The ProStar 325 UV/VIS detector with dual wavelength
and extended range capabilities.
Introduction
The Varian ProStar 325 UV –Vis HPLC detector is a state of the art detector the can be used for
both analytical and preparative applications. It was designed and built in a collaboration of the
Varian Inc. divisions in Melbourne, Australia and Walnut Creek, California, and combines the
optical expertise of the designers and builders of Cary spectrophotometers and the ProStar 310
UV-Vis detectors. The innovative design combines the best features of an analytical and prep
HPLC detector in a single unit.
The Varian ProStar 325 UV-Vis detector is designed for outstanding performance in both
analytical and prep applications. In addition, it has dual wavelength and wavelength ratioing
capabilities without compromise.
Figure 1: ProStar 325 detector
Optical design
Basic ProStar 325 optical design
Outstanding optical design is key to the performance and capabilities of a UV-Vis detector. The
Optical design for the ProStar 325 is shown in Figure 2.
Figure 2: Optics design for the ProStar 325 detector
The basic optical design involves passing the light from the UV and/or Visible lamps through the
flow cell before it goes into the dispersion element. This design is called a reverse optics design.
The UV lamp is a prealigned high intensity Hamamatsu Deuterium lamp which has a maximum
UV output in the 240 to 260 nm range and a 2000 hour lamp life. This provides the lowest noise
in that region of the spectrum. The lamp is cooled by a fan at the rear of the module which sucks
air from the rear of the units and blows the air over the lamp housing. This keeps the lamp
housing within 8 to 10 C of the ambient temperature, making detector warm-up quick.
The UV light passes through the focusing lens and then to a beam splitter which direct about ½ of
the light to the sample (lower) path and ½ of the light to the reference (upper path). The beam
splitter, which is placed at a 45-degree angle to the direction of the light, contains a hexagonal
pattern of transmission and reflection. This provides a much more efficient and more
manufacturable design than the older ProStar 320 beam splitter.
After passing through the beam splitter and some transfer mirrors, the white light in the sample
path hits mirror N1 and the reference path hits mirrors N3 and N2. The two beams pass through
an entrance mask which shapes the beam to the geometry required before entering the flow cell.
Upon leaving the flow cell, they again pass through a mask before entering the monochromator.
The masks help determine the resolution of the detector optics.
Light passes into the monochromator and is dispersed. The monochromator grating is rotated so
that the correct wavelength passes out of the exit masks and onto the two silicon photodiodes.
The two paths of light that go through the flow cell are vertically displaced on the monochromator.
The visible lamp is located above the beam splitter. When it is turned on, light from it is also split
and directed through the two flow cell paths. An example of the path of the visible light through
the optical system is shown in Figure 3:
Figure 3: Path of the visible light through the flow cell and into the monochromator.
Single wavelength and dual wavelength operation.
Under normal single wavelength operation, the monochromator stays on the wavelength selected
by the user and data is collected at the maximum rate and averaged to give 20 Hz data. (The
data can be further bunched to reduce date file sizes.) When the detector shifts to another
wavelength in single wavelength mode, the monochromator moves as fast as possible from its
present position to a position 5 nm lower than the target wavelength. It then moves at a fixed rate
to the target wavelength. For example, if the present wavelength is at 254 nm and the detector is
required to move to 280 nm, the monochromator will move as fast as possible to 275 nm and
them move at a controlled rate to 280 nm. If it is at 350 nm and it is programmed to go to 280
nm, it will go to 275 nm and then go back up to 280 nm. In this way, the detector achieves not
only high wavelength precision for all wavelength changes but also wavelength accuracy.
In dual wavelength operation, it is necessary to continually move between two wavelengths taking
a point every time the monochromator stops. In order to do this in the fastest possible way so
that the detector can spend as much time taking the data as possible, the detector uses a
different scheme for changing wavelength. The detector moves directly between the
wavelengths. If the two wavelengths are 254 and 280 nm, it will move directly between 254 and
280 as quickly as possible.
