The New ProStar 335 Diode Array Detector –
Analytical to Preparative considerations
Introduction
The Varian ProStar 335 DAD is the latest exciting product to come
off the production line at Melbourne. This state of the art detector
has all of the usual features you would expect from a diode array
(1024 diodes, benchmark noise specifications etc…) which will excite
the Analytical chromatographer, however it also incorporates a
unique feature for Analytical to Preparative work – “The Dual Array”
Dual Path Cell
When Varian bought the HPLC business of Rainin Instruments in
1997, we acquired a unique technology, the dual path flow cell. In
the UV-1 and later the ProStar 320, the sample flowed through both
a sample and reference side of different path lengths. When the
sample absorbance exceeded approximately 1 Abs, the detector
automatically switched from measuring the absorbance through the
long path (and using the shorter path as a reference) to just
measuring the absorbance through the shorter path in single beam
mode. Chemists could determine the entire peak shape for both the
smaller peaks and the larger peaks in a chromatographic analysis,
and the feature enabled the system to be used as an analytical or a
preparative tool.
Figure 1: Schematic of the Varian ProStar 320 dual path flow
cell.
Combining Technologies
Although the UV-1 and 320 detectors incorporated this unique and
powerful feature, the benchmark specifications you would expect for
noise/drift , and in turn sensitivity for Analytical applications, were
compromised.
With the introduction of the ProStar 325 UV-Vis detector, Varian has
combined the best analytical specifications with significant
enhancements on the dual path design( i.e flow-cell independent of
tubing volume) into a single unit that has outstanding performance in
both Analytical and Prep applications. In addition, we have added
dual wavelength and wavelength ratioing without compromising
either performance or prep capabilities.
The 335 Diode Array
Figure 2: ProStar 335 detector
The ProStar 335 detector, the latest UV-Vis detector from Varian,
takes these principles a step further .Designed and built in a
collaboration of the Varian Inc. divisions in Melbourne Australia and
Walnut Creek California, it combines the optical expertise of the
designers and builders of Cary spectrophotometers and the ProStar
310 UV-Vis detectors. It’s innovative design combines the best
features of an Analytical and Prep HPLC detector in one single unit.
Optical design
The incorporation of the dual path design meant the requirement of a
second array hence dual path “Dual array”. If we use the 9x1 cell as
an example, the larger 9mm path uses the sample photodiode array
and the smaller 1mm path uses a reference photodiode array.
Figure 3: Optical design
The Flow Cell
In this design( the same as the ProStar 325), 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.
Figure 4: Flow-Cell Schematic
Diode Array for Prep?
There is often confusion when it comes to the application of a diode
array detector for Preparative chromatography, why would you need
one when all the prep chromatographer wants to do is purify
“x”grams of material as simply as possible. Prep people are usually
given suitable chromatographic conditions to work with by their
analytical colleagues( or produce this data themselves on an
analytical system) and this involves using a single wavelength.
Therefore the dedicated preparative chromatographer has little use
for a Diode Array.
However, the 335 diode array detector is unique because it allows a
user or a multitude of users to use the system for
“Analytical to Prep” applications.
These users fall into 2 categories:
1.”Scale up” chromatographers
The scale up chromatographer (can be academic, from industry or
even pharmaceutical) would like the ability to work up his analytical
methods from the beginning. Once the method is developed, an
optimum wavelength discovered and a loading study completed, they
would then like to use the same system to purify their desired
amount of material (Believe it or not, of all the HPLC companies
around, there are not many who can do this. Within this small group
of companies, all of them have to change some components to
achieve this flexibility)
Column ID
4.6mm
10mm
1”
2”
Scale Up Factor
(flow rate)
1
4.7
21.6
81
Typical Loading
1-4mg
5-25mg
22-88mg
81-324mg
2.Multi user setup’s
In a multi user setup, often academic institutions, the customer
wants to get the most for their money and has a team of people who
use the system for a diverse range of work. This means the widest
flow-rate range possible, the largest injection volume range possible,
a detector that can cope with small/large flows and small/large
concentrations and appropriate valving to house different size
columns. This will enable them to switch a valve to select a column,
select a valid method for this column and run their samples. They
also require these features with the minimum of hardware changes.
There are few companies that can provide this type of system( Varian
being one of them) and a large customer base requirement. Many
customers buy two systems for twice the cost because they do not
know that this technology is available. Once the availability of “
Analytical to Prep” systems is known a customer will find this an
attractive prospect that will save them money.
