ and bisintercalator binding to the Holliday junction Sr in minor grooves N N N O N N N N O N Sr Brogden, A.l., Hopcroft, N.H., Searcey, M. and Cardin, C.J., Angewandte Chemie International Edition, 2007, 46, 3850-3854
- 41. Bruker SAXS Nanostar instrument
- 42. SAXS Instrument Bruker Nanostar AXS
- 43. The SAXS technique • SAXS is an accurate, non-destructive technique that is used to determine the micro and nano-scale structure of particle systems and samples that are analysed by SAXS are typically sized in the range 0.5 tp 50nm1. The material under investigation can be solid or liquid and can also contain solid, liquid or gaseous domains. Minimal amounts of sample are required and the technique can look at anything including colloids, polymers, proteins and much more. It can also employ lab based or synchrotron sourced X-rays. • When SAXS is used, it is the elastic scattering of the X-rays by a nano- scale inhomogenous sample measured across a narrow range of angles that is recorded. The data gained from the angular range, which can be as narrow as 0.1 to 10o can deliver information on the macromolecular structure, including distances of partially ordered material and pore sizes. As dictated by Bragg's Law, the diffraction information about structures with large d-spacings lies in the region covered by this technique. Therefore SAXS is commonly used for probing large length scale structures such as biological macromolecules.
- 44. SAXS applications • Some practical applications of SAXS include the characterisation of transient intermediates in Lysozome folding with time-resolved small-angle X-ray scattering.3 In this experiment SAXS was combined with a mixed flow monitoring technique to observe (on the ms timescale) the folding of the lysozome protein from a denatured state (GdHCl) to the native state. SAXS allowed the researchers to gain information on the geometry of the protein at different stages of the folding process. The lysozome protein consists of 129 residues and contains 4 di-sulphide bridges. The native protein consists of α-helix and ß-sheet regions. The study showed that it takes approximately 2 seconds for the total re-arrangement of the protein. By improving the time resolution of the SAXS experiment and combining data from continuous and stopped flow experiments under identical conditions, it was possible to monitor the formation and subsequent decay of a helical intermediate.
- 45. Thermal Analysis Dr Becky Green (Pharmacy) • Hot Stage Microscope Mettler Toledo FP900 • DSC (solution) TA instruments - Nano DSC • DSC (standard) TA instruments - Q2000 DSC • TGA (1) TA instruments - Q600SDT • TGA (2) TA instruments - TA –Q50 • ITC Microcal - ITC200 (low solvent volume)
- 46. Differential thermal calorimeter - the Nano DSC from TA instruments
- 47. DSC - Characteristics and uses • Provides information on thermal changes which do not involve a mass change in the sample. • Sample and reference are maintained at the same temperature through a thermal event, and the energy required to do this is measured.
- 48. DSC (standard) TA instruments - Q2000 DSC • Studies molecules in their native state without labelling and in solution, or suspension – Provides thermodynamic profile that gives information in mechanisms of unfolding: enthalpy of unfolding, change in heat capacity of denaturation, ultra-tight molecular interactions… – It can help elucidate factors that contribute to folding, such as hydrophobic interactions, hydrogen bonding and conformational entropy • Perfect for probing protein stability and folding, membranes and lipids, antibody domain structure, biopharmaceutical formulations, …
- 50. Q2000
- 51. Q600 – Combined DSC and TGA
- 52. Q600 Dual instrument – an advantage
- 53. Isothermal titration calorimeter - the Microcal ITC200
- 54. ITC Microcal – Nano ITC and ITC200 • Enables direct measurement of binding thermodynamics (ΔH, ΔG, ΔS) • Can give insight as to number and types of binding site involved in an interaction • Label-free, no immobilization and can study interactions in buffered solution • Compatible with turbid or coloured solutions and particulate suspensions • Nano ITC – 1 ml volume cell – Aqueous solvents only • ITC200 – Low volume cell – less sample required – Improved compatibility to organic solvents
- 55. ITC – What does it do? • In a typical experiment, the macromolecule is placed in a sample cell of the calorimeter and the ligand loaded into an injection syringe. A reference cell is filled with the solvent. The ligand is titrated into the sample cell as a sequence of 5-10 μl injections. Raw data are obtained as a plot of heating rate (μcal s-1) against time (min). These raw data are subsequently integrated to obtain a plot of observed enthalpy change per mole of injected ligand (ΔHobs, kJ mol-1) against ligand concentration (mM) or molar ratio (ligand:macromolecule). From this integrated plot, complete thermodynamic data including enthalpy (ΔH), entropy (ΔS), free energy (ΔG), association constant (Ka) and stoichiometry (i.e., number of binding sites, n) are provided. ITC is the only technique for studying interactions that directly measures enthalpy. Therefore, ITC extends and complements the existing range of experimental techniques for the characterisation of molecular interactions (i.e., spectroscopy, surface plasmon resonance, atomic force microscopy, etc.).
- 56. ITC – An illustrative example
- 57. ITC - Applications • (All reversible reactions involve changes in enthalpy) • ITC sensitively detects changes (nanomole quantities) in heat of interacting species in solution • Interactions of proteins/lipids/nucleic acids with small molecules • Protein-lipid, protein-nucleic acid, protein-protein interactions • Enzyme kinetics • Polymer-surfactant interactions • Binding to solid particulate surfaces • Micellar/aggregation processes
- 58. ITC - An example Changes in lysozyme structure on addition of a surfactant
- 59. ITC - The instrument
- 60. Hot Stage Microscope Mettler Toledo FP900 • This enables imaging and video imaging at high magnitude. • With temperature stage can visualise melting and changes between crystalline and amorphous states (-100 to 400 oC). • Heating rate and imaging rate can be varied to suit system to be studied. • Plenty of applications without using the temperature stage.
- 61. Molecular spectroscopy Dr Adam Squires FT-IR Microscope Perkin Elmer Spotlight 400 FT-IR Perkin Elmer Spectrum 100 FT-Raman Thermo Fisher NXR9650 (633 &1064nm) Raman Microscope Renishaw InVia Reflex (532; 633 & 785nm) Fluorescence Varian Cary Eclipse (Peltier variable temp. -10 – 100 °C) UV/Vis Varian Cary300
- 62. FT-IR Microscope Perkin Elmer Spotlight 400 • Works both for imaging and for single spot spectroscopy. • Has wavelength range extending to 690 cm-1 (total spectrometer range 7800 cm-1 to 370 cm-1 ). • Spectral resolution better than 0.5 cm-1; spatial resolution 10μ, but extended to 3μ for ATR using Ge „atmosphere“.
- 63. Raman microscopy - the Renishaw InVia Reflex
- 64. Fluorescence Varian Cary Eclipse (Peltier variable temp. -10 – 100 °C) • Allows excitation down to 275 nm • Time-resolved studies available with a minimum gate-time of 1 μs
- 65. State of play • Brochure being printed • Website being prepared • Interim management committee has met twice • All instruments functioning, contact the section head or Dr Andy Russell • Visit by Andy Russell and John McKendrick followed by a tour has been arranged for Feb 10th