Moving the monochromator in this way produced some backlash. If the monochromator were to
try to stop at 254 nm coming from 280 nm, it would actually back up to between 254.5 and 255.
Therefore, the algorithm driving the wavelength change in dual wavelength will try to set the
wavelength of the lower wavelength to 0.7 nm below the target wavelength. In the above
example the detector will drive the monochromator to 253.3 nm. Since the backlash in each
motor is not the same, the wavelength accuracy achieved in dual wavelength mode will not be as
good as the accuracy achieved in single wavelength mode. The precision, however, will be just
as good in dual wavelength mode as in single wavelength mode.
Another consequence of running the detector in dual wavelength mode is that the achieved data
rate in dual wavelength mode will not be as high as in single wavelength mode. The highest
achieved data rate in dual wavelength mode for wavelength transitions between two wavelengths
less than 200 nm apart is 5 HZ per wavelength. For a transition between 254 nm and 280 nm,
the data rate will be 5 HZ.
For transitions between wavelengths further apart than 200 nm, the data rate is 2.5 Hz per
channel. These are fixed data rates (there is no data bunching). Also, the data rate is calculated
on the largest transition in a particular method. If for part of the time, the two wavelengths are
254 and 280 and then, through a time program, the two wavelengths are modified to 254 and 800
nm, the data rate will be 2.5 HZ for the entire run. It is necessary to keep a constant data rate for
an entire chromatographic run.
The flow cell
The flow cell design provides true dual beam operation for low noise and drift in the analytical
mode and extended range capability for preparative HPLC. All of the flow cells are made of three
blocks of Titanium with Teflon® gaskets between the blocks.
The layout of the flow 9x0 cell is shown in Figure 4. This is called a 9x0 flow cell because the cell
has a 9 mm sample path and a 0 mm reference path. No sample flows through he reference
path.
The light beam is directed through both the sample and reference side of the flow cell. Liquid
goes into the flow cell body at the inlet as shown in the diagram and through a proprietary thermal
equilibration system that minimizes band spreading (patent applied for) while adjusting the
temperature of the liquid in the flow cell. This minimizes refractive index effects. The liquid then
goes through the 9 mm cell path and out through the outlet. The liquid moves between the inlet
and outlet and the flow cell itself through galleries created in the Teflon gaskets between the
blocks.
Figure 4: General design of the 9x0 flow cell.
For the preparative flow cells, the path is illustrated in Figure 5
Figure 5: Flow path for the 9x1 preparative flow cell.
In this design, the mobile phase first flows through the sample side and then across and back
through the reference side. This is similar to the ProStar 320 flow cell.
The preparative flow cell design is different in several ways from the analytical flow cell design.
The preparative flow cells have much larger diameters than the analytical flow cells. This
provides much lower pressure drops in the flow cell and therefore much higher maximum flow
rates. They also do not have any internal thermal equilibration system. They are more
susceptible to RI effects from flow variations. Because these are preparative flow cells, they are
not normally used near the detector noise level.
The main advantage of the dual path flow cell design is ability to use the extended range feature.
In normal operation, the reference path is used as a true dual beam reference. This minimizes
the noise and drift created by lamp fluctuations. This also reduces the peak height of the
chromatographic peaks. Effectively, in the 9x1 flow cell, the flow path is 8 mm when the
reference is used. That is because the peak is also going through the reference side.
When the peak reaches a preset absorbance of 0.9 AU, the detector begins to blend the
absorbance from the both of the paths together. This provides a smooth transition as the peak
increases in absorbance. When the peak reaches an absorbance of 1.1 AU, the shorter path
generates the entire signal. When a preparative flow cell is installed in the detector, it will always
operate in extended range mode.
Detector electronics and optional interfaces
A single board computer with surface mount electronics controls the operation of the ProStar 325
detector. This main board controls all of the functions of the detector. It contains Flash RAM
which allows the firmware which controls the detector, to be updated in one simple process.
Features that will be added later to the detector itself will be easily upgraded to all existing
detectors in the field.
Other electronics in the detector include the power supply, a front-end board which has the two
photodiodes and the board which contains the IR port and the front status lights.