VARIAN ARE THE ONLY COMPANY THAT CAN PROVIDE THE
COMPLETE BENCHTOP ANALYTICAL TO PREP SYSTEM WITH NO
HARDWARE CHANGES FOR EITHER DUAL WAVELENGTH UV OR
NOW DIODE ARRAY
ANALYTICAL TO PREP HARDWARE
Pumps
The ProStar 210/218 with 25ml heads provides accurate gradient
flow-rates from 0.1 to 25ml/min ( 2mm to 1 inch columns)
The PrepStar SD1 with 200ml heads provides a range of 0.1 to
200ml/min (2mm to 2” columns)
(NO HARDWARE CHANGES AND NO COMPROMISE)
Injectors
By combining a manual valve with an autosampler you can span an
automated large volume range and a manual small volume range or
vice versa
i.e ProStar 410 with 100ul loop (1-100ul) + 7725i w/5ml loop (100ul5ml)
Or 410 prep (200ul-10ml) + 7725i w/100ul loop (1-100ul)
Or even better, you can fit a second valve next to the 410 enabling a
“dual loop” concept – this valve can have 2 loops installed and you
can get the 410 to suck through either loop – giving automated
analytical and prep injections with NO HARDWARE CHANGES AND
NO COMPROMISE
Detectors
ProStar 325 with 9/1 cell – Easily covers 4.6mm to 2” ID columns
Or
ProStar 330 ( Existing solution) – This required a ProStar 330 + extra
semi-prep/prep flow-cell + analog output board for peak fraction
tracking
ProStar 335 Solution: The 335 with its built in Analog outputs and
dual path cell/array eliminates the need for any hardware changes
Below are 2 examples of very powerful Analytical to Prep
Configurations
Example 1
A 210/218 binary gradient system with 25ml heads, 410
autosampler/7725I manual valve, 701, CVM 500 and the 335 would
allow the user to perform:
•Diode array Analytical method development
•Single/Dual wavelength Preparative runs and collections
•Analytical purity check of fractions with DAD
•1ul to 5ml inject vols(1-100ul on 410, Manual 5ml loop)
•Optional dual loop valve
•0.1-25ml/min flow-rates at 4600/6000psi
•Automatic 3/6 column selection
•NO HARDWARE CHANGES REQUIRED
Example 2
The same as above but using a Binary SD-1 system with 200ml
heads.
Case Study
A company in the UK called Hexcel Composites manufactures carbon
fibre and resin coatings for aircraft wings. Its principle customers are
Boeing and Airbus.
The end user was looking to purchase an Analytical system and after
discussing “ Analytical to Prep” he described how this type of system
would be perfect.
“We don’t have a diode array on our existing analytical system, so a
Diode array implemented onto a system that I can use for method
development AND do purification would be fantastic – I’d have to see
it to believe it though”
Hexcel Composites
This customer duly came for a demo and was amazed at the
flexibility. We ran a competitor resin analytically, scaled up onto a 1”
column and isolated several fractions for GC-MS analysis.
The GC-MS results helped them to identify 2 unknown components
that they could now experiment with in their own product line.
Example 335 Data
Below is a chromatogram showing a 1ml injection of 3 parabens onto
a 2” column. The flow-rate was 40ml/min through a 9/1 dual array
335 . Note the y axis at nearly 15AU!
Figure 5:Dual Array 40ml/min Parabens
Competition
Who seriously competes in this arena?
Waters, Gilson, Agilent, Varian
All competition require separate flow-cells( Waters do have a dual
inlet but still have to physically change the fitting from one to
another but with compromises on the pathlength).
Waters are probably the best equipped in that they have the
pumping/injector and detector range( with flowcell changes)
They, like the Gilson and Agilent, also have the ability to Inject and
collect on the same XYZ bed – this is Varian’s only weakness.
The flowcell change from analytical to 0.15mm is tricky and you need
a screwdriver to do this.
Gilson struggle at the lower pumping range (2-200ml/min) and have
to change flow-cells.
Agilent also struggle at the lower pumping range (5-100ml/min) and
also have to change flowcells.
Conclusion
Varian command a strong position in the Analytical to Prep arena.
We have always been strong with respect to pumping systems after
the 1997 Rainin acquisition and now with the enhancement of the
Dual pathlength cell on the 325 and its unique implementation onto
the 335 Dual array we can push even harder in this area.
Mr Frankie Button
HPLC Product Specialist UK