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Large dynamic range small-angle diffractometer D22
Small-angle scattering is a technique that measures the deviation to small angles (much less than one degree to several degrees) of an X-ray or neutron beam due to structures of small size in the sample. “Small” means dimensions of a few tenths to about 100 nanometres, such as clusters in alloys, polymers, or biological macromolecules.
Stopped-flow
D22 presents:
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Introduction
ILL’s high flux SANS spectrometers open a new field of experiments with real time measurements and rapid kinetics. Real time signifies a movie sample after a disturbance, pH or temperature jump, dilution, mixing, application of a magnetic field... Rapid implies short acquisition times, of the order of 100 ms, in order to detect the early stages of formation of a sample. Indeed, the knowledge of intermediary phases or structures is sometimes crucial to understand and modify the properties and characteristics of the steady state.
Principle of real time measurements with Stopped-Flow apparatus
The interest of a Stopped-Flow apparatus is to control a reaction by the mixing of several suspensions in a short time (10-50 ms) and to know precisely the time between the beginning of mixing and the observation. The SFM-3 from the Bio-Logic company handles three stainless steel syringes of 20 ml each, driven by independent stepping motors and two mixing chambers. The vertical syringes are filled from the top of the apparatus. This geometry simplifies the evacuation of air bubbles formed sometimes in the syringes during the filling. A valve system drives the path of the solution. The maximal flow rate may reach 6 ml/s per syringe and the minimal volume for a good reproducibility is estimated at 20 ml. A rectangular Hellma cell of 1 mm path and 0.2 ml has been adapted. It is opened on the both sides and fixed between two teflon supports to assure tightness.
Volumes and mixing times are computer controlled. At any time of the defined sequence of injection, the Stopped-Flow electronics may send a TTL signal to the instrument workstation starting the sequence of acquisition. A sequence of acquisition defines the number of spectra and the time of each one. The scattered intensity is recorded on the D22’s 2D gas filled detector. The new electronics card especially designed for rapid kinetics experiments can store temporarily up to 450 spectra, with a maximal preset per run of 391 s.
- The Stopped-Flow apparatus at the sample position and the Schematic Overview
Some features of the Stopped-Flow apparatus
A. The observation head
The observation head has been especially drawn for D22's geometry and Small Angle Neutron Scattering. On the detector side, the metallic part of the observation head is opened with a 20° angle to increase the solid angle. The smallest sample to detector distance (D = 1.4 m) with beam centered can be used without shadow on the detector.
A standard Hellma cell (1mm path, 200 ml volume) has been adapted to the head size. Its is fixed between two teflon supports to assure tightness.
Take care of it ! (costs: cell and teflon supports: 4 656 F; metallic head: 13 500 F)
- Stopped-Flow observation cell at the sample position
B. Control of the mixing process
A first mixing chamber is situated after the syringes 1 and 2. After this comes a delay line whose volume determines the time constant. The second mixing chamber is found at the end of the delay line, mixing the solution coming from the mixer 1 with that from syringe 3. Then, the liquid is filled in the observation cell.
The two relevant times are tET, the evolving time between the two mixing points and tDT, the dead time between the middle of the cell and the last mixing point.
These two times depend on the flow rate, on the delay line used and on the cell geometry. Working with two solutions, the minimum time for observation is achieved using syringes 1 or 2 and syringe 3. tmin varies between 50 and 200 ms.
<table class="standard_table" style="width: 100%; text-align: center; vertical-align: middle;" align="justify" cols="2" width="80%"><tbody><tr><td width="50%">
</td> <td> 
</td> </tr> <tr> <td> figure 1: use of syringes 1 and 2 </td> <td> figure 2: use of syringes 1(or 2) and 3 </td> </tr> </tbody></table>
Intermixer volumes of the available delay lines precisely determined by Biologic
<table class="standard_table" style="width: 100%;" align="justify"><tbody><tr><td width="80">Delay Line (DL) number</td> <td> 1 </td> <td> 2 </td> <td> 3 </td> <td> 4 </td> <td> 5 </td> </tr> <tr> <td width="80">Intermixer volume (ml)</td> <td> 47.4 </td> <td> 63.8 </td> <td> 120.4 </td> <td> 172.3 </td> <td> 220.6</td></tr></tbody></table>
Calculation of flow rate and time after mixing
* The suspensions are in syringes 1 and 2. Syringe 3 is used to rinse the cell (figure 1).