Figure 6 – Back panel of the ProStar 325 detector.
The interface connectors are located on the rear panel of the detector (see Figure 6). There are
5 interface connectors as shown in the Figure. Connector J1 is used to connect the ProStar 325
to a computer or to a computer network through an Ethernet cable. If you are connecting the unit
to a PC directly, use the cross over cable supplied with the detector. If you are using a computer
hub, use a standard patch cable.
The Sync signals on connectors P9 and J10 provide all of the signals associated with the ProStar
modules including READY, START and FAULT inputs and outputs. In addition, contact closure
inputs for LAMP OFF and AUTOZERO are also found on these connectors. This makes the
detector extremely easy to interface to other manufacturer’s HPLC modules.
To make connecting to the sync signals easier for the customer, an I/O adaptor board, (shown in
Figure 7) is available. This board provides connectors for connecting bare wires to the ProStar
325. In addition, each of the functions is clearly labeled so that it will be easy for a customer to
make the correct connections. If a customer is going to use the sync signals, they must purchase
the I/OP adaptor board.
Figure 7: I/O adaptor board for the sync signals.
Connector J4 provides 4 time programmed contact closures and one peak sense relay output.
The contact closures can be programmed to close and open at any time. This could be used to
provide synchronization between the HPLC system and any other modules. The peak sense
relay will give pulsed outputs based on time programmable single of multiple pulses. This could
be used to drive a simple fraction collector. A relay adaptor board, shown in Figure 8, makes
interconnecting the relays easy.
Figure 8: Relay adaptor board
The relay board uses either Molex connectors or silvered wires.
The final connection on the back panel is the Analog Output. This provides signal connections for
the two analog channels. An individual cable with labeled silvered wires for both channels is
available if the signal has to go to another device that needs the analog signal. This could be a
fraction collector or CIM.
Front panel status lights and IR control.
Status lights and the IR port for communications to the hand held computer are located on the
front of the ProStar 325. There are three status lights labeled as shown in Table 1.
LED label
Color
Status
Power
Initialising
Orange flashing
Power On
Green
Lamp
Initialising
Green flashing
Lamp On
Green
Fault
Red
Ready/Run
Not Ready
Off
Ready/Stopped
Green
Method Running
Orange
Table 1: Status lights on the front of the ProStar 325 detector.
The window to the right of the status lights is the IR port which is used by the hand held computer
to communicate with the detector. The hand held PC can control all aspects of the detector in a
manner similar to the Star or Galaxie drivers except that it cannot store raw data and it cannot
calculate the absorbance ratio from two wavelengths.
The range of the IR communications is 1 to 3 feet between the detector and the hand held
controller. An example of one of the screens is shown in Figure 9.
Figure 9: Status screen for the hand held computer control of the ProStar 325
Not only can status be read and methods be built and downloaded, but real time chromatograms
can also be displayed on the hand held computer. It should be noted that it is not advised to
control the detector with both the hand held computer and either the Star workstation or Galaxie.
If both instruments download methods at the same time, the user may not be fully aware of what
program the detector will use. The main use of the hand held computer is for monitoring detector
status and for controlling the detector when no other computer workstation is controlling it. For
more information on the capabilities of the hand held computer, see the ProStar 325 Operator’s
manual.
Computer Control of the ProStar 325 Detector
The ProStar 325 detector can be controlled from either the Star workstation or the Galaxie
workstation. It can perform all of the standard actions that are expected of a UV-Vis detector.
This is shown in the following three screens, two method editor screens and the status screen.
Figure 10: ProStar 325 Method editor screen for single wavelength
Figure 11: ProStar 325 Method editor screen for absorbance ratio
Figure 12: The Star workstation status screen for the ProStar 325 workstation showing
wavelength ratioing.
Either the Star or Galaxie software can control the ProStar 325 with the following method
parameters:
1.
Time programmed wavelength, attenuation and autozero control of 1 or 2 channels
2.
Time programmed control of up to 4 relays
3.
