Introducing the new ProStar 335 Photodiode Array
Detector with extended range capabilities
Introduction
The Varian ProStar 335 DAD HPLC detector is a newly designed state of the art detector that can
be used for both analytical and preparative applications. It builds on the highly successful ProStar
325 UV-Vis detector and Varian Analytical Melbourne’s optical expertise along with the proven
capability of PolyView? photodiode array detector software to produce the best detector system
on the market for peak purity, peak identity and peak quantitation.
With the introduction of the ProStar 335 photodiode array detector, Varian has significantly
upgraded its offering in the area of information rich detectors.
Figure 1 ProStar 335 detector
The ProStar 335 has many common parts with the ProStar 325 UV-Vis detector. The flow cells,
lamps and cables are all the same for both products. In addition, many of the service parts
including the main CPU board and the power supply are the same in both products. This
commonality allows customers to interchange parts as needed and to stock fewer parts for the
product. This commonality also allowed the development and introduction of a new photodiode
array detector only nine months after the previous introduction of the ProStar 325. Note, there is
one difference on the front panel of the ProStar 335 from the ProStar 325. The square
opening for control by the hand held computer is not present in the ProStar 335. There is
no hand held computer control in the ProStar 335.
For further technical information on the parts that are common between the ProStar 325 and the
ProStar 335 (flow cells, etc) please read the ProStar 325 technical paper.
Product Overview
The ProStar 335 Photodiode array detector uses either a single 1024 diode array for analytical
work or a dual diode array system for preparative work in the extended range. It covers the
wavelength range of 190 to 950 nm with standard UV and visible lamps. The ProStar 335 is
controlled by either Star or Galaxie software and with Star takes advantage of PolyView spectral
processing software, the best data mining software available.
Optical design
Overview
The mechanical layout for the optics is shown in Figure 2.
“Figure 2 Mechanical optical diagram
This is essentially the same layout as the ProStar 325 optics.
Lamps
The UV lamp is located at the extreme right hand side of the instrument. It is a deuterium lamp
that puts out light from below 190 nm to about 350 nm in significant amounts and then into the
visible. It also has intensity peaks at 656 nm and to a lesser extent at 486 nm. It is accessible
though the front panel with an interlock to prevent it from being removed from its housing while
plugged in. This prevents the operator viewing the UV lamp directly while it is turned on.
The UV lamp is completely enclosed in a large housing to prevent any drafts from affecting the
stability of the lamp. This housing is cooled to about 10 °C above ambient temperature by a fan,
which pushes air from the rear of the unit through the front door. Keeping the lamp housing cool,
allows the rest of the detector to run cooler. The flow cell usually is 2 or 3 C above ambient
temperature. This means that the liquid coming into the flow cell can quickly come to thermal
equilibrium, therefore minimizing any potential refractive index effects.
Light throughput is proportional to source brightness, so the deuterium lamp with the brightest arc
is used. These have a small arc diameter, so some magnification is required to fill the system
aperture. A two-element focusing lens (see Figure 3) is used to image the 0.5 mm diameter
source arc to overfill the 1 mm diameter system apertures. Overfilling minimizes noise and drift in
the presence of mechanical movement induced by ambient temperature changes or vibration.
Figure 3 Optical layout, another view
The visible lamp is located above the flow cell and accessed from the front panel. The Vis source
is a 10 W tungsten halogen lamp with a filament width just over 0.5 mm to match the D2 arc size.
It is imaged onto the aperture pair with a similar two element lens. For the diode array detector,
an attenuator reduces the Vis intensity peak near 700 nm to match the D2 peak intensity near 250
nm so that the sampling time (gain setting) of the array can be maximized in the UV without
saturating the visible range. The attenuator is located between the visible lamp and the focusing
lenses.
Both beams have to be merged and focused through both flow paths. This is done with the same
design as the 325. The splitter plate is a silica window coated with circular dots of reflecting
aluminum covering approximately half the area. Light from the UV lamp that goes through the
reference path passes through the transmitting surfaces and light that goes to the sample path is
reflected. The opposite happens for the visible lamp.
Flow cell optics
To get the lowest noise and best linearity, the highest light throughput possible is required. To
minimize refractive index effects which cause baseline upsets, the light must not hit the walls of
the flow cell.