The Total Flow Rate F through the cell is the sum of the flow rate f1 and f2 imposed by the two syringes:
F (ml/s) = f1 + f2
The Evolving Time, tET between the two mixers is: Intermixer volume/F
The Dead Volume is the volume from the last mixing point to the center of the observation area, i.e. the half volume of the cell (vc/2). The Dead Time, tDT is the dead volume divided by the total flow rate:
tDT( s) = (vc/2) / F
Finally, the minimal time after the mixing that may be achieved, using syringe 1 and 2 is:
tmin = tET + tDT
Example: DL no1; Flow rate, F=10 ml/s; tmin=12.24 ms
* The suspensions are in syringes 1(or 2) and 3. Syringe 2(or 1) is used to rinse the cell (figure 2).
The total flow rate F through the cell is the sum of the flow rate f1(or 2) and f3 :
F (ml/s) = f1(or 2) + f3
There is no Evolving Time before the cell filling.
The Dead Volume is the volume from the last mixing point to the center of the observation area, i.e. the half volume of the cell (vc/2). The Dead Time, tDT is the dead volume divided by the total flow rate:
tDT (s) =( vc/2) / F
Finally, the minimal time after the mixing that may be achieved, using syringe 1(or 2) and 3 is:
tmin = tDT
Think about the syringes that you use !
Note: in such real time neutron scattering experiment, a large aperture is used before the cell to have the maximal flux. Thus, the age of the sample viewed by the beam is not homogenous. The time difference from the mixing between the bottom and the top of the observation window is: Dt = heL / F, with h the height of the aperture; e, the cell thickness; L the width and F, the flow rate. Typically, h=10 mm; e=1 mm; L=10 mm, F=10 ml/s; Dt=10 ms.
More about stopped-flow and real time
Further developments
Temperature control.
Resistance to acidic and basic solutions.
New cells with smaller or larger path.
Faster detector to increase the count rates and decrease the acquisition time.
Many thanks to
R. May for scientific help; F. Descamps, J. Ratel and J.-A. Vidal-Garcia for the electronics developments; M. Roure for computing; M. Bonnaud and P. George for technical support.
Fields of research
Soft condensed matter: growth of inorganic particles in organic matrix, micellar and vesicles growth, growth of inorganic material such as mesoporous structures.
Biology: protein binding, changes of quaternary structures during function.
Publications about real time, stopped-flow, neutron and x-ray small angle scattering
How does ZrO2/surfactant mesophase nucleate? Formation mechanism (ILL, D22)F. Né, F. Testard, Th. Zemb, I. Grillo Langmuir (2003), in press
Formation and growth of anionic vesicles followed by small angle neutron scattering (ILL, D22)
I. Grillo, E.I. Kats, A.R. Muratov Langmuir (2003), 19 (11) 4573-4581
Millisecond-range time-resolved small-angle X-ray scattering studies of micellar transformations
Schmoelzer S., Gräbner D., Graszielski M., Narayanan T.; Phys. Rev. Letters (2002) 88 (ESRF, ID2)
Vesicle formation as studied by means of highly time-resolved stopped-flow experiments (ILL, D22)
D. Gräbner, M. Gradzielski, I. Grillo ILL Annual report 2001
Detection and characterization of an intermediate conformation during the divalent ion-dependent swelling of tomato bushy stunt virus
Pérez J., Defrenne S., Witz J., Vachette P.; Cell. Mol. Biol. (2000) 46, 937-948 (LURE, D24)
Roessle M., Manakova E., Lauer I., Nawroth T., Gebhardt R., Narayanan T., Heumann H.; ESRF Newsletter (1999) 33 (ESRF, ID2)
Né F., Testard F., Zemb Th., Petit J.-M.; ESRF Newsletter (1999) 33 (ESRF, ID2)
Egelhaaf S.U., Schurtenberger P.; Phys. Rev. Letters (1999) 82, 2804-2807 (ILL, D22)
Egelhaaf S.U., Olsson U., Schurtenberger P., Morris J., Wennerström H.; Phys. rev. E (1999) 60, 5681 - 5684 (ILL, D22)
De Moor P., Beelen T., Komancheck B., Diat O., Van Santen R.; J. Phys. Chem B (1997) 101, 11077 (ESRF, ID2)
Egelhaaf S.U., Schurtenberger P., Morris J., Olsson U., Wennerström H. ILL Annual Report (1997) (ILL, D22)
Egelhaaf S.U., Schurtenberger P.; P. Physica B (1997) 234-236, 276-278 (ILL, D22)
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