Time programmed control of the peak sense threshold which is used for absorbance
rationing.
4.
Time programmed control of pulsing the peak relay
5.
Control of data bunching, the noise monitor length and response time and data
collection end time.
6.
Control of 2 analog outputs
7.
Control of absorbance ratioing including setting the threshold above which ratios are
calculated.
8.
Control of the peak-sensing relay.
Through the status screen, the user can control the following:
1.
The user can turn the lamps on or off
2.
The user can set the flow cell and the cell path length ratio
3.
The user can control the operation of the sync signals
4.
The user can Autozero the detector at any time, modify the run end time and reset
the detector.
5.
The user can observe the chromatogram in real time.
6.
The user can also get the status of the detector parameters in a continuous mode
both through text and lights.
7.
The user can read the GLP information such as when the detector and the lamp were
installed, how many hours has the lamp been used, and the MAC and IP addresses.
Absorbance ratioing
Ratioing of the absorbances at two different wavelengths is a function of the Star and Galaxie
drivers. In figure 12, an example of the real time plot of the ratio is displayed. For the first peak,
there is an obvious shoulder on the front of the peak. The ratio clearly points out that this is not a
single peak. For the second peak, a flat ratio indicates that the peak is probably pure.
Absorbance rationing is done very simply. A threshold in AU is set by the user in the timeprogrammed section of the method. This threshold is used to detect peaks on channel 1. When
a peak has exceeded the threshold on channel, the driver will go back 40 points and find the
lowest point. This will be designated as the baseline and the absorbances on channel 1 and 2 at
this time will be subtracted from all of the absorbances above the threshold and the resultant
absorbances will be ratioed. Mathematically, the ratio is calculated in the equation below.
EQ 1: Ratio = Absorbance (1) – Baseline absorbance (1)
Absorbance (2) – Baseline absorbance (2)
Figure 13 shows graphically how the driver takes a 40 point buffer and finds the lowest point in
those 40 points to determine the baseline to subtract.
Figure 13: Graphical representation of the determination of the point for baseline
subtraction.
The ratioing of the absorbances at 2 different wavelengths is not an ideal way to tell whether a
peak is pure. It is necessary to choose the 2 wavelengths for ratioing so they there are significant
absorbance at each wavelength and so that you are not on the side of an absorbance peak.
Also, it is necessary to set the threshold in the method to a low enough level that it will be able to
find the true baseline while not too low so that it will be ratioing the baseline noise. Finally, if the
detector lamp is old and wanders significantly on the time scale of the peak, the absorbance ratio
could easily identify a peak as impure when it is only a matter of baseline wander.
Detector Performance
The ProStar 325 UV-Vis detector is a high performance analytical and optimized prep detector all
in one package. It is only necessary to change flow cells to convert between the analytical and
the prep detector.
Detector Noise:
The key performance measure of good analytical performance in an optical detector is low noise.
The ProStar has extremely low noise in all conditions. Noise is affected by the following factors:
1.
The response time (digital filtering)
2.
Single or dual wavelength operation
3.
Data bunching
4.
Wavelength and lamp
5.
The mobile phase
Response Time
The response time parameter controls the digital filtering that reduces the noise on the detector.
This has the most significant effect on the noise. While in the past, detector noise was reduced
using analog time constants; today digital filters are used to reduce the noise. The filter used is a
boxcar filter with some number of stages and some number of points per stage. The filters that
are used when different Response Times are entered into the method are shown in Table 2
Response time setting
Single wavelength
filter
2 sec
1 sec
0.5 sec
0.05
4 x 33
4 x 19
4 x 10
2x3
Dual wavelength filter
for less than 200 nm
transition
4x9
3x7
3x4
1x2
Dual wavelength
filters for greater than
200 nm transition.
4x5
3x4
3x2
1x1
Table 2: Filters used to reduce noise. The first number is the number of stages and the
second number is the number of points per stage.
The digital filters have three effects on the data. First, they reduce the noise significantly. Figure
14 shows the effect of filters on the noise measurement using flowing Acetonitrile at 1 mL/min at
254 nm using a 9x0 flow cell as well as a dry 0x0 test cell.