To ensure the beam is clear of the cell walls, limiting apertures are placed before and after the
cell and cell optics. One of these is the polychromator entry mask located on the optics housing
just after (to the left of) the flow cell and the other is a mask that precedes the cell and is also
mounted on the beam splitter block to the right of the flow cell. There are two apertures on each
side of the flow cell, one for the sample side and one for the reference side. These minimize light
hitting the cell walls.
To obtain maximum light throughput, the cell imaging is crucial, and occurs when source and
aperture images of maximum size are formed at each end of the cell creating a pencil beam that
just clears the cell walls. This is very difficult to do mechanically unless the cell windows form
part of the imaging system so lenses are used as cell windows.
Figure 4 Flow cell optics
Doublet lens are used on each side of the flow cell (one of each pair is in the flow cell window) to
better focus the light through the cell. This is a very similar design to the ProStar 310/Star
9050/and UV-100/200 system. Images of the limiting apertures are formed at each end of the
flow path, clearing the walls by approximately 0.15 mm to minimize the effects of changing liquid
refractive index. While doublet lenses are more expensive than single lenses, they reduce optical
aberrations and reduce the cell volume needed to maintain the critical clearance between beam
and cell walls. In the Reference path of each cell, the shorter or zero liquid path length is spaced
out with a silica rod to equalize the optical path lengths.
Figure 5 Polychromator optics
Slits
After the light passes through the flow cells, it passes through a slit wheel and then the
polychromator mask. The ProStar 335 allows a slit selection of 1, 2, 4, 8, or 16 nm. A smaller slit
provides more optical resolution for the spectrum but increases the noise. A larger slit minimizes
the noise while lowering the optical resolution. In most cases, a lower optical resolution does not
lower the quality of the spectra because most organic compounds have a broad natural
bandwidth in the UV part of the absorption spectrum.
Slits are 2 mm long at a radius of 45.57 mm. Motor steps are 7½ 0 (6 mm at this radius). The slit
to slit separation in each pair is 9 mm, so this scheme interleaves sets of slits. Microstepping is
used to more accurately locate the slit pairs, but the increment from pair to pair is a whole step to
maintain uniformity. The interleaving is used to mix pairs which allows a 0% T correction to be
made using 16 nm Reference and blocked Sample slit. There is a cross slit used when the unit is
first powered on to calibrate the slit sector in much the same way as zero order is used for the
325 grating calibration.
Polychromator
After passing through the slit wheel, the light is reflected off a spherical collimating mirror and
onto the grating. Focal length of the polychromator is 110 mm and the ruled grating is 25 mm
square, with 300 lines per mm. This produces a spectral range on the array of just over 190 to
950 nm at 0.75 nm per diode. Allowance is made for some lateral misalignment and for focal
length variations. This allows accurate wavelength calibration in the factory and wavelength
adjustment in the field.
After the light is dispersed in the polychromator, it is reflected off a toric mirror. This mirror is
specially designed to minimize optical aberrations. Then two fold mirrors reflect the light upwards
to the photodiode arrays. These mirrors also separate the two channels in space so that the
arrays can be conveniently mounted on a single circuit board in an unrestricted plane above the
grating. This plane is tilted relative to the slit/grating plane to optimize the focus of both ends of
the spectra on the arrays.
Second order light is always a problem with diode array detectors. Second order light can be
created by second order reflection in the polychromator. For example, light from 250 nm can be
diffracted in the polychromator to hit the diode that is measuring 500 nm. If 250 nm light hits the
500 nm diode, then anything that absorbs light at 250 will also cause absorption at 500 nm giving
an incorrect spectrum.
There are several ways to deal with this problem. A UV cutoff filter can be rotated in and out of
the light path to block light from 250 nm from striking the 500 nm diode while it is being read but
that cuts down the overall time of sampling each diode and will increase the noise. A physical UV
cutoff filter can be placed directly before the diode array covering the high wavelength region of
the array. The disadvantage of this second method is that the optical filter refracts light passing
through it and consequently there is a disruption to the wavelength scale at that region of the
spectrum corresponding to the transition between the filter and the unobstructed light path.
