Frequently Asked Questions for PiStar Instruments
Lamps
CD Measurements
- Why is the CD spectrum noisy in the far UV?
- Why is the amplitude of my CD spectrum wrong?
- How do I calibrate Pi-Star if I want to measure CD?
- Why is my CD baseline so far from zero?
Absorption/Fluorescence Measurements
- Why doesn't my fluorescence signal move when I apply an offset?
- How can I improve the light throughput in fluorescence and absorbance mode?
- Do I have to buy the TF.2 fluorescence accessory to perform fluorescence measurements ?
- Which filters does Applied Photophysics Ltd. supply for fluorescence work?
Kinetics
- What volumes do I need to perform a kinetic experiment?
- Which pathlength do I use for kinetic experiments?
- How can I reduce the vibrational artifacts on kinetic traces?
- What is this 2ms plateau at the beginning of my trace?
Software
- How can I use the software in emulator mode?
- How do I change the AutoPM target voltage?
- Is the offset facility for PiStar used in the same way as on the SX.18?
General
- Why is the KSHU so difficult to move?
- Why is there a slow drift on my signal?
- Do I need to turn off the room lights when removing a detector?
- How do I drain the thermostat liquid out of the SHU?
Which lamp should I use?
There are 2 lamps available. The Xenon (Xe) and the Mercury-Xenon (HgXe) lamp. The Xe lamp has a more or less constant light output from the far UV well into the visible. Therefore it is the lamp of choice for any spectra being acquired. The HgXe lamp focuses on certain "wavelength peaks" which makes it the choice for a lot of kinetic experiments. It is best to compare the lamp profiles and find out which lamp has better performance at the wavelength desired. The lamp spectra can be found under /Public/Examples/SpecLamp/. It is important to overlay spectra which have been acquired with the same bandwidth.
What is the life of the lamp?
The Xe lamp has a guaranteed lifetime of 1000 hours and lasts on average for 2000 hours. The HgXe lamp's guaranteed lifetime is 500-1000 hours with an average lifetime of 1000-2000 hours.
How do I align the lamp?
The lamp is pre-aligned at the factory. Any attempt to align the lamp will result in a poorer performance.
Why is the CD spectrum noisy in the far UV?
For CD spectra only the E-SHU should be used because the housing is optimised for the requirements of CD spectrum acquisition. For spectra which are intended to go below 200 nm the system should be purged with nitrogen for at least 1 hour (10L/min=open the gauge fully) and with 5L/min thereafter. The purging should persist while acquiring data.
It is important to keep the absorbance of the sample low (ideally around 0.8 AU) in order to assure sufficient light reaching the detector. Thus it is important to reduce the buffer concentration sufficiently (e.g. 10 mM phosphate buffer). Chloride ions should be avoided.
Another means to reduce the absorbance is to reduce the pathlength of the cell. This will effect the amplitude however. Typical pathlengths for CD spectra are 1 mm or less. Generally the slit widths both for entrance and exit are 2nm, although these can be increased to increase the quantity of light passing through the sample.
Why is the amplitude of my CD spectrum wrong?
It may be that the CD calibration value needs to be checked and reset. This is usually as a result of changing slit widths or changing sample handling units.
Also it is very important to make sure that you have the AutoPM box ticked. This will ensure that you will have the PMT high voltage adjusted at each wavelength visited to create a uniform output signal.
How do I calibrate Pi-Star if I want to measure CD?
Typically the calibration chemistry should be chosen to suit the wavelength range of the intended spectrum. Pantoyllactone is the choice for the far UV and is calibrated at 219 nm whereas camphorsulfonic acid (CSA) is used for the near UV (calibration at 290.5 nm).
In the CD mode of the software the calibration is performed in the "Calibrate" subwindow. First set the slit widths to those required for later measurements. Then a baseline should be taken for the respective wavelength or, to save time, for the complete wavelength range of interest including the calibration wavelength. After opening the "Calibrate" subwindow, enter the desired value into the upper box. The lower box shows the calculation factor to be calculated. Clicking the "Measure" button will perform the calculation.
The value should be between 1200 to 1500. If the value is completely different then either the wrong wavelength has been defined, or the wrong desired value has been entered. Clicking on "Apply" will enable the calibration. The calibration should be performed when interchanging the K-SHU and E-SHU.