Peak to peak noise
Effect of filtering
50
45
40
35
30
25
20
15
10
5
0
0x0 single
0x0 dual
0x0 dual ex
9x0 single
9x0 dual
9x0 dual ext
0.05
0.5
1
2
Response Time Setting
Figure 14: Effect of filtering of noise.
The filtering makes a significant difference in the noise level. At a 0.05 filter (virtually no filtering)
the noise is between 25 and 45 micro AU peak to peak. With a 2 second response time, the
noise for all of the conditions, is in below 10 micro AU (+/- 5 micro AU peak to peak) for the noise.
The second effect is to delay the peak. A larger response time results in a longer delay in the
peak appearance, both on the real time plot and in the data file. This is shown in Figure 15.
Figure 15: Change in retention time of a peak as a function of filtering
This time change is plotted in Figure 16.
Retention time change with filter
Retention time in
seconds
205
203
201
RT 1
199
RT 2
197
195
0.05
0.5
1
2
Filter
Figure 16: Change in retention time with changing filter setting.
This change amounts to only about 2 sec when going between each of the different filter setting.
The change is very precise. Data should be taken at the same filter setting for both the standards
and the samples so that there is no disparity in retention times.
The last effect of the filter setting is a change in peak width. For larger filters, the peak width
increases. In the example above, the peak width as calculated by the Star workstation varies
between 5.2 seconds for the 0.05 response time filter and 5.7 seconds for the 2 sec response
time filter.
Effect of single and dual wavelength operation on detector noise
The second largest effect on noise is whether you operate in single and dual wavelength.
Because you have to cycle between two wavelengths in dual wavelength mode, there are less
points generated. When the distance between the two wavelengths is less than 200 nm, the
detector will take data at 5 Hz per channel. Therefore, to keep delay due to the response time to
approximately the listed response time, there is less filtering. Table 2 shows that the filter used
when a 2 sec response time is set uses about ¼ of the points as the filter for single wavelength.
Therefore, the noise will be higher for dual wavelength than single wavelength. This will be
greater when the different between wavelengths is greater than 200 nm. Figure 14 shows the
effect of filtering on noise for dual wavelength and extended dual wavelength operation.
Effect of data bunching on detector noise
Data can be bunched together to reduce the data rate. Also, bunching has been used in the past
to reduce the noise. In the ProStar 325, bunching will always reduce the data rate, giving you
smaller data files, but it will reduce the noise only when using the smaller response time. A plot
of the bunching vs. noise at different response times is shown in Figure 17
Noise
Effect of bunching on noise
30
25
20
15
10
5
0
2 sec
1 sec
0.5 sec
0.05 sec
Bunch 1
Bunch 4
Bunch 16
Bunch 64
Bunch size
Figure 17: The effect of data bunching on noise.
As you can see from the plot, there is a significant reduction in noise by bunching only when
using the 0.05 sec response time. There is a small but measurable decrease in the noise from
bunching when using the 0.5 and 1 sec response time and essentially no change in noise when
using the 2 sec response time.
The dependence of noise on wavelength and lamp
The noise specifications for the ProStar 325 detector are set at 254 nm. This wavelength was
chosen because it is the common wavelength used for noise specifications, it is the most used
wavelength in HPLC and the noise is optimum in that area of the spectrum. A combination of the
optimization of the grating and the deuterium lamp output make this an excellent wavelength for
setting the specification. However, the detector can be used down to 190 nm and up to 900 nm.
The noise that you can expect varies with wavelength. A plot of this noise with and without the
visible lamp is shown in Figure 18.
Noise dependence on wavelength
Noise in micro AU
1000
100
UV only
UV and Vis
10
1
150
350
550
750
950
Wavelength
Figure 18: Noise dependence on wavelength measured using a 0x0 flow cell.
