Rapid-Kinetics and Spectroscopy: Stopped-Flow; Fluorescence, Absorbance, Circular Dichroism, Anisotropy Spectrometry

Stopped-Flow - the Bio-Logic technology

Power and ease of use.

Since 1984, we have continuously improved the Stopped-Flow and Quench-flow technology.

Our SFM-400Stopped-Flow/Quench-Flow and our Quench-flow QFM-400  are often considered as the new benchmarks. The SFM-20 is high performance single mixer stopped-flow.

Our MOS-450/AF-CD is THE reference for CD-Stopped-Flow experiments.

What do we do for science? See a list of published papers.

Please take a look at our FAQ section.

Do you want to know more about our Stopped-flow and quench-flow? Talk directly to our application specialists (English/French) or contact us by email or fax.

First class Stopped-Flow and Quench-Flow instruments

Single, double, triple mixing Stopped-flow & Quench-Flow with intermediate ageing.
Independently driven programmable syringes.
High precision variable mixing ratio and dilution (density and viscosity independent).
Sub millisecond dead time (down to 0.25 ms).
µvolume operation.

• Stopped-flow & quench-flow instruments - click here

High sensitivity fast Spectrometers

For absorbance, fluorescence, fluorescence anisotropy, light scattering or circular dichroism.
Steady state spectral recordings
Fast kinetics transient recordings

• Optical systems - click here

Rapid-Filtration instrument

• click here

Bio-Logic develops and manufactures Rapid-Kinetics instrumentation, associating quality with innovation to produce instruments a step above the rest

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Over 400 Bio-Logic rapid kinetics instruments have been installed worldwide. The reason for our success lies in the quality of our instruments.

We understand that investing in instrumentation is not a trivial matter. What arrives in your lab today is going to be part of your research program for years to come. Our instruments are designed to last and grow with your research program. We are dedicated to supporting our instruments, be it the initial or the most recent instrument to come from our factory. Our first instruments are still in use today !

Over the years our instruments have grown and changed, fueled by customer / manufacturer interaction.
The Bio-Logic development team is dedicated to listening to client feedback and seeing which direction research is moving. Our instruments evolve with client needs and in anticipation of them.

This tradition continues on today with our current line of rapid mixing instruments. 

The Bio-Logic SFM

The Bio-Logic Stopped-Flow Modules (SFM) are a fusion of technology, design and innovative control. Each element of the fusion compliments the others making the Bio-Logic SFM most powerful and flexible rapid mixing instruments existing today.

All Bio-Logic SFM instruments are based on our innovative use of stepping-motors to drive the syringes.
With modern microprocessor techniques, stepping-motors offer control and power unmatched by any other fast driving system.

The high performance of each SFM and the high speed of the stepping-motors is achieved because of the quality of the power-supply.

Each Mixer Power-Supply (MPS) unit contains independent power-supplies for each syringe, each driven by their own microprocessor. The sequence of impulses sent to the stepping-motors is stored in the memory of each motor board. One main microprocessor board synchronizes all the power supplies, and communicates with the computer via a serial interface.

Stepping motors advantages
The use of stepping-motors and a high quality power supply, gives each SFM the following advantages.

• Completely Variable Mixing Ratios:
  Because the flow rate and volume delivered by each syringe is independent of the others, mixing ratios are not fixed by syringes sizes. Mixing ratios are defined by the user, on-line.
• Precise Flow Rate and Volume Control:
  The flow rate and volume delivered by each syringe is chosen by the user, not defined by the instrument.
• Reproducibility and Regularity:
  The reproducibility and regularity of the linear translation of the syringes and the absence of pressure artifact allow optical recording during liquid flow.
  This capability greatly facilitates the determination of the initial phase of the reaction being monitored during stopped-flow experiments and makes the equipment suitable for accurate, continuous flow experiments.
• No Overpressure Artifacts:
  Syringe movements are completely controlled by microprocessor and can be halted in a fraction of a millisecond. This abolishes the need for a stop syringe during stopped-flow experiments and eliminates the stop artifact present in most conventional stopped-flow systems.
• Mixing of viscous solutions:
  Syringe velocity is programmed and thus is independent of the solution viscosity. Very asymmetric mixing such as pure glycerol against water can therefore be achieved.

