SX20 Stopped-Flow Spectrometer
Performance Information
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Light Sources
Photometric accuracy
Cell design
Absorbance
Fluorescence and inner filtering
Lamp stability
Dead-time determination
Low temperature work
Three types of lamp are available for the SX20 lamp housing: a 150W xenon arc (ozone-free), a 150W xenon arc with spectrosil envelope (ozone-producing) and a 150W mercury-xenon (ozone-producing). All lamps provide outstanding stability and durability conforming to Applied Photophysics's tight specifications. The ozone-free xenon lamp is supplied as standard and is suitable for most applications. The ozone-producing xenon lamp has a much higher light intensity below 240nm. The mercury-xenon lamp has strong Hg emission lines over the xenon spectrum at wavelengths that can be of interest for specific applications in fluorescence and circular dichroism. The figure below shows the output profiles for a xenon lamp (red) and a mercury-xenon lamp (blue).
The lamp housing includes a nitrogen purge facility (for use with the ozone producing lamp) and allows external lamp adjustment. Lamps can be readily interchanged but if interchanging the lamp (e.g. between Xe and Xe-Hg) is likely to be regularly required, we recommend purchasing a second back-plate for the lamp housing – this will enable lamp to be changed in a few minutes without the requirement to handle the lamp itself or re-align the lamp when it is fitted.
The lamp power supply unit (PSU) is optimised to provide a stable lamp output. Lamp ignition uses a ‘SafeStart’ igniter system which will not interfere with sensitive electronic equipment.
The use of spectrophotometry as a tool to follow concentration changes requires that certain criteria are met. With respect to absorption measurements, there must be conformance to the Beer/Lambert law so that absorbance is directly proportional to concentration (i.e. A = e.c.l where e = extinction coefficient, c = concentration and l = optical path). Accurate absorbance measurements in the UV region require that adequate account be taken of spectrometer stray light error. Potassium dichromate is a useful reference material for assessing spectrometer performance. The spectral plots shown in the figure below indicate that the standard SX20 stopped-flow spectrometer has good photometric accuracy down to ~240nm (green and blue traces) whereas using the double monochromator configuration of option AM.1 (red trace) there is excellent photometric accuracy to 2AU at 200nm.
The ozone-free lamp has no emission below 230nm and so the apparent absorbance spectrum in this region with a single monochromator (green trace) is entirely due to stray light error. The ozone-producing lamp does emit below 230nm and so the measured spectrum in this region with a single monochromator (blue trace) is a combination of real sample absorbance and stray light error. When the ozone-producing lamp is used in combination with the double-monochromator configuration (option AM.1) there is effectively no stray light error and the true absorbance spectrum of potassium dichromate can be recorded down to 200nm. Comparison of these spectrum with those recorded with a single monochromator indicate that good photometric accuracy can be achieved to at least 240nm using a single monochromator.
The standard 20uL volume quartz cell has dimensions 10mm x 2mm x 1mm, and provides optical pathlengths of 10mm and 2mm. Black quartz is used to mask the optical windows. To switch optical pathlengths, the user simply relocates the detector and light guide – as task of about 1 minute.
Fluorescence Measurements and Inner Filtering
The SX20 cell is uniquely optimised for fluorescence detection because the ‘fifth’ side of the cell is dedicated to this purpose. This means that:
- The cell is able to incorporate a light pipe specifically designed to maximise collection of fluorescence emission thereby increasing sensitivity.
- The Inner filtering effect (see below) can be low without having to compromise sensitivity by reducing the cell volume.
- No reconfiguration is required when switching between absorbance and fluorescence detection.
With respect to fluorescence measurements, the inner filter (or self-absorbance) effect must be considered before assuming that the measured signal is directly proportional to the concentration of a chemical species. The inner filter effect is caused by progressive absorption of the fluorescence excitation light as it penetrates the solution being studied, thereby producing progressively less fluorescence signal. Hence a change in the total absorbance during the reaction can produce non-exponential fluorescence kinetics. The effect is minimised by using low sample concentrations and/or low optical pathlength cells. With the SX20 20ul cell, sample excitation via the 10x1mm window (2mm port) gives a low optical pathlength for fluorescence measurements (1.5mm – assuming scattering from the centre of the cell chamber). Excitation via the 2x1mm window gives a higher value (5.5mm). In both cases the entire sample volume can be irradiated. Compare this with a similar stopped-flow cell where the fifth side of the cell cannot be used: here the optical pathlength will be 6mm irrespective of which port is used for sample excitation. This higher value limits the range of sample concentrations that can be used.