This plot indicates that for operation below 350 nm, the UV lamp is sufficient. For operation
above 350, the Visible lamp would be desirable if high sensitivity is needed. The noise on this
plot was measured using a 0x0 flow cell (no mobile phase) so that the potential absorbance from
the mobile phase would not be a factor in measuring the noise. Obviously, if the mobile phase
absorbs light, the noise will be higher.
Other factors that effect noise
There are many other factors that affect the noise of the ProStar 325.
1.
An old lamp which has a lower light output will usually result in higher noise. A lamp
that has over 1500 hours of operation will fall into this category although lamps with
less time might also have lower output.
2.
Using a mobile phase which absorbs light at the wavelength being used will result in
higher noise.
Detector warm up and drift
Detector drift is caused by the system thermally equilibrating. When you first turn the system and
the lamps on, everything has to come to thermal equilibrium. Because the ProStar 325 operates
close to ambient temperature, it is fairly quick to warm up. Our experience has been that the unit
will settle down to its drift specification in about 20 minutes. Because it is a dual beam system, it
will achieve its noise specifications fairly quickly.
If you turn off the lamp, it will also cool down quickly because the fan will continue to run.
Therefore, if you turn off the lamp, even for a short period of time, it will drift when the lamp is
turned on again.
Wavelength accuracy in both single and dual wavelength operation
Wavelength accuracy has been tested across the entire spectrum and found to meet
specifications. Because of the different way the monochromator is controlled in dual and single
wavelength operation, the wavelength achieved in single wavelength and dual wavelength
operation will be different. In both cases it will be extremely precise (<0.1 nm precision) but it will
be different.
Linearity
Linearity has been tested in both extended range and normal range. Linearity is due to the
presence of stray light in the detector. The linearity of the optical products from Melbourne has
always been very good and this product is no exception. The gradient in Figure 19 was
generated using a ProStar 230 pump (note the presence of the 50/50 cross over point in the plot).
Red food coloring was used 0.2 % and 2 % in water. Several wavelengths were run with 210 nm
being the best wavelength as that was the wavelength at which the food coloring absorbed the
best.
Figure 19: Linearity using red food coloring, 9x0 flow cell, UV lamp on only.
The ProStar 325 also has the ability to operate in extended range. This was described
previously. Extended range was investigated with both the 9x1 flow cell and the 4 x 0.15 flow
cell. The experiments with the 9x1 flow cell used conditions similar to the 9x0 tests but the
concentration of the dye was 2%. Again, the 230 pump was used to proportion water vs. water
with 2 % dye and the linearity was measured at several different wavelengths. Three
wavelengths are shown in Figure 20.
Figure 20: Linearity in extended range at three different wavelengths.
Using 210 nm, the linearity was about 25 AU for the 9x1 cell. This is predictable considering the
linearity of the 9x0. The linearity plots at the other two wavelengths were limited by the
absorbance of the dye at those wavelengths. In both cases, the detector was linear over the
entire range. Also, there was no evidence of any disturbance when the detector changed modes
between dual beam and single beam operation.
Flow cell performance
The flow cells are a critical part of the system. They can make or break the performance of a
detector. There are several characteristics of a flow cell which are important to their overall
performance.
Flow cell volume and band spreading
A flow cell should not spread the chromatographic bands as they pass through the through the
system. To do this, most manufacturers minimize the volume of the flow cell. However, a low
volume flow cell is not sufficient to minimize band spreading. The flow cell also has to be
designed so that the mobile phase can sweep the sample into the cell and out of the cell without
having anywhere in the sell for the sample to collect. This is usually referred to as unswept
volume. Good flow cell design tries to eliminate unswept volume.
Band spreading in the flow cells were measure by injecting 1 L of acetone at a concentration
that would give about 200 mAU of absorbance into 45 cm of 0.0025 in id. PEEK tubing. The
pump was run at 200 L per minute and the peak width at half height was measured. The
variance was then calculated. The variance along with the volume for each flow cell is shown in
Table 3. The variance for the ProStar 310 flow cell was about 16 L2.