Mixer technology
The mixer is the heart of a stopped-flow instrument. Although being often referred as "T-mixer" a straight T-connection between 3 tubes is capable of mixing two solutions only in limited cases (aqueous solutions, symmetrical 1:1 mixing, limited range of flow rate). The mixer used in the Bio-Logic Stopped-Flow are all based on the "ball mixer" technology which is a proven design for creating turbulence in the most stringent conditions. Because of it the Bio-Logic Stopped-Flow can operate in a wide range of fluidic conditions such as : high dilution ratio (up to 1:100), high range of flow rate (from 1 to 20 mL/s), mixing of highly viscous solutions against water (viscosity up to 2000 centipoises or 2 pascal.second have been achieved).

Applications

The application of the Bio-Logic SFM family of instruments is large and evolving. We have made our instruments as versatile as possible so that they are not the limiting factor in your research.

Here is a sampling of how our instruments have been used:

Stopped-Flow Experiments Protein Folding Conformational Changes Substrate Binding Enzyme Kinetics Substrate Transport in Vesicles	Measured by: UV/VIS Absorbance Light Scattering Fluorescence Circular Dichroism Fluorescence Anisotropy FTIR X-ray scattering Conductivity Mass Spectrometry
Quench-Flow Experiments Substrate Binding Enzyme Kinetics Protein Folding Kinetics Conformational Changes Second Messenger Studies	Measured by: Radioactive labeling Hydrogen/Deuterium Exchange followed by NMR or Mass Spectrometry
Freeze Quench Methods Trapping of kinetic intermediates by ultra-fast freezing	Measured by: Solid-State NMR EPR X-ray scattering EXASF

The range of application of our instruments extends beyond the several examples listed here and is growing every day ! 

If you have questions about your specific application and would like to know how it can be supported by a Bio-Logic rapid-kinetics instrument, contact us !. We would be happy to discuss your application with you.

Mechanical Design

Each SFM is designed to be a versatile rapid-mixing instrument. It is not confined to a single functionality, but adaptable and expandable to a variety of rapid-mixing experiments.

This is due to the "open" mechanical design of an SFM. The syringes and motors are housed in a body that accepts attachments created for different types of experiments. Every SFM can be configured for Stopped-Flow, Quench-Flow, Optical-Quench and many other experimental conditions.

Switching from one mode of operation to another is just a matter of replacing one attachment with another. This operation requires only a few minutes and no specialized tools.

Design Features

Vertical mounting of the SFM syringes facilitates purging of bubbles which might interfere with experiments. Bubbles can be evacuated from the instrument with a few pushes of each drive syringe.
The syringes, valves, and observation chamber can be very uniformly thermoregulated through the use of a circulating temperature bath connected to the SFM. The coolant flows through two internal circuits: one around the injection and reservoir syringe ports and the other through the valve block and observation head. This thermoregulation prevents the occurrence of temperature artifacts on a very wide temperature range, and permits rapid-kinetics studies even at low temperatures.

Quality Construction

Every SFM is carefully constructed of high quality materials. The parts in contact with the sample and the buffers are all machined out of materials selected for their inert characteristics

Instrument control

The Bio-Kine32 software operates under the latest version of Windows environment (Win95, Win98, WinME, WinNT, Win2000, WinXP, WinVista)

The software is easily configured for any of our SFM instruments. Through the use of various menus and windows, the Bio-Kine32 software allows the user to: 

• know the volume of the solution in each syringe
• perform manual or automatic movement of the syringes
• create a driving sequence with complete control of the volume delivered and flow rate of the syringes
• save or recall driving sequences
• start or stop experiments
• program the synchronization pulse used to trigger the acquisition system
• load the spectrometer data acquisition software: Bio-Kine

Dead and ageing time estimations are updated anytime the driving sequence or configuration parameters are changed.
These estimations are only possible because the user has complete control over syringe flow rate and volume delivered.

