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

Stopped-Flow micro cuvette

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.