Varian, Inc. has developed a new approach to solving this problem. This is called Seamless
Spectral Coverage Technology (Patent applied for). The optical transmission characteristics of
the window are not uniform but are specifically varied so that only light having wavelengths lying
in an appropriate wavelength range can pass through and be detected. An interference filter
having the required light transmission characteristics is fabricated directly onto the window. This
is done by depositing thin films of the required thickness directly onto to window to create the
required interference filter. The window is masked prior to the deposition of the film to ensure
that the interference filter is fabricated only above those pixels that should receive only those
wavelengths that are transmitted by the filter.
Dual and Single arrays
The ProStar 335 is available in two versions, a single array version and a dual array version. The
single array version is designed for microbore to analytical work while the dual array is designed
for analytical to preparative work. (Using only a single array, lowers the cost of the unit and
therefore the price to the customer.)
In the single array unit, the reference is a pair of 2.4 mm square diodes centered on 250 and 600
nm. The size of the diodes gives them a bandwidth of about 72 nm. If only the D2 lamp is on, the
250 nm diode is used as a reference for the whole wavelength range. If only the Vis lamp is on,
the 600 nm diode is used over the whole range. If both are on, the 250 nm diode references
wavelengths below 400 nm and the 600 nm diode references wavelengths above 400 nm, with
"blend region" between the two to reduce spectral artifacts induced by slow wavelength drifts
Optical alignment and calibration
The optical system is aligned at the factory. This is done in three stages. First, the arrays are
positioned in the detector and the deuterium lamp is used to move the arrays to the correct
position.
Secondly, a Hg lamp is inserted into the deuterium lamp holder and the detector and the optical
components are adjusted to give the best positioning of the spectrum on the array. Various
aspects of the optics are adjusted using the Hg lines.
Thirdly, a wavelength calibration is then done using the same Hg arc lamp and the diagnostic
software. During this calibration, three Hg lines (253.656, 546.074 and 912.297 nm?) are used
as standards. The difference between the diode where each line should be theoretically found
and the actual diode at which it is found is determined. This error is then fitted by a quadratic
curve and a calibration table is made up relating the diodes to their wavelength. This procedure
can be performed by a Varian service person if they have a Hg lamp.
Fine tuning the optical calibration
When the lamp is first turned on, the ProStar 335 will go through a procedure that will result in a
“fine tuning” of the optical calibration. Once the deuterium lamp is on, the detector will find the
656 nm line of deuterium and adjust the calibration table for any changes in the position of the
line. This is done automatically and is not dependent on the solvent in the flow cell or how the
cell is aligned although a cell must be in the detector and sufficient light must pass through the
system at 656 nm.
Signal Processing
After the light is captured on the photodiode array, it goes through a series of signal processing
steps.
1.
2.
3.
4.
5.
6.
7.
8.
9.
The diode array is sampled periodically and digital intensity data is produced.
A correction to this data is made for the dark current.
Another correction is made for 0%T.
A filter is used to adjust the digital data to reflect the value of the slit bandwidth.
The signals from each diode are then converted to wavelengths.
The digital data is converted to absorbances.
The response filter is applied to the data.
A zero absorbance correction is performed
If the detector is a dual array detector and the flow cell is a prep flow cell and the
absorbance is greater than 0.9 AU, the detector goes into extended range mode.
10. The data is bunched together based on the data bunching parameter and the data is sent
to the workstation.
All of these steps are done automatically without any intervention by the user. However some
user inputs can affect data processing at several of the steps.
Step 1 – Diode Sampling
Each diode array can be sampled 1, 2, 4, 8 or 16 times every 100 msec. The rate is set by how
much light intensity is falling on the diode array. The more light that hits the array, the more
frequently the array will have to be sampled or else the array will be saturated and problems such
as negative peaks or “flat” baselines can occur. If only a small amount of light is hitting the array,
the gain on the amplifier will have to be increased to get a larger signal and minimize the noise.
To make this happen, the detector gain needs to be calibrated. In the ProStar 335, the detector
gain calibration is a user function. When a new lamp is installed in the detector, the user will be
instructed to go through a gain calibration procedure. Once the gain is calibrated, it will be
remembered in the non-volatile detector memory. Only when the lamp has aged significantly so
that there is a significant loss in lamp intensity and therefore a significant increase in noise,
should the gain calibration be performed again.