Up to 20 phases can be included in a driving sequence, allowing experiments involving complicated steps and washings to be executed with ease.

Automation of experiments is possible through the Bio-Kine32 software.
The Bio-Kine32 software is also used to automate experiments and acquisitions. Multiple shot averages can be made with a single button push.

Stopped-Flow optics

The Observation Head
The observation head is the interface between the Stopped-Flow modules SFM-20/300&400 and the recording spectrometers for optical Stopped-Flow experiments. The observation head houses the flow cell (cuvette). A variety of flow cells have been developed to optimize the optical conditions specific to each mode of observation. Installation and exchange of observation cell is an easy operation that requires a couple of minutes without any optical readjustment.

It is also a unique feature of the SFM instrument, that the observation head allows insertion of standard 1 cm x 1 cm cuvette for steady-state measurements and calibration of the optical system.

The optical ports used for detection can be equipped with lens or fiber optics to direct the transmitted or emitted light wherever it is needed.

In most standard configurations, the optical head is set directly on the top of the Stopped-Flow instrument. This ensures mechanical and optical stability and also the lowest volume between the drive syringes and the last mixers. However, for some applications it is necessary to have a physical separation between these two parts of the instrument. This is achieved by the Umbilical link.

Observation cuvettes

Feature

• Allow observation of very fast reaction in stopped-flow mode. Dead time:
  • down to 0.25 ms with SFM-300 & SFM-400
  • down to 0.4 ms with SFM-20.
• Quick attachment to an SFM
• Built-in temperature jacket

The microcuvette accessory is a specially designed observation head that allow observation of very fast Kinetics.

Optical Connections

The microcuvette accessory is built in the same fashion as our stopped-flow observation head with four optical ports (1 x illumination and 3 x detection). The microcuvette accessory is 100% compatible with the optical connections for all Bio-Logic Optical systems, minimizing setup time. The microcuvette accessory can also be connected to third party optical systems using fiber optic light links.

This series of test was designed to demonstrate the fastest dead time that can be achieved with the SFM-300 or SFM-400 instruments.

The test below uses the 2001 version of the SFM-300/400 with its new hard stop valve (V3), in addition it uses the new microcuvette µFC-08 and its associated micromixer.

This cuvette has a volume varying from 1 to 3 µL depending on the point of observation. The experiments below were executed at the median position of 2 µL The optical light path of the microcuvette is of 0.8 mm, it has 3 observation windows that allows it to be used either in absorbance or fluorescence modes.

Instruments used for the test experiment

Stopped-flow : SFM-300 equipped with µFC-08 cuvette. All syringes were the standard 10 mL syringes

Spectrometer : MOS-250 in absorbance mode.

• Illumination wavelength : 525 nm
• Bandpass 5 nm
• 150 W Xe lamp

Test reaction

Reduction of DCIP by ascorbic acid at acid pH

Description of the experimental procedure

Syringe contents :

• Syringe N°1 = water
• Syringe N°2 = 50 mM Ascorbic acid
• Syringe N°3 = 750 µM DCIP

Flow rate was adjusted to 8 mL/s per active syringe (16 mL/s in total)

Absorbance was measured at 525 nm and data were recorded at a rate of 50 µs per point.

In a first serie of shot DCIP was mixed with water and the absorbance of DCIP diluted by ½ was recorded by using syringes N°1 and N°3

In a second series, DCIP was mixed with ascorbic acid by using syringes N°2 and 3.
Reduction of DCIP was observed and the measured absorbance  is expressed as % of the absorbance measured when mixing DCIP with water.

Results are shown in the figure below

The time axis is set to zero at the time of hard stop closure. Data points are in red and the trace is fitted with an exponential corresponding to a rate constant of 1850 s -1.

From this value of the rate constant and from the observed absorbance amplitude (62 %) a dead time of 0.25 ms can be calculated. A 0.25 ms dead time would allow observation of 1/3 the amplitude of a reaction having a rate constant of 4400 s -1.