Applied Photophysics Ltd

Frequently Asked Questions for SX Instruments

How do I know if my lamp needs replacing?

The Xenon lamps used by Applied Photophysics Ltd. have a rated life of 1000 hours. The manufacturers of the lamp state that they must be changed after 125% of rated life. Some lamps may not last that long. A lamp may need replacing earlier than 1000 hours if it exhibits any of the following symptoms:-

  1. It may be more difficult than usual to start.
  2. It may take longer to stabilise.
  3. The light output may jump around erratically.
  4. There may be a ripple on the lamp that wasn't there before.
  5. A visual inspection of the lamp may show a brown deposit on the inside of the quartz envelope.
  6. A visual inspection of the electrodes may show deposits of metal on the ends of the electrodes.

The lamps have a shelf life of around three years. During this time the Xenon inside them slowly seeps out. If your replacement lamp has been sitting in a draw or on a shelf for several years it may not perform as well as it should and may not last as long as it should.

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How do I choose the correct slit widths on my monochromator?

There is, unfortunately, no simple answer to this question. The only thing that is certain is that the exit and entrance slit on a monochromator should be the same. The size of the slit width affects several things. It affects the bandpass of the monochromator, which is 4.65 nm/mm. It affects how much light reaches your sample. It can affect the percentage stray light. All of these things need to be considered when choosing a slit width. Narrow slits mean smaller bandpass and less light at the sample. This results in less photochemistry but the photomultiplier will have to work harder giving a poorer signal to noise ratio.

As a general starting point the following slits widths often give good results:-

  • 0.5 mm on a single monochromator system performing absorption experiments.
  • 0.5 mm to 1.0 mm on a single monochromator system performing emission experiments.
  • 1.0 mm on monochromator 1 and 2.0 mm on monochromator 2 for absorption experiments where the two monochromators are in series.
  • 2.0 mm on monochromator 1 and 4.0 mm on monochromator 2 for CD experiments where the two monochromators are in series.
  • 1.0 mm on monochromator 1 and 3.0 mm on monochromator 2 for fluorescence experiments where the two monochromators are acting independently (one for excitation and one for emission).

Which filters do Applied Photophysics Ltd. supply for fluorescence work?

All stopped-flow system supplied by Applied Photophysics Ltd. include two standard cut-off filters, with values of 305nm and 320nm. The 305nm filter is most often used with tyrozine and the 320nm filter is most often used with tryptophan. Other cut-off filters are available with the following cut-off values; 295nm; 335nm; 360nm; 375nm; 395nm; 400nm; 420nm; 455nm; 475nm; 495nm; 515nm; 530nm; 550nm; 570nm; 590nm; 610nm and 645nm.

Applied Photophysics Ltd. can also supply ultra thin bandpass filters which can be used in conjunction with a cut-off filter to isolate a particular wavelength region. The plot below shows the transmission curves of the three filters we can currently supply.

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Why does Applied Photophysics Ltd. use Acorn computers?

When Applied Photophysics produced the world's first pneumatically driven, vertical, stopped-flow spectrometer, back in 1989, the best value, high performance desktop computers were manufactured by Acorn. However, the price performance equation has shifted in recent years and our current spectrometers are controlled by PCs operating under Windows™ XP.

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Can I connect the Acorn computer to my Local Area Network?

Using a piece of generic client software called Lanman98, an Acorn computer, fitted with an Ethernet card, can be made a client of a LAN Manager server. This uses TCP/IP as its underlying protocol. For this reason, the Acorn's TCP/IP stack must be activated and correctly configured. Also, the PC that you wish to connect to must be running TCP/IP.

Under these circumstances the Acorn will be acting as a client (i.e. it receives the service of the PCs filing system which is acting as a server). The Acorn will not be acting as a server for this protocol so its harddrive and printer are not accesible to other computers on the network.

When connecting to a Microsoft standard TCP/IP network it is only possible to connect to a specific PC server directly, not to a workgroup on the network. It is necessary to know the name of the server and a share on that server along with its password (this will have been previously set up on the PC network, a directory or possibly the whole of a drive will have been set to be shared under a name that isn't necessarily the same as the directory or drive, and a password will have been assigned to the share, although this might be null). Full instructions for connecting an Acorn computer to a PC via a network can be found by clicking here.

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How can I check that the detection system is being triggered
correctly?

In order to prevent faulty triggering a complex electronic circuit is used to trigger the data collection. This circuit incorporates "anti-bounce" circuitry and "false trigger" elimination. If the triggering circuitry fails it will do so in one of two ways. Either the trigger will be permanently on, resulting in triggering the moment "Acquire" is clicked, or the trigger will be permanently off, resulting in no triggering and the detection system timing out.

If the system is triggering incorrectly a systematic approach is required to identify the faulty part of the circuit. There are many possible causes of faulty triggering. The two most common causes are described here. If neither of these solutions cures your problem then contact the Technical Support Department who will send you further tests to isolate the fault.