Since the gain calibration is fixed, the sampling rate is adjusted to make sure that the maximum
signal is always being acquired. This is one of the reasons why the ProStar 335 has a very low
noise level for a photodiode array detector.
Step 4 – Slit bandwidth
The user can set the optical bandwidth from either Star or Galaxie. A small bandwidth (1 or 2
nm) will give a higher optical resolution but a higher noise. A larger bandwidth will give poorer
optical resolution but lower noise. Since most compounds have larger natural bandwidths in the
UV, larger bandwidths are appropriate. However, the photodiode array reads every diode
individually. To reflect the optical bandwidth setting, a bandwidth filter is applied to the data
baaed on the actual slit width. This process contributes to the low noise level while preserving
the optical resolution of the data.
Step 5 – The signal from each diode is converted to a wavelength.
In general, each exact wavelength will fall between pairs of diodes. To produce as accurate a
wavelength as possible, the signals from adjacent diodes will be combined proportionally to
produce the intensity at that exact wavelength. For example, if one diode is at 250.6 and the next
diode is at 251.6, then 60% of the intensity of the first diode and 40% of the intensity of the
second diode will be combined to produce the intensity at 251 nm.
Step 7 – After the absorbance has been calculated, the signal will be further filtered based
on the response time set by the user. This filter is a box car type filter specially designed to
remove random noise while preserving the shape of the chromatographic peak. If a response
time of 0.05 sec is set, there will be no filtering. If a response time of 2 sec is set, the most
filtering will be applied. For most chromatographic peak, the preset of 2 sec is a good choice of
filter.
Step 10 – Data bunching
After all of the signal processing has been applied, the data can be reduced in the time domain by
bunching. The base data rate of the ProStar 335 is 10 Hz or 10 spectra per second. In many
cases, this is much more data than needed. Good area quantitation can be obtained with as little
as 15 points per peak. By using the bunching parameters, the size of the data file and the
amount of network traffic can both be reduced significantly.
The flow cell
The same flow cells are used in both the ProStar 325 and he ProStar 335. 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 6. 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 the 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 6 General design of the 9x0 flow cell
For the preparative flow cells, the path is illustrated in Figure 7.
Figure 7 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.
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.
Flow rate considerations for flow cells
All of the flow cells have a pressure limit of 1000 psi and therefore maximum flow rate limitations
that vary depending on the diameter of the flow cells and their connections will apply. These are
listed in the ProStar 335 data sheet. Practically, the two preparative flow cells also have
minimum flow rate limitations. For a dual path flow cell, the mobile phase first goes through the
sample side and then through the reference side. There is a small but finite connecting volume
between the sample and the reference side. Because spectra are collected on both the sample
and reference side and compared, especially through the blend region, the flow rate should be
high enough so that the same part of the peak is essentially in both the sample and reference
paths. This can be thought of as a concentration offset. This offset is not particularly a problem
for pure peaks or for peaks whose absorbances are significantly below the blend region of 0.9 to
1.1 AU. However, for partially resolved peaks or when going though the absorbance blend
region, a low flow rate can create a shoulder on a peak. As a recommendation, the 9x1 flow cell
should be used at flow rates greater than 10 to 20 mL/min especially when looking at high
absorbances.
Detector electronics and optional interfaces
A single board computer with surface mount electronics controls the operation of the ProStar 335
detector. This main board controls all of the functions of the detector. It contains Flash RAM
allowing 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 on all existing detectors in the
field.
Other electronics in the detector include the power supply, a front-end board which has the two
photodiode arrays (for the analytical to prep detector) or a photodiode array and two reference
photodiodes (for the microbore to analytical detector) and the board which contains the front
status lights.
Figure 8 Back panel of the ProStar 325 detector
The interface connectors are located on the rear panel of the detector (see Figure 8). There are
five interface connectors as shown in the Figure. Connector J1 is used to connect the ProStar
335 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 manufacturers’ HPLC modules.