9x0
4x0
9x1
Estimated pressure drop at 100 mL/min 50psi
750 psi
100 psi
water
Maximum recommended flow rate
100 mL/min
20 mL/min
500 mL/min
Cell volume of long path
15L
1.6L
30L
Band spreading
15 L2
2L2
20L2
Table 3: Flow cell variance parameters for the 4 ProStar 325 flow cell.
4x0.15
5 psi
10 L/min
15L
NA
Flow cell backpressure
Another parameter of the flow cells that is important is the backpressure generated by the flow
cell for a given flow. For preparative HPLC, high flows are needed and the cells need to have a
minimum of pressure drop in order to accommodate these high flow rates. Each of the flow cells
is speced for a maximum of 1000 psi. Therefore, by measuring the backpressure generated by
water at various flow rates, the maximum recommended flow for each flow cell can be
determined. These are also listed in Table 3. It should be noted that the 4 x 0.15 flow cell
pressure drop was measured at 100 mL/min water. It was not measured at 10L per minute but
the low pressure drop at 100 mL/min flow rate indicates that it will be able to operate at the high
flow rate.
Best Practices for the ProStar 325 detector
Like any product, there are ways to operate the ProStar 325 which will give the best results and
there are ways to operate the product that could compromise the results. This is a list of best
practices for the ProStar 325 detector.
Retention time reproducibility
When controlled by the Star workstation, the ProStar 325 will normally be started by a signal on
the Ethernet communications link. For most situations, this will produce a sufficiently precise start
signal so that retention times are reproducible. We have operated for months through a hub to
which the workstation and the detector are attached. However, it is conceivable that the network
through which the detector is connected to the workstation could experience periodic surges in
network traffic that could prevent the start signal from reaching the detector immediately. This
could definitely be true when the workstation and the detector are on different subnets. If it turns
out that retention times are not very reproducible, especially if a small percentage of them are
significantly different than the rest, it could be network traffic problems. In that case, you can use
the sync system to provide a start signal directly from the pump or AutoSampler. This will require
the sync I/O board for the ProStar 325 and whatever is needed for the other module.
Use of the hand held computer with either the Star or Galaxie drive.
The hand held computer can completely control the ProStar 325 in a manner similar to the Star or
Galaxie driver. If you are controlling the detector from a computer, the hand held is an excellent
way to be able to read status and see a real time chromatogram. However, if you try to download
methods from the hand held to the ProStar 325 while it is being controlled from one of the two
workstations, the detector can become confused and run an incorrect method that could be a
hybrid between the two methods. Also, the workstation may not even know that the hand held
has downloaded something. This can lead to incorrect results. As a general rule, only use one
device to control the detector. You can look at detector status from the hand held at any time.
Dual and Single Channel operation
As described above, a different algorithm moves the monochromator between wavelengths for
single and dual wavelength operation. Although these two algorithms provide wavelength
accuracy within specification, they produce wavelengths that are up to 0.5 nm different. Each
algorithm, however, is very precise. Therefore, it is not advisable to try to compare the detector
response for a peak in single wavelength mode to the same peak at the same wavelength in dual
wavelength mode. There could be significant differences which could lead to incorrect result
calculation. It is best practice to run both standards and samples in either single wavelength or
dual wavelength modes.
Dual channel operation
Although there is virtually complete flexibility in the operation of the ProStar 325 in dual channel
mode, there are two things that you cannot do in dual channel mode. First, you cannot program
the same wavelength for both the first and second channel. The driver software will not let you
do that. Second, you should not “flip flop “ the two channels on consecutive lines of time program
in the driver. For example, you should not program channel 1 = 254 nm and channel 2 = 280 nm
immediately followed by channel 1 = 280 nm and channel 2 = 254 nm. This could cause the
driver to incorrectly assign points to a particular channel.
High temperature pulsed flow operation
The ProStar 325 analytical flow cells have a patented internal thermostatting system that
equilibrates the temperature of the mobile phase to the temperature of the detector block. RI
effects in the flow cell will be minimized when the temperature of the mobile phase is constant
while passing through the flow cell. If the mobile phase is at a significantly different temperature
than the flow cell (which operates about 2 to 3 C above ambient temperature) AND the pump is
pulsing significantly, you may observe a cyclical baseline that may be as high as 30 to 40 AU.
