saxs and sans facilities and experimental practice · pdf filex-ray or neutron beam sample ......
TRANSCRIPT
![Page 1: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/1.jpg)
SAXS and SANS facilities and experimental practice
Clement Blanchet – EMBL Hamburg
![Page 2: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/2.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
2
Small Angle Scattering experiment
2θ2θ sX-ray or neutron
Beam
Sample
Buffer
Detector
The beam hits the sample, X-rays/neutrons interact with the sample and are scattered, providing structural information on the sample.Same formalism but different scattered particles Different instrument.
![Page 3: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/3.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
3
Outline
• X-rays / neutrons
• SAS instruments
• Sample environment
• Sample requirements and collection strategy
![Page 4: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/4.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
4
X-rays and neutrons
![Page 5: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/5.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
5
X-raysRoengten, 1895
![Page 6: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/6.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
6
Electromagnetic wave
![Page 7: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/7.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
7
How are X-ray produced?
• Brehmstrahlung– When a charge is accelerated charge,
electromagnetic radiation is produced (from Maxwell equation)
![Page 8: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/8.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
8
X-ray sources - Synchrotron
• Synchrotrons
![Page 9: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/9.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
9
X-ray sources - synchrotron
• Synchrotron radiation – Insertion devices
![Page 10: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/10.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
10
Insertion devices
Dipole bending magnet (APS) Undulator (PetraIII)
![Page 11: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/11.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
11
Synchrotrons around the world
![Page 12: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/12.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
12
X-ray sources - FEL• Free electron laser
– Electrons are accelerated and send to a long undulator (several 100s meters)
– Self amplified spontaneous emission:electrons group themselves into small bunches.
– Production of very short and intense X-ray pulses
![Page 13: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/13.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
13
X-ray sources - FEL• Free electron laser
![Page 14: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/14.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
14
Lab sources
![Page 15: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/15.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
15
Lab sources
• Principle : electrons, produced by heating a cathode are accelerated in an electric field and projected on a metallic anode.
– Brehmstrahlung
– Fluorescence
![Page 16: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/16.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
16
X-ray sources
• Lab source (rotating anode, liquid jet)
![Page 17: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/17.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
17
![Page 18: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/18.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
18
Neutron
λ=h/mv James Chadwick
![Page 19: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/19.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
19
Neutron production
• Nuclear reaction
![Page 20: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/20.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
20
Neutron production
• Spallation source– Accelerated protons hit a heavy metal target.
![Page 21: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/21.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
21
Neutrons Facilities
![Page 22: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/22.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
22
SAXS and SANS Instruments
![Page 23: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/23.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
23
Optics
Polychromatic divergent beam from the source
Monochromatic focused (parallel) beam for SAS
![Page 24: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/24.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
24
Monochromatic X-ray
• Bragg diffraction on a crystal
nλ = 2dsinθ
![Page 25: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/25.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
25
Monochromator
• Before• Polychromatic
• After• One wavelength + harmonics
![Page 26: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/26.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
26
Focusing/low divergence
• Small beam at the detector position• Small beam at the sample position
2θ2θ
![Page 27: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/27.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
27
Focusing X-ray
• Compound refractive lenses
• X-ray mirrors
![Page 28: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/28.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
28
Focusing X-ray
• Focussing mirror
• Reflectivity
Energy [eV]10000
Tran
smis
sion
0,0
0,2
0,4
0,6
0,8
1,0
0.15 Degree0.25 Degree1 Degree
![Page 29: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/29.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
29
Focussing mirror – harmonics filter
Monochromatic, focused x-ray beam
![Page 30: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/30.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
30
Monochromatic neutrons
• De Broglie equation: λ=h/mvThe wavelength of a neutron is related to its velocity.
• Velocity selector∆λ/λ=5-10%
• For pulsed source, TOF
![Page 31: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/31.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
31
Collimation neutrons
• The collimator is used to obtain a parallel beam
![Page 32: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/32.jpg)
Get rid of parasitic scattering: slits
Beam defining slits Guard or anti-scatter slits
![Page 33: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/33.jpg)
Hybrid slits
• Idea: use a crystal for the tip of the blade: no scattering but diffraction
6/20/2016 SAXS and SANS facilities Clement Blanchet
33
![Page 34: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/34.jpg)
Hybrid slits
• On the P12 beamline
6/20/2016 SAXS and SANS facilities Clement Blanchet
34
![Page 35: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/35.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
35
Sample environment
![Page 36: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/36.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
36
Flight tube
![Page 37: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/37.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
37
Beamstop
• Prevent the direct beam from hitting the detector
– Big enough to stop the direct beam– Small enough to collect the small angle
• Measure transmitted beam
![Page 38: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/38.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
38
Active Beamstop
• SAXS images needs to be accurately scaled to allow for proper buffer subtraction and extraction of the solute SAXS pattern
![Page 39: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/39.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
39
Detectors
![Page 40: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/40.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
40
CCD detector
![Page 41: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/41.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
41
![Page 42: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/42.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
42
Single photon counting detector principle
![Page 43: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/43.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
43
Single photon counting detectorPilatus
– High dynamic range– No background noise– (relatively) Fast framing
Ideal for SAXS
![Page 44: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/44.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
44
Neutron detection
• He3 detector:n + 3He → 3H + 1H + 0.764 MeV
![Page 45: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/45.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
45
Sample environment
![Page 46: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/46.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
46
Samples
Metal alloys
Nanomagnetic materials
Sufactants PolymersTissues
Bio-macromolecules in solution
SAS applicable to many type of samples.