To make connecting to the sync signals easier for the customer, an I/O adaptor board, (shown in
Figure 9) is available. This board provides connectors for connecting bare wires to the ProStar
335. 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/O adaptor board.
Figure 9 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 10, makes
interconnecting the relays easy.
Figure 10 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 are located on the front of the ProStar 335. 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
Firmware
The firmware in the ProStar 335 has been significantly upgraded from the firmware in the ProStar
325 while keeping the best aspects of the ProStar 325. Many of these changes will be
incorporated in the next release of firmware for the ProStar 325.
Similar to the ProStar 325, the firmware in the ProStar 335 can still be downloaded from a
computer if an update is needed.
Performance
The ProStar 335 specifications are listed in the ProStar 335 data sheet. Here we are going to
discuss the parameters that effect the performance specifications.
The most important performance parameter of any HPLC detector is noise. The noise
determines the limit of detection and the limit or quantitation. There are three factors that most
significantly affect noise in the ProStar 335, the slit width, the filtering and the wavelength.
Effect of slit width on noise
As the slit width increases, the amount of light that passes through the optics and on the
photodiode array increases. This in turn decreases the detector noise. Figure 11 shows the
effect of bandwidth on noise and response time.
Effect of slit width on noise at 254 nm
+/- Peak to Peak Noise
120
100
0.05 sec
0.5 sec
1.0 sec
2.0 sec
80
60
40
20
0
1
2
4
8
16
Slit width (nm)
Figure 11 Effect of slit width on noise
Here you can see that increasing the slit width has a significant effect on peak to peak noise.
This is especially true when the response time is very low. For a response time of 0.05, there is a
10 fold decrease in noise going from a 1 nm slit width to a 16 nm slit width. For a 2 second
response time, the decrease is only a factor of 5. Therefore, whenever possible, use a slit width
of 4 nm or greater. Since most compounds have natural bandwidth in the UV of 40 nm or more,
you will not be losing any spectral information using a larger slit width. This data was take at 0.25
mL/min water with a 9x0 flow cell and both the UV and visible lamp on.
There is also a significant effect of wavelength on noise. Figure 12 shows the effect of
wavelength on noise at different slit widths.
+/- Peak to Peak Noise
Effect of wavelength on noise
90
80
70
60
50
40
30
20
10
0
2 nm
4 nm
8 nm
16 nm
195 210 240 254 280 350 450 600 700 950
Wavelength (nm)
Figure 12 Effect of wavelength on noise
It is clear from this chart that for a slit of 8 and 16 nm, the detector has very low noise even at 195
nm and 950 nm. It is interesting to note that the 8 nm slit is better than the 16 nm slit at the
extreme wavelengths. This is probably due to the lack of light below 190 nm and above 950 nm.
As the bandwidth widens at the extremes of the detector ranges there is not much of an increase
of light. This data was taken at 0.5 mL/min water.
All other factors involved with the noise operate the same as in the ProStar 325.
Optical resolution
Optical resolution is of importance in HPLC only when the compound of interest has narrow
bandwidths. Most compounds have a broad natural bandwidth so we recommend that you use a
bandwidth of 4 to 8 nm to minimize noise. However, when a compound has a small natural
bandwidth a detector with a narrow optical band is important for accurately identifying the
compound.
The 335 has a choice of slits. Figure 13 shows a spectrum of benzene in water.
Figure 13 Benzene in water with 1 nm slit
As you increase the slit width the optical resolution will decrease. Figure 14 and 15 shows the
gradual decrease of resolution with increasing slit width.
Figure 14 Benzene in water with a 1 and 2 nm slit
Figure 15 Benzene in water with a 4, 8 and 16 nm slit
A good simple way to get a measure of the optical resolution of a photodiode array detector is to
measure the height of the main absorbance peak compared to the valleys separating that peak
from the next two peaks. This is shown in Figure 16.
Figure 16 Measurement of optical performance of the ProStar 335
The numbers on the plot show the heights of the central peak and the two valleys next to it. The
ratios of the peak height to the valleys are 2.2 for the low wavelength side of the peak and 2.58
for the high wavelength side of the peak.