This is due to the fact that, as the pump pulses, it changes flow rate and, at lower flow rates, the
mobile phase will equilibrate to a different temperature than it will at high flow rates. In practice
we have only observed this when the mobile phase in the column is greater than 20C above
ambient and the pump is operating at lower pressures.
If you observe this cycling baseline, there are two things you can do. First, you can increase the
backpressure on the pump to minimize the pulsations. Secondly, you can get the temperature of
the mobile phase closer to the temperature of the flow cell. If you cannot, lower the temperature
on the column, then connect the column to the detector with stainless steel or Titanium tubing.
Metal tubing will help the mobile phase equilibrate with the ambient temperature.
Use of the preparative 9x1 cell for analytical work.
The 9x1 cell can be used for both analytical and extended range preparative applications with
some limitations. There are three main differences between the analytical 9x0 and the prep 9x1
flow cell.
1.
The 9x1 has a different effective path length than the 9x0. In the 9x1 cell, the mobile
phase passing through the 1 mm path essentially “subtracts” 1 mm of the signal
because it is used as a reference signal. Therefore the effective path length of a 9x1
flow cell is 8 mm. Peaks will have a lower height, a smaller area and a smaller signal
to noise ratio. When used for preparative applications, this is not a problem.
2.
The 9x1 has a wider diameter and a slightly larger band spreading than the 9x0. The
wider diameter gives a lower backpressure allowing it to operate at higher flow rates.
In the analytical mode, peaks will be slightly broader because of this increased
volume.
3.
The 9x1 does not have the extra internal thermostatting that the 9x0 has. In
preparative applications, most peaks are large and no one will notice a slight
pulsation on the baseline. The lack of the internal thermostatting features again
creates less backpressure and therefore allows increased flow rates in prep
applications.
4.
The 9x1 always operates in the extended range mode. As the absorbance crosses
over between 0.9 and 1.1 AU, the system will start to use the reference channel for
operation. The linearity will be better in the 9x0 than the 9x1 in the range of 0.9 to
1.5 AU because the 9x0 will still be using a reference.
The bottom line is that, if a user is going to operate in both trace analytical mode and preparative
mode, they should have 2 flow cells. It is VERY easy to change the flow cell to get the optimum
results from both the analytical and the prep applications.
Removing the flow cell and operation of the ProStar 325 without a flow cell.
The flow cell is very easy to remove (see the video on replacing the flow cell). However, when
you do remove the flow cell, you will disturb the flow of light through system. This may cause an
error message to be displayed in the message log of the Star workstation. This will not hurt the
detector in anyway so you can replace the flow cell with the lamp on. Just expect the error
message. However, you will not get results with the flow cell out. You cannot measure the noise
of the detector without a flow cell in place!
Turning off the lamp without turning off the detector.
If you are not going to use the detector for an extended period of time, like the remainder of the
day, you might want to turn the lamp off. This will extend lamp life, especially for the UV lamp. If
you do this, you should also turn the detector off. Warm up for the ProStar 325 is dependent only
on the lamp being on, not the detector itself being on. If the lamp is off, the detector warm up
time is the same whether the detector was on or off. If you turn the UV lamp off and then back on
without turning the detector off, you will get an error message when the detector goes through its
gain calibration procedure. This error will not stop the instrument from running and will only affect
results above 600 nm. Turning the instrument off and then back on can fix the problem again.
Using the UV and Visible lamp together.
If the detector has a visible lamp installed, it is best that it is only turned on when the user is going
to operate above 350 nm. Below 350 nm, the lamp does not improve the noise of the detector. It
does add heat to the system and shortens that life of the visible lamp. Therefore, operate the
visible lamp only when you need to. Also, when the customer is only going to operate in the
visible, they should only turn on the visible lamp. If they are going to operate both above and
below 350 nm, the system can be operated with both lamps on however, for best results, operate
the system with only one lamp at a time.