The most common cause of faulty triggering is the trigger mechanism itself. This is located under the stop syringe and can become corroded due to chemical leakage. When performing an acquisition (in SX mode and INT TRIG turned 'OFF'), the computer is given the signal to begin collecting data when the leaf trigger switch is closed by the stop syringe plunger. To check that this is working correctly, remove the trigger lead that connects the AutoStop to the back of the Sample Handling Unit. Connect a continuity meter to the two pins inside the plug at the end of the lead. Ensure that the stop syringe plunger is not in contact with the trigger. The continuity meter should read open circuit. If it doesn't then the trigger is shorting to the stop. Assuming that you do have open circuit, manually close the leaf trigger switch and check that the continuity meter now shows closed circuit. If it doesn't then clean the contacts on the auto-stop leaf switch with fine abrasive paper. If this fails then check the condition of the soldered connections inside the Auto-stop trigger plug. Also check the soldered connections at the other end of the trigger lead.

The second most common cause of faulty triggering only affects users of DX.17MV and SX.17MV spectrometers fitted with a flat ribbon, multi plugged, data cable. These cables are fitted with a 20 way rectangular plug which attaches to the Digital I/O socket on the Analogue to Digital Converter card fitted in the back of the computer. Only two pins are used; pins 6 and 19. Often one of these pins will be pushed back inside the connector on the ribbon cable, so that it does not make good contact with its mate inside the ADC card. To check this, remove the 20 way connector from the Digital I/O socket on the ADC card and visually inspect pins 6 and 19. They are on opposite sides of the connector. If it looks as if one of them is not fully forward in the connector then you will need to disassemble the connector and re-seat the pin.

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What is this 2ms plateau at the beginning of my trace?

Explanation of Pre-Stop Information on APL Stopped-Flow Spectrometer

The Applied Photophysics Stopped-Flow Spectrometer starts to acquire data 2ms before the flow is stopped. This allows for extrapolation of fitted curves to before the point the flow stopped, ideally to the mixing point for the chemistry.


Click to open larger view in new window

Starting from the left hand edge of the diagram above, the computer sends out a signal to the sample handling unit to fire its drive ram. After a period of time the drive solenoid opens and the pressure behind the drive ram builds up enough for the ram to start moving. This is the point at which the flow starts. The flow of chemistry from the two drive syringes passes through the mixer and this, newly mixed, chemistry flushes out the old chemistry from the observation cell, replacing it with newly mixed chemistry. Eventually the cell is completely flushed with newly mixed chemistry and there is continuous flow of new chemistry through the cell. This shows up as a horizontal signal, the length of which is determined by the volume of chemistry flowed.

During this continuous flow the detection system is triggered and data acquisition starts. On the diagram above this is the point the trace changes from green to red. None of the green part of the trace is seen on the computer display (unless pre-trigger is selected).

2ms after the data acquisition has started the flow of chemistry through the cell stops. This 2ms period is called the pre-stop period. It is important to bear in mind that this 2ms period is an arbitrary amount and in no way relates to the dead time of the instrument. With enough chemistry we could flow much larger quantities for much longer times through the cell. The time taken for the chemistry to get from the point that it has mixed to stationary in the cell (dead time) is independent of the amount of time we allow this flow to continue. This is because the flow rate is constant during this continuous flow.

Once the flow has stopped the course of the reaction can be monitored. After the data has been acquired it can be fitted. It is suggested that the left hand fit range is set slightly to the right of the stop position to allow for the transition from flowing chemistry to stationary chemistry. The X-Datum can be set to correspond to the true time zero for the reaction. This true time zero is the time that the reaction starts and is the dead time before the stop position. Alternatively an X-Shift can be set in the New Data section of the software to automatically move the zero on the X-axis to correspond with the true time zero. In the above example the X-Shift would need to be set to 1000µs. This needs to be done before data is acquired. The fitted curve will extrapolate back to either the X-Datum or zero on the X-axis. The calculated delta signal will be from this extrapolated point to the calculated end point. It is worth noting that the calculated rate constant is not affected by the position of the extrapolation point.

If multiple traces with different rate constants are overlayed, then all of the extrapolated fits will intersect at the true time zero and this is one way to check the dead time of the instrument.

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How do I get rid of artefacts?

1) Random Artefacts.

Random artefacts have three major causes. The first is random fluctuations in the light source, commonly called "Arc Wander". This will result in random spikes in your data traces. It is generally an indication that your lamp needs replacing. The second cause is gas bubbles passing through the optical path. These bubbles may have been introduced with your chemistry, they may be forced out of solution by the mixing action (in which case degassing may help), or they may be generated as a by-product of your reaction (in which case holding the drive pressure on during acquisition may help). The third cause is particulate matter passing through the optical path. This generally means that you have not cleaned the observation cell as thoroughly or as often as necessary.

2) A Single, Reproducible, Artefact Between 30 and 100 Milliseconds.