Linearity
Linearity in a HPLC detector is important because is it the basis for limiting the number of points
on a calibration curve and it provides a wider dynamic range so that the detector can analyze
compounds of widely varying concentration. In absorbance detectors, stray light causes nonlinearity. If you minimize stray light you will get a larger linear dynamic range.
The typical procedure for measuring stray light is to use an accurate HPLC pump to add some
kind of absorbing compound, in this case acetone, to a non absorbing compound, in this case
water, in known steps and determine the linearity. This is the method outlined by the ASTM.
This was done for the ProStar 335 and is plotted in Figure 17.
Linearity Plot
3.5
3
2.5
Abs
2
1.5
1
0.5
0
0
20
40
60
80
100
120
%C (C=2% acetone)
Figure 17 Measurement of linearity for the ProStar 335
Specifications for detector linearity are given as a pair of numbers, 1% deviation at 1.5 AU or 5%
deviation at 2 AU. This can be confusing when comparing the specifications of two detectors.
However, since the cause of non-linearity is stray light, the deviation from linearity is related to
actual amount of stray light in the system. Figure 18 shows a plot of the effect of stray light on
the theoretical absorbance linearity with 0, 0.1% and 0.05% stray light.
Figure 18 Effect of stray light at different absorbances
It the plot, the black line is the theoretical absorbance with no stray light the red line is the
theoretical absorbance with 0.05% stray light and the blue line is the theoretical absorbance with
0.1% stray light. The two inserts show an expansion of the plot at 1.5 AU and 2 AU. IN the upper
left, for a stray light of 0.1%, there is a deviation of about 1% at 1.5 AU (1.5 AU to 1.485 AU). IN
the lower right, for the same stray light, there is a deviation of about 2% at 2 AU (2AU to 1.96
AU). Essentially a spec of 1% deviation at 1.5 AU is the same as a spec of 2 % deviation of 2
AU.
Since the deviation from linearity is dependent on the amount of stray light in the system, there is
a simpler way of measuring detector linearity than having a pump produce a series of steps or a
linear gradient. If all of the light passing through the flow cell is absorbed, then the only light left
is the stray light. Experimentally, this is done by injecting a 20% solution of acetone in water into
the 9x0 flow cell and measuring the absorbance. This can then be compared to the plot in Figure
19 to determine the stray light.
Determining stray light
Maximum absorbance
4.5
4
3.5
3
2.5
2
1.5
0.00%
0.05%
0.10%
0.15%
0.20%
0.25%
0.30%
0.35%
0.40%
% Stray light
Figure 19 Plot of stray light vs maximum absorbance after blocking all light through the
flow cell
Once the stray light is determined, then absorbance deviation at any absorbance can be
calculated. For the ProStar 335, if the absorbance maximum is above 3 AU, the stray light is less
than 0.1% and the instrument will have less than 1% deviation at 1.5 AU.
Wavelength accuracy and wavelength precision
Wavelength accuracy in a photodiode array detector is determined by a combination of
wavelength calibration and wavelength stability or precision. The wavelength calibration
procedure performed at the factory was described above. A fine tuning of the calibration,
described above, is done every time the detector lamp is powered on using the 656 nm light
emission maximum of the deuterium lamp. This is similar to the process used n the ProStar 310
detector.
Once the lamp is turned on, and this system warms up, the wavelength calibration can shift
slightly due to thermal effects. This is most pronounced when the detector is first built. As a
result, the detector goes through a burn in period before it is initially calibrated. However even
after production, there may be slight wavelength drift when a unit is initially turned on. Figure 20
shows the wavelength drift of several ProStar 335 detectors after the lamp is turned on.
Wavelength stability
487
D2 peak max
486.8
486.6
486.4
Proto 4
486.2
Proto 8
486
Pilot 7
485.8
Pilot 9
485.6
Pilot 10
485.4
485.2
485
0
1
2
3
4
Hours
Figure 20 Effect of detector warm-up upon wavelength stability
Here we looked at the deuterium peak at 486 nm after turning on the lamp and automatically
calibrating it at 656 nm. The chart shows a wavelength accuracy of +/- 1 nm (from 485 to 487).
All of the detectors easily meet the criteria. For two of the detectors, there is some effect of
warm-up on the actual wavelength. When proto 8 is initially turned on, it had a wavelength drift of
about 0.25 nm for the first hour. After 2 hours of warm-up, the wavelength had stabilized to about
+/- 0.01 nm.
