Stopped Flow Support FAQs
How often should I replace the lamp?
- The recommended lamp lifetime is approximately 1,000 hours for optimum performance. However, there can be variation in performance between lamps. As lamps age, they become less stable, less intense and more difficult to ignite. It is very important that lamps are not operated beyond the manufacturers maximum lifetime (normally 2,000h).
How do I change the lamp?
This video demonstrates the correct procedure for lamp replacement and alignment
How often does the instrument need to be serviced?
- Depending on how heavily the instrument is used, servicing is recommended at different intervals. The most common service interval is every 12 months.
What is the minimum sample volume required for a stopped-flow measurement?
- For single mixing experiments a minimum of 120 µL is the recommended total drive volume. Users may choose to reduce drive volume to improve sample economy under certain experimental conditions. Further advice is available from our support team.
How do I adjust the total drive volume of the instrument?
- Adjust the total drive volume by rotating the knurled adjuster ring underneath the return cylinder (the bottom piece of the autostop assembly). A clockwise rotation will increase the drive volume, and a counter-clockwise rotation will decrease drive volume. A full turn of the adjuster is equivalent to approximately 45 µL.
How do I clean the cell and flow circuit?
- Flushing the instrument with water will normally be sufficient. More stubborn materials can be removed using a 2M nitric acid solution. If there is heavy contamination, remove the cell and treat individually with a more concentrated solution of nitric acid.
What materials are used in the flow circuit?
- The flow circuit is largely made of polyether ether ketone (PEEK) with a quartz optical cell and glass syringes.
What is the instrument dead-time and how can I measure it?
- The standard instrument deadtime is around 1-1.5 ms. This can be measured electronically with a sequential mixing drive ram or using a published chemical method.
What is the purpose of the Pressure Hold function?
- This function relieves an artefact that may appear in kinetic traces due to the release of the drive pressure by the instrument during the early phase of an experiment (typically around 50 ms).
Are there any circumstances under which you do not recommend the use of pressure hold?
- Do not use Pressure Hold for reactions lasting longer than 10 seconds.
What is oversampling?
- Oversampling is a noise-reduction technique employed by the instrument. Each time point is the plotted average of a series of 12.5 µs samples.
When should I use the Logarithmic and Split Timebase functions?
- Use these options to study reactions with multiple steps. These timebases provide a convenient means of plotting fast and slow reaction phases with a sufficient number of data points.
What is the maximum number of data points?
- The instrument can handle up to 10,000 data points. For most experiments 1,000 data points should be more than sufficient.
What is the temperature range of the stopped-flow?
- Using the standard configuration, the drive syringes are suitable for operation from -20 to 60°C. Alternative syringes may be suitable for operation at the extremes of this range.
- Bubbles create artefacts in your data, but also take a few sample drives to work their way out of the system, so they are costly in terms of precious samples. The bubble will often appear as a rapid increase followed by slower signal decay in your data.
How do I remove bubbles from the flow circuit?
- Firstly, take care not to introduce bubbles with the sample during loading. If a persistent bubble has made its way into the flow circuit, flush the flow circuit with ethanol. A pressure hold may also help prevent the migration of bubbles.
What is the best way to store the instrument when not in use?
- Use water for day-to-day storage of the flow circuit. For prolonged periods of inactivity, store the flow circuit in 20% ethanol to prevent biological growth. It may also be wise to drain the water bath and remove the front panel to prevent biological growth in the water bath over prolonged periods of non-use.
Can I measure absorbance and fluorescence at the same time? Can I measure two fluorescence signals at the same time?
- Both options are possible for instruments with two data acquisition channels. Dual detection is possible if your instrument has two PMT detector cables connected to the electronics rack. If you only have one PMT detector cable, dual detection is not possible without a dual detection upgrade.
What cell pathlengths are available?
- Standard cell (20 µL): 2 mm and 10 mm pathlengths. Rapid kinetics cell (5 µL): 1 mm and 5 mm pathlengths.
What are symmetric and asymmetric mixing?
- Symmetric mixing uses two identically-sized drive syringes, such as the standard 2.5 mL syringes provided with the instrument.
Asymmetric mixing uses two syringes of different sizes to mix a volume ratio other than 1:1. For example, a 10:1 ratio (common for refolding experiments) can be achieved using a 250 µL syringe in place of one of the 2.5 mL syringes.
What ratios are available for asymmetric mixing?
- 2:1 (5.0 mL and 2.5 mL), 5:1 (2.5 mL and 500 µL), 10:1 (2.5mL and 250 µL), 25:1 (2.5mL and 100 µL).
Are any adjustments required for an asymmetric mixing experiment?
- Reduce the drive pressure on the external regulator (attached to the back of the instrument) to 2 bar/30 psi. Failure to do so will cause the syringe, or worse the cell, to fail.
What are single and sequential mixing?
- Single mixing is the mixing of two reagents (only one mix occurs, a “single” mix).
Sequential mixing, also known as double mixing, is when there are two mixings. The first mixing pushes two reagents, A and B, into an unobservable aging loop for a prescribed delay time, before being pushed into the flow cell with a flushing agent. A third reagent is pushed directly to the flow cell at the time of the flush.
Are adjustments required for a sequential mixing experiment?
- Use the brake on the autostop assembly to stabilize the stop syringe during the ageing period.
What is the recommended start-up procedure for the instrument?
- Start the lamp first. Switch on the power supply for 10-15 seconds before pressing the ignition button. Then turn on the electronics and allow them to initialise before starting the software.
Wait 30 minutes for lamp output to stabilise. Use this time to set up other elements of the instrument, such as water bath temperature and gas pressure (8 bar/120 psi at the source and 4 bar/60 psi on the instrument regulator at the rear of the KSHU).
What is the recommended shutdown procedure for the instrument?
- Closing the software before switching off the electronics. Shut down other elements in any order.
Is the software licensed?
- The Pro-Data SX software including the Pro-Data instrument control software, Pro-Data Viewer and Pro-Data Converter are licensed for installation on multiple PCs on one site without restriction.
The Pro-K global analysis software is licensed for installation on single PCs up to a specific number of licenses agreed at point of sale.