This artefact is caused by the drive pressure being released during data acquisition. Normally removing the drive pressure during data acquisition is perfectly acceptable so there must be an additional problem to cause the artefact. This could be any one of the following:-

  1. The trigger spring is too far away from the stop. This would result in the spring pushing the stop syringe plunger upwards when the drive pressure is released, back flushing a small amount of chemistry through the observation cell. The solution is to bend the trigger spring so that it is closer to the stop. Ideally it should be less than 0.5mm from the stop, but not actually touching the stop.
  2. There are gas bubbles in the stop syringe. These bubbles will expand when the drive pressure is released and cause a small amount of chemistry to be back flushed through the observation cell. The solution is to remove all of the gas bubbles from the stop syringe.
  3. The flow tubes which connect the cell to the stop syringe have become elastic due to old age. This would result in these tubes expanding when under pressure and contracting when the drive pressure is released, again resulting in chemistry being back flushed through the observation cell. The solution is to replace these flow tubes.
  4. Microscopic gas bubbles in the chemistry are expanding in the optical path when the drive pressure is released. This is a particular problem when using solvents like DMSO. Degassing the solvent may help, otherwise the only solution is to maintain the drive pressure during data acquisition.
  5. Very light solvents with a relative density of 0.8 or less, such as acetone or acetonytrile, change their optical density as a function of pressure. The drop in pressure that occurs when the drive pressure is released results in a reduction in the absorption of the solvent. The only solution to this problem is to maintain the drive pressure for the duration of data acquisition.

In all cases, maintaining the drive pressure for the duration of data acquisition may help. In some cases, increasing the flow volume may help.

3) A Single, Reproducible, Artefact at Around 2ms in Fluorescence Mode.

This artefact is caused by the fluorescence cutoff filter moving due to a physical shock caused by the flow being stopped suddenly. The solution is to hold the filter in place. Two small pieces of putty (Blue Tack or Sticky Tack) are ideal for holding the filter in place.

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Where is this ripple coming from?

There are three sorts of ripple that you may observe on your data.

1) A Decaying Sinusoidal Ripple

This ripple is caused by vibration resulting from the shock caused by the drive rams firing and the flow being suddenly stopped. It is a very strong indication that the optical system is not properly aligned.

2) A Mains Frequency (50 or 60 Hz) Ripple

This has the following causes:-

  1. Vibration from the fan in the lamp power supply. If your workbench conducts this frequency well, then it is quite likely that the fan in the lamp power supply will cause a vibration ripple to be displayed on your traces. The solution is to isolate the lamp power supply from the workbench. An easy way to do this is to place one or two mouse pads or other foam material underneath the lamp power supply.
  2. Light leakage. Fluorescent lights in your laboratory oscillate at mains frequency. If light from this source can get into your optical system then it will show up as a mains frequency ripple on your traces. The most common reason for light leaking into your system is that one of the blanking plugs, used to cover spare ports on the sample handling unit cell block, is missing. The easiest way to test for light leakage is to turn off all of the lights in your laboratory and see if the ripple is still present.
  3. Lamp ripple. If your Xenon lamp is near to the end of its useful lifetime it may cause ripples to be observed on the traces. The solution is to change the lamp.
  4. Other electronic noise. An electronic component may have failed on your system, resulting in mains frequency noise being injected into your signal. Diagnosis of this problem is difficult and should be done in consultation with the Technical Support Department.

3) A Slow Frequency Ripple (Between 0.5 and 2 Hz)

This ripple is called parasitic oscillation. It is due to a power matching problem between the lamp power supply and the lamp. It is always present around the edges of the arc and may indicate that your optical systems requires aligning. If this does not help then adjusting the power balance may help. If your lamp power supply has a scale on the front then adjusting the power knob to run the lamp at a slightly higher power may help. If your lamp power supply does not have a scale on the front then you will need to adjust the potentiometer on the back of the supply using a trim tool (a small, insulated, flat bladed screwdriver.). If adjusting the power balance does not help then you may need to replace the Xenon Lamp.

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How can I check if my flow circuit is leaking?

  1. Ensure that the flow circuit is flushed through with distilled water
  2. In the "NewData" section of the control software, set the timebase to 5 seconds
  3. Ensure that the drive valves are in the drive position and the the drive syringes are full (i.e. that the sample handling unit is ready to be fired.)
  4. Click on "Acquire" with the right hand mouse button.
  5. The AutoStop will go through an empty cycle and the stopped-flow will fire. Because you used the right hand mouse button the drive pressure will be held on for the duration of the 5 second data acquisition.
  6. As soon as the stopped-flow fires watch the pistons of the drive syringes. They should come to a complete stop. If they continue to move forward during the 5 second data acquisition period then there is a leak in your flow circuit.

If the drive syringe piston do keep moving then you will need to track down the leak. This will involve draining the thermostat water bath and removing the front cover. The following places should be monitored. All flow tube connections; the tops of the drive syringes, the reservoir syringes (if one of the drive valves has worn out then liquid will leak past the valve spindle back into the reservoir syringe); the waste line (if the stop valve has worn out then liquid will leak past the valve spindle back into the waste line.). Once the leak has been found appropriate action can be taken.

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Why doesn't the thermostat bath fill up with water when I turn on the circulator.

The thermostat water bath has been connected the wrong way round. That is the outlet from the circulator is connected to the wrong thermostat port on the back of the sample handling unit. The solution is to reverse the connection to the thermostat bath.

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If you have a question that is not answered here please e-mail the Technical Support Department.