This small variation would normally not be an issue except for the Purity Parameter in the Star
PolyView2000 software. The Purity Parameter is sensitive to very small changes in wavelength.
This is what enables it to distinguish between compounds with very similar spectra. A change of
0.25 nm can be large in terms of a purity parameter change. Therefore, if using the purity
parameter to confirm the purity and help identify peaks, it would be best to allow the detector to
warm up at least 1 hour before doing sensitive work. This may not be needed on all detectors.
Extended Range Capabilities – Single and Dual Array
The ProStar 335 detector is manufactured in both a single and dual array model. As mentioned
above, the single array model is used exclusively for microbore to analytical work while the dual
array can be used for analytical to preparative work in a scale up mode. The details of the prep
capabilities of the dual array detector are described elsewhere. However, it is important to realize
that the microbore to analytical detector (single array) cannot be used with either a 9x1 or 4x0.5
detector. Because the reference side of the detector uses only two photodiodes, any kind of
extended range operation would not be possible on the single array detector. Therefore
customers should only use the 9x0 and 4x0 flow cells in single array detectors. They should NOT
use the 9x1 or 4x0.15 flow cells in the single array detectors.
Computer control of the ProStar 335 detector
The ProStar 335 can be controlled by either The Star Workstation or the Galaxie Workstation or
Chromatography Data system. All of the capabilities of the detector are controlled by both
systems. For detailed information on operation of the detector from either Star or Galaxie, please
refer to the Star or Galaxie driver operator’s manual. Here we will describe only some of the
controls and features of control.
Star workstation control
The Star workstation controls the ProStar 335 in a similar manner to how it controls the ProStar
325 detector. Figure 21 shows the Star method editor screen for the ProStar 335.
Figure 21 Star Method Editor Screen for the ProStar 335
Photodiode Array Spectra Acquisition
The right hand side of the screen provides the controls for the acquisition of spectra into the Star
workstation software from the ProStar 335. The range of wavelengths being acquired is listed as
the Min and Max wavelengths. These can range from 190 to 950 nm with 1 nm increments.
Spectra are acquired at a base frequency of 10 Hz or 10 spectra per second. This can lead to
very large data files. Two parameters can reduce the size of the data files acquired, the Detector
bunch rate and the Sampling Wavelengths. The detector bunch rates controls how many spectra
are taken per second. If the chromatographic peaks of interest are broad, the bunch rate can be
used to reduce data file size. For example, for good quantitation with the Star workstation data
handling algorithms, 15 data points across a peak are sufficient. If the narrowest peak of interest
is 10 seconds wide at half height, then 15 points are needed across the entire peak of about 30
seconds. This would be a point every 2 seconds or a bunching rate of 16. This will reduce the
size of the data file by a factor of 16.
A second way of controlling the size of a data file is the sampling wavelengths parameter located
in the lower right hand corner of the screen and shown in Figure 22.
Figure 22 Sampling Wavelengths Entry
This allows the chromatographer to select which wavelengths to store. He can choose to store all
wavelengths, every 2 nm (e.g. 250, 252, 254, 256 etc), every 4, 8 or 16 nm. This significantly
reduces data file size when the chromatographer has limited electronic storage.
Chromatogram Acquisition
The ProStar 335 can output one or two time programmed chromatograms on the analog channel
outputs on the rear of the detector similarly to the ProStar 325 detector. Also like the ProStar
325, the time programmed wavelength table in the method can control the chromatograms that
are stored in the .RUN file in the Star workstation. If no entries are made in the Commands
Method entry of PolyView 2000, then the chromatogram(s) designated in the time programmed
ProStar 335 method will be stored with the .RUN file.
Display Mode
The real time display can be controlled through the method by the display mode entry located on
the center left of the screen and shown in Figure 23 below.
Figure 23 Chromatogram Display mode
With the display mode entry, the chromatographer can choose to display either one or two
wavelengths and, if required, a real time plot of the purity parameters. The purity parameter is
controlled by the entries on the right side of the screen in the Min Purity Wage length and Max
Purity Wavelength boxes and the threshold level set in the time programmed part of the method.