The calibration value will be slightly different for different slit widths and so should be performed with the slit widths set to the value that will be used during measurements.
Why is my CD baseline so far from zero?
The CD detector end window will have some birefringence. It will give slightly different baselines depending on its orientation (i.e. rotational angle). This angle will have been set to the best position during testing at the factory. To get the best CD baselines it is important to ensure that the CD detector has been mounted with the red dot on the top if using the KSHU or aligned with the line marker if using the ESHU.
Why doesn't my fluorescence signal move when I apply an offset?
The software automatically adds the offset back in to return the signal to the set position. Offsetting allows acquisition at higher gains, but the relative position of traces remains constant.
How can I improve the light throughput in fluorescence and
absorbance mode?
For measuring low levels of absorbance or fluorescence the polariser (necessary for CD) can be taken out of the light beam by loosening the retaining screw and slowly moving the polariser downwards. The self-absorbance of the polariser is subsequently removed and will allow more light to reach the detector.
Do I have to buy the TF.2 fluorescence accessory to perform
fluorescence measurements?
No. Fluorescence measurements can be performed using the CD detector, although this will be at a lower sensitivity.
Which filters does Applied Photophysics Ltd. supply for
fluorescence work?
All stopped-flow systems 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.
What volumes do I need to perform a kinetic experiment?
For a 1:1 mixture about 100 µl total volume need to be pushed. This equals 2-2.5 turns on the AutoStop volume adjuster. In ratios other than 1:1 the volume to be pushed needs to be increased. About 200 µl are required for a 1:10 mixture.
Which pathlength do I use for kinetic experiments?
There are two pathlengths (2mm and 10mm) which are chosen by rotating the cell block using the attached lever. For CD experiments in the far UV the 2mm cell is the choice in order to decrease sample absorption. In the visible enough light is produced by the lamps to work with the 10 mm pathlength (e.g. 403 nm for Soret band of metalloproteins).
For fluorescence measurements the 2mm pathlength should be used in order to avoid self-absorbing (inner filter) effects.
How can I reduce the vibrational artifacts on kinetic traces?
Check that the cell block is in the middle of its available travel on the soft coupling. It may be necessary to reduce the thermostat flow rate by partially clamping the inlet line.
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.
How can I use the software in emulator mode?
By default, the software will initialise the electronics when you log on. To stop this from happening hold down the "Escape" key on the keyboard whilst logging in so that the initialisation will be skipped. Afterwards by clicking the middle mouse button on the PiStar icon the "choices" feature will be highlighted and the respective tick box can be activated.
How do I change the AutoPM target voltage?
Click on the little square in the top right hand corner of the PCU control box and enter the new value.
Is the offset facility for PiStar used in the same way as on the
SX.18?
The offset facility on the PiStar instrument is not quite the same as on the SX.18 instrument. The effective vertical resolution on the PiStar is 16 bit whereas on the SX.18 it is 12 bit therefore PiStar has 16x more resolving power.
This means that when making fluorescence measurements (even when you have a small signal superimposed on a large non-specific fluorescence background) the available resolution should be sufficient to correctly digitise all of the fluorescence, even a small signal. In order to display just the small signal of interest, it is intended that the one should perform a "rubber band" zoom when the data trace is on display. This should provide data at a suitable resolution.
In order to manually use the offset facility you would need to switch off the automatic gain control and then select the gain setting that you require. The offset that you key in will now not be reset to zero when the acquisition takes place. However, PiStar is different to the SX.18 in that the offset that you have applied in order to access the signal using a particular gain setting, gets added back in when the data file is created so that you still see the whole of the signal.
Why is the KSHU so difficult to move?
Because the release screws on the base have not been unscrewed.
Why is there a slow drift on my signal?
This may be due to either photochemistry or diffusion. For a photolabile sample, ensure that the slits are as closed as possible and that dense chemistry is always in the left-hand drive syringe.
Do I need to turn off the room lights when removing a detector?
No. The detectors are safe in ambient light as long as the PM high voltage is turned off.
How do I drain the thermostat liquid out of the SHU?
The KSHU is fitted with a bleed screw that allows air into the thermostat area. This enable the thermostat liquid to drain back into the circulator prior to removing the front cover for syringe replacement or reconfiguring for sequential mixing.
If you have a question that is not answered here please e-mail the Technical Support Department.