Large dynamic range small-angle diffractometer

D22 presents: SANS measurements with a Stopped-Flow apparatus, the sample movie

Isabelle Grillo
Institut Max von Laue - Paul Langevin, F-38042 Grenoble Cedex 9


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.

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.

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)

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.

Intermixer volumes of the available delay lines precisely determined by Biologic

Delay Line (DL) number 1  2 3  4  5
Intermixer volume (ml)47.463.8120.4172.3220.6

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. 


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)

The stopped-flow apparatus is completely controlled by PCby a software called Mps.exe. All the following parameters are given for a standard use. For any unusual use (personnal cell,...) please ask your local contact.

All the parameters present in the Config menu have to be correctly defined to ensure the rigth calculation of the flow rate, evolving and dead times.

Syringe initialization

Turn the syringe valves to R. Set the syringes to their uppermost (empty position) manually directly on the MPS module or with the MPS software. When the uppermost position is reached, the motor vibrates with noisy pulses.
On the software, open Syringes Command: Load

Reset individually the syringes by pushing the Reset button or all the syringes with Reset All.

Manual filling

* Verify that the three syringes are completely empty (upper most position) and that the valves are on position R.
* Attach the syringes containing the sample to the syringe reservoir port on the top of the SFM.
* Select the syringe number on the VMS module. Use the arrow down to fill the syringe.
* Eliminate any bubbles in the SFM syringe by driving it up and down several times while it is connected to the reservoir syringe.
* Turn the syringe valves to C.
* Empty of one or to step the syringe to definitely eliminate the bubbles.
* Repeat all these operations for the to other syringes.

Filling through the MPS software

* Verify that the three syringes are completely empty (upper most position) and that the valves are on position R.
* Attach the syringes containing the sample to the syringe reservoir port on the top of the SFM.
* In the Syringe Command window, select the syringe. Use the arrow down (simple arrow:1 motor step; double arrow:10 motor steps)  to fill the syringe.
* Eliminate any bubbles in the SFM syringe by driving it up and down several times with while it is connected to the reservoir syringe.
* Turn the syringe valves to C.
* Repeat all these operations for the to other syringes.

A sequence file defines to the electronics module different operations such as moving the syringes, sending a trigger, activation of the hard stop...
Open Sequence Files: New for a new file or Sequence Files: Load if the sequence already exists.

Fill the description of the syringe content:

* Enter the volume of each syringe and the time of the injection. The injection may be repeat several time to ensure a good cleaning of the cell.
* Activate/desactivate the trigger signal (Synchro 1 on/off).

The software calculates:

* The volumes used and the flow rate.
* The dead time related to the cell volume and the evolving time ( or ageing time), in function of the delay line chosen.

Before starting the sequence, check:

* The flow rate: 0.045-6 ml/s/syringe
* The minimum flow rate for an efficient mixing: 1 ml/s (total flow rate through each mixer)
* The minimal volume: at least the cell volume.

At this stage, the stopped-flow apparatus is ready for injection. Turn to D22's workstation to prepare the frames.

The acquisition program needs a one-column text file containing the time of each run. The time (in seconds) has to be converted into ticks the electronics time unit (1 tick=90.9 ns). The default file extention is .tic.

* Open the pilot program GEORGE on the workstation to create the frames. Press Setup and chose Kinetics files.

Five options are available. All other suggestions are welcome!

* Constant acquisition time. If the total time is not a multiple of the preset time, the file stops at the closest and lowest value of the total time.

* Geometric series: tn+1=atn=an+1t0, Total time=t0(1-an+1)/(1-a). Again, the file stops at the closest lowest value of the total time.

* Personal choice for time acquisition: do what you want!

* Concatenation of files. Addition of 2 or 3 files already created.

* Reading a file already created and posting on screen the preset times (if you do not remember what you have done...)

Example: Geometric series

Save the file and press do to check your file

D22's workstation standby, waiting for TTL signal

In the Mad window write the command kin slicename.tic x s/n, return or use the GEORGE dial.
x is the number of repetitionn is no saves means save.
The VME acquisition card is in a standby mode, waiting for the TTL signal to start the acquisition.
Turn again to the Stopped Flow software.

In the window Stopped Flow Program, select Single or Multiple. The number of possible shots according to the sequence file and the remaining volume of suspensions is calculated.

If READY !!, use the arrow to start the sequence. the black square stops the experiment at any time if necessary.
Single allows only one shot. Close the window Program run to return to the Stopped-Flow Program window. Chose Single or Multiple again to continue with the same sequence files or define another sequence file.
In the Multiple configuration, the arrow execute the sequence file until the Number of shots equals 0. 

The data acquisition starts when the trigger is activated and during the 0 to 5V part of the TTL signal. During the acquisition, the data are directly saved on the acquisition card. This avoids the transfer from the electronics to the workstation and the dead time between two spectra. At the end of the slice file, the spectra are transferred and saved on D22’s workstation.

Real time experiment may creates many thousand of 2D-spectra. It will be maybe necessary to compress the files during the experiment to save place on the Unix station hard disk.
* Open an x-term window
* c(hange)d(irectory) /users/data/
* compress 075* (for example)

The data reduction is really hard work, be patient and rigorous. Further data reduction programs will be developed, in function of the user's needs. Nowadays, the available programs are described in the ILL report “A computing Guide for Small Angle Scattering Experiments”, R.E. Gosh, S. U. Egelhaaf and A.R. Rennie and in the LSS's web pages. Do not hesitate (in a reasonable way!) to ask the help of your local contact.

Stopped-Flow module cleaning and storage

* Remove the remaining samples and buffer from the syringes.
* Turn the valve to R and fill the three syringes with pure water. Turn the valves to C and empty the syringes to wash the flow lines and the cell.
* Repeat the procedure with ethanol.
* Dry the syringes and flow lines with a wash of air, with the same procedure as described in point 2.
* Turn the valve to R and move the syringes to their lowest position.
* Turn the valve to C and turn off the MSP.

Data storage

The raw data are store both on D22’s hard disk and on a general ILL’s server, under the directory /usr/illdata/data/d22/. They are saved for years under this last path.
The treated data are also saved under your directory on d22sgi for several months. But save your data on zip or CDs before leaving. 

Months or years later…Data are treated and understood. Do not forget when you write your paper that all your results would not have been possible without the beam time allocated by the ILL and the help of your local contact even if he/she has left the ILL.