Whenever the absorbance exceeds the threshold, the purity parameter will be calculated and
displayed. In this way, the chromatographer can get information about peak purity as the run is
progressing.
No Run File
If the ProStar 335 is being used for preparative chromatography with the analog signals being
used for fraction collector control and as input to the Dynamax Module in the Star workstation, the
Chromatographer can choose to check this box not to save the spectral data. This will save
significant room on the hard drive, especially for preparative chromatography.
System control
The Star system control screen is shown in Figure 24.
Figure 24 Star System control screen for the ProStar 335 detector
This screen is similar to the ProStar 330 system control screen. A real time Isoabsorbance plot is
located in the upper right hand part of the screen. Clicking on this will automatically access the
Aurora application where various graphical plots can be manipulated in real time.
The bottom of the screen is reserved for chromatographic plots and a spectral display. The
content of the chromatographic plots (one or two wavelengths and the purity parameter) is
determined by method inputs described above. The layout of the plots are controlled by the
display options box located above the center of the display and shown in Figure 25 below.
Figure 25 Plot and Spectral display control
Here the display can be arranged side by side as shown in Figure 24, above or below each other
or the real time spectrum display can be completely removed from the display. This gives the
chromatographer complete control of what they see on the screen.
Options box
Because so much room has been taken up by the graphical data displays, many of the functions
normally on the control screen have been put under the Options Button (Figure 26).
Figure 26 Options Screen
The Options screen now contains the information which used to be directly on the main screen for
turning the lamps on and off, setting the flow cell characteristics, changing the output gain, and
controlling the sync signals. In addition, two other functions have been added to the Options
screen.
Calibrate Lamps
When the ProStar 335 is first powered up, it automatically goes through a wavelength calibration
similar to most other absorbance detectors but it does not go through a gain or lamp calibration
as the ProStar 325 does. The gain or lamp calibration is used to set the amount of time that each
diode is allowed to collect light before it is scanned. The ProStar 335 tries to use as much time
as possible to minimize noise without having any of the diodes saturate and cut off a peak. It
therefore needs to have an estimate of current lamp intensity. Since the UV and visible lamp
intensities do not decrease rapidly, this calibration is a user option. When a new lamp is put in or
when the noise on the detector has significantly increased, the chromatographer can choose to
flush the flow cell with a non absorbing solvent and perform a lamp calibration. Our experience
has shown that this will probably not have to be done more than once when a new lamp is
installed and once or twice during the life of the lamp. If the user in looking at preparative peaks,
they may only have to do it upon lamp installation. A detailed procedure for doing lamp
calibration is in the ProStar 335 Operation manual.
Ethernet Settings
The ProStar 335 normally uses a procedure called BootP to assign an IP address on the host
workstation for establishing communications protocols with the detector. This means that a
different IP address could be assigned every time the ProStar 335 is powered on and in some
laboratories that could be contrary to the laboratory protocol. If there is a desire to have a fixed
IP address for each ProStar 335, Star Workstation can assign an IP address to a particular
ProStar 335. See the ProStar 335 Photodiode Array detector Control Software manual for more
details.
Manual Control
Time programmed wavelength outputs and relays can be manually controlled during a run using
the Insert Method Line Screen (Figure 27).
Figure 27 Insert Method Line screen
On this screen, the chromatographer can set one or more wavelengths and attenuations and
change one or more relay setting and then immediately send this method line to the ProStar 335
detector. The detector will then execute the line immediately, changing the state of the relays
and the rear panel analog output. This allows a chromatographer doing preparative work to
change wavelengths as needed to more clearly visualize the peaks and to better control the
automated fraction collector program by changing wavelength going into the fraction collector.
Galaxie Workstation and CDS Control
All of the same control features that are present in the Star Workstation driver are also present in
the Galaxie driver. Shown in the next five figures are several of the Galaxie screens to illustrate
some of the Galaxie driver capabilities.
Figure 28 Galaxie method for acquisition of spectral data
Figure 29 Galaxie method for real time purity parameter display
Figure 30 Galaxie Control for real time method line insertion
Figure 31 Galaxie 4 view display of benzene spectra from the ProStar 335