![Page 47: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/47.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
47
Rapid mixing device
Heating stagesMagnetic field system
Sample changers
Sample environment
Example ID02 (ESRF) multipurpose beamline
![Page 48: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/48.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
48
Sample environment
• Bio-macromolecules in solution are weakly scattering sample.
• For biological macromolecules in solution: – fragile– Preferably in vacuum– Thermostated
![Page 49: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/49.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet 49
Sample cell• Cell material: low absorption and scattering
– Mica, quartz, polycarbonate• Sample thickness (t): compromise between
scattering and absorption– Scattering α t– absorption α exp(-ut)
• For neutron, cell are rather thin (<1mm to avoid multiple scattering
![Page 50: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/50.jpg)
Solution SAXS
6/20/2016 SAXS and SANS facilities Clement Blanchet
50
10 years ago:Manual sample loading
Buffer and sample should be measured in the same cell
Difficult to implement in vacuum 10-15 minutes per measurement High sample consumption Non-optimized cleaning procedure Tedious, energy and attention
consuming
![Page 51: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/51.jpg)
SAXS sample changer @EMBL Hamburg
6/20/2016 SAXS and SANS facilities Clement Blanchet
51
![Page 52: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/52.jpg)
SAXS sample changer
6/20/2016 SAXS and SANS facilities Clement Blanchet
52
![Page 53: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/53.jpg)
Sample changer performances
• Large storage capacity• Full cycle time (loading, exposure,
flushing, cleaning, drying) ≈ 1 min• Volume 5-20 microliter• Very efficient cleaning• Flow measurement
6/20/2016 SAXS and SANS facilities Clement Blanchet
53
![Page 54: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/54.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
54
Online size exclusion column
![Page 55: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/55.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
55
SEC + SAXS
Definedbuffer region
![Page 56: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/56.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
56
Experimental practice
![Page 57: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/57.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
57
Buffer subtraction
![Page 58: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/58.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
58
Buffer subtraction
• Biological sample scatters very weakly, SAXS curves collected on the buffer should be carefully subtracted– Exactly matching buffer (dialysis, elution
buffer)
– Sample and buffer measured in the same cell
![Page 59: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/59.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
59
Monodispersity
• SAS is very sensible to aggregation, the sample should be monodisperse
![Page 60: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/60.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
60
Monodispersity
• Check the monodispersity of your sample before coming to the beamline.(native gel, dynamic light scattering,
ultracentrifugation,…)
• Use online chromatography
![Page 61: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/61.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
61
Inter-particle interactions
![Page 62: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/62.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
62
Inter-particle interactions
• Change solution (pH, salt concentration) to limit interactions
• Measure different concentrations and extrapolate
![Page 63: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/63.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
63
Measure also water and/or standard protein…
• … to estimate the molecular mass of your sample using the forward scattering
– For data on an absolute scale (water measurement)
• M=I(0)*NA/(C*ν∗∆ρ)
– Using a protein standard• M=MBSA*I(0)/IBSA(0)
![Page 64: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/64.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
64
X-rays - Radiation damage!!!
• With intense synchrotron beam: radiation damage:
• Monitor radiation damage: collect several frames and compare them.
• Limit the radiation damage
H2O OH·
Free radicals: oxidize proteins which leads to their aggregation
H ·
![Page 65: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/65.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
65
X-rays - Radiation damage!!!
Flow measurement
Beam attenuation
Use of additivesRnase – Static measurement
![Page 66: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/66.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
66
Contrast in neutron
• Neutrons interact with the nucleus of atoms
• Each atoms has its own scattering length:
H D C N O P S
-.3742 0.6671 0.6651 0.940 0.5804 0.517 0.2847
![Page 67: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/67.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
67
![Page 68: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/68.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
68
Labeling• Protein and nucleic acids have different
scattering length densities and the different components can readily be studied by SANS
• To study protein-protein complex, one of the components needs to be deuterated (hydrogen exchanged with deuterium) to changed its scattering length density
• A deuterated protein is obtained by producing the protein in a deuterated medium.
![Page 69: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/69.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
69
Neutrong collection strategy
• Solvent matching– Find the solvent composition that match the
contrast of the component you want to hide– Measure the sample in this solvent
• Contrast variation– Measure the sample in solvent with different
D/H ratio, different scattering length.– Using Stuhrmann analysis you can access the
curves of the different components.
![Page 70: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/70.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
70
Summary: SAS sample
• Protein concentration: 0.1-10 mg/ml
• Volume: 5-50 microliter (SAXS), 200-300 microliter (SANS)
• Time: • lab source: 5-60 min• Synchrotron: seconds• Neutrons: 30 minutes - hours
![Page 71: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/71.jpg)
6/20/2016 SAXS and SANS facilities Clement Blanchet
71
Summary: SAS sample
• Pure and monodisperse sample• Exactly matching buffer• Measure concentration series• For SAXS:
– Be aware of radiation damage• For SANS:
– Carefully design your experiment, think of your collection strategy.
![Page 72: SAXS and SANS facilities and experimental practice · PDF fileX-ray or neutron Beam Sample ... • Sample environment ... SAXS and SANS facilities and experimental practice Author:](https://reader033.vdocuments.mx/reader033/viewer/2022051320/5a9f38437f8b9a7f178c84a6/html5/thumbnails/72.jpg)
Conclusion• To optimally use your beamtime: carefully plan your
experiment, prepare your sample and characterize them before coming to the beamline
• SAXS is now widely used. Dedicated instruments with low background and high level of automation, high quality lab instruments.
• SANS more difficult: consume more time and sample, requires good planning of your experiments and collection strategy, but can provides unique information.
6/20/2016 SAXS and SANS facilities Clement Blanchet
72