Light source stability is a critical requirement to ensure that small signal changes can be detected and that kinetic traces are reproducible. Lamp stability should be examined for quality in several time domains. Around 100ms will show up ripple originating from the lamp power supply with a frequency related to that of the line supply voltage. The 100ms time period will also show photo-multiplier shot noise due to insufficient light flux. Different types of lamp instability (i.e. plasma instability and arc wander) are revealed between 1 and 10 seconds with long term drift showing over periods of 1000 seconds or more. The traces below illustrate the performance of a typical SX20 instrument with an ozone free xenon arc lamp. The instrument had been allowed to fully stabilise prior to recording these measurements. An absorbance change of 0.001AU represents a signal voltage change of 0.25%.
 Over 0.1 seconds. |
 Over 1.0 seconds. |
 Over 10 seconds. |
 Over 1000 seconds. |
The instrument dead-time can be defined as the earliest time at which valid measurements of the reaction can be made. A short dead-time is an important requirement for measurement of very fast reactions and its value is therefore an indicator of the both instrument’s kinetic performance and the overall design quality of the sample handling unit.
The fluorescence quenching reaction between N-acetyltryptophanamide (NAT) and Nbromosuccinimide (NBS) provides a useful tool for measuring the dead-time[1]. To obtain the data shown here, NAT was mixed with a range of NBS concentrations in a 1:1 and 1:10 drive ratio. The reagent concentrations (after mixing) were 10-5M (NAT) and 5x10-5 to 5x10-3M (NBS). Excitation was set at 280nm and the fluorescence signal was isolated using a 305nm cut-off filter.
The dead-time can be calculated as the slope of a linear plot of ln(initial signal) vs. rate constant. The figure below displays the plots and the corresponding dead-times for 1:1 and 10:1 mixing experiments. It can be seen that using the 5µL cell (option RC5.1) provides a significant improvement in the dead-time allowing measurement of rates in excess of 3500s-1.
Software Controlled Dead-Time Determination. With option SQ.1, the dead-time can also be measured directly from the instrument control panel simply by doing a stopped-flow drive; the time course of the drive together with the total volume dispensed, the final velocity and the calculated dead-time are recorded. This is a useful feature for checking the instrument’s performance and for assessing the effects of parameters such as drive pressure, reagent volume and reagent viscosity on the dead-time without recourse to more lengthy chemical methods as shown above.
[1] Peterman, Anal. Biochem., 1979, 93, 442.
The standard SX20 instrument operates over the temperature range +60°C to -20°C with no requirement for additional accessories. No instrument reconfiguration is required when operating at low temperatures and the sequential mixing facility (option SQ.1) can also be utilised over this temperature range. The SX20 control software can also be linked to a programmable circulator bath to enable acquisition of an automated series of kinetic measurements of a range of (user specified) temperatures.
Measurements of absorbance changes in the alkaline hydrolysis of 2,4-DNPA (2,4-Dinitrophenylacetate) in methanol are shown here to demonstrate the SX20’s performance over the temperature range -22°C to+24°C. 100mM 2,4-DNPA in MeOH was hydrolysed in a 1:1 symmetric mix with 0.3M sodium methoxide (NaOMe) in MeOH. The stopped-flow kinetics were recorded at 360 nm. A Neslab RTE-200 circulator with water:ethylene glycol (50:50) circulating coolant was used for thermostatting.
The figure (above) shows the kinetic traces acquired at the upper and lower limits of the temperature range. The recorded traces at each temperature were analysed to create an Arrhenius plot (below) from the measured individual rates. The linearity of the data points demonstrates that the SX20 retains its excellent performance even at these low temperatures.
SX20 - the ultimate gold standard in stopped-flow
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