1. Sterilization Device for Liquid Chromatography Solvents
Design Team
Nick Roulleau, Michael Vose
Michael Racette, Michael McKay
Our Project is entitled:
This project is sponsored by Waters Corporation
My Name is…
The other members of the design team are…
Advisor
Professor Mohammad Taslim

2. Introduction Background Problem Statement Past Art Design Requirements
Design Concepts
Prototype Design
Component Analysis
Recommendations
In the next few minutes we will…
… give a brief summary of the background of chromatography
…Introduce the problem that the team seeks to solve
…Discuss the impact that successful design could have on the field
…Give an overview of some of the existing methods and approaches to the problem
…talk about the requirements and constraints that have been established for the design
…introduce some of the design approaches the group has considered
…and present some next steps for moving towards delivery of a solution

3. What is Liquid Chromatography?
A substance comprised of components A and B is dissolved in a solvent and enters the analytical column, where it is separated
A substance made up of a number of components, say, A and B is dissolved in a solvent and passes into a column
The components pass through the column at different rates based on their relative chemical properties.

4. Basic Components of an HPLC System
So the sample is injected into a stream of solvent and is passed through the analytical column
As it passes through, the components separate, and are individually detected as the exit the column into the detector.
The output is a chromatogram which shows a peak for each component and the time it took to elute
CLICK TO ACTIVATE ANIMATED CIRCLE
The problem we seek to address is apparent HERE in the inlet of the column
From

5. Problem
So the sample is injected into a stream of solvent and is passed through the analytical column
As it passes through, the components separate, and are individually detected as the exit the column into the detector.
The output is a chromatogram which shows a peak for each component and the time it took to elute
CLICK TO ACTIVATE ANIMATED CIRCLE
The problem we seek to address is apparent HERE in the inlet of the column
From

6. Design Goal
To mitigate the risk of blockage at the inlet frit due to bacterial contamination and extend the useful life of the UPLC column.

7. Existing Solutions In-line filters Guard columns and cartridges
Some of the methods that companies in the field of chromatography use for reducing column clogging include:
……A recommended practice is pre-filtration of solvents, which would not protect from contamination which comes from the bottle itself, or from post-filtration handling.
…In-line filters and Guard columns are commonly used for removal of any particulates between the pump and analytical column, but they add dead-volume to the flow path which reduces the resolution of the system. Any decrease in resolution is undesirable for UPLC.
Pre-filtration of samples and mobile phase liquids

8. Product Requirements Mandatory: Desirable:
Must be adaptable for use worldwide
Must extend the useful life of columns
Must meet safety standards (ISO, UL and CE)
Must operate for 1 year w/o user intervention
Desirable:
Should be able to filter two bottles simultaneously
Should meet customer acceptance criteria
Low-maintenance
Easy to use
Cost
What is mandatory for the design to accomplish is that…
…it must be adaptable for worldwide use
…it must extend the useful life of the column
…it must meet Safety standards, including ISO requirements for medical devices, as well as UL and CE requirements
What Waters would LIKE the design to do is…
…to be adaptable to multiple bottle sizes
…and to be acceptable to the customer in cost, need for maintenance and ease of use. Any solution that is drastically different from what users are used to will not be as acceptable

9. Constraints Cannot change the chemical composition
Of the solvent
Of the sample
Cannot create risk of causing pump cavitation
Cannot hinder bottle accessibility
Cannot negatively impact system resolution
The things that any solution to the problem cannot do are:
To impact any aspect of the chemical composition of the solvent or the sample: pH, detectable impurities
We cannot create a risk of mis-priming of the pump which leads to cavitation
We can’t make the bottles any less accessible, since they are already very high for shorter technicians
And we cannot have any negative impact on the system’s resolution

10. Initial Design Concepts
UV Probe
Pump/filter--Cap enclosure
Pump/filter--External enclosure

11. Preliminary Design – UV Probe
Inexpensive
Simple Design

12. Why Not Use Ultraviolet Radiation as a Primary Solution?
Degradation of organic solvent modifiers (Low Risk)
Degradation of aqueous additives (Low Risk)
User safety from UV-C exposure (Medium Risk)
UV can inactivate but not remove bacteria
There is the Potential for photo-oxidation or degradation of organic solvent modifiers as well as additives to the aqueous buffers, however, these risks are low and would require significant energy transfer (high power, long exposure)
Our biggest concern is the potential for releasing cytoplasmic molecules such as proteins which could have significant impact on the analytical result, especially for users involved in proteomics.
The risk of exposure to dangerous UV-C radiation is a moderate risk, but mitigation measures would add cost to the solution.

13. Filter Sizing How many bacteria could be generated per year?
Logarithmic growth:
Assuming worst case
100% replicating
Short generation time
Neglecting lag phases and cell death
Filter capacity = 107 CFU/cm2

14. Filter Sizing
With logarithmic bacterial growth, filter area becomes exceptionally large in a short period

15. Current Design External Filter Enclosure with UV
Dual-head brushless DC pump
UV lamp with multiple sterilization lines
Pall AcroPak 200 filters

16. Filter Selection Membrane with material compatibility
Sufficient capacity to contain 1 year of inactivated bacteria

17. Pump Selection Micro-diaphragm pump Dual pump heads Ability to run dry
DC brushless motor for long life

18. Pump Pressure Requirements
Pump must deliver sufficient differential pressure (Δp) to move fluid through filter
Darcy’s equation for porous media:
L = membrane thickness
p1 = pump-side pressure
p2 = outlet pressure
Q = flow rate
k = permeability constant for filter
A = effective filter area (EFA)
µ = viscosity

19. UV Block Design-Initial Concept

20. Dose = Irradiance x time
UV Block Design
99.99% inactivation requires a UV dose of at least 40 mJ/cm2 for nearly all species of bacteria
Dose is a function of the irradiance (mW/cm2) and time of exposure (in seconds)
Dose = Irradiance x time
The Dose for any particle is a function of the Irradiance and the time exposed. The residence time, or time of exposure, required to obtain a Dose of at least 40mJ/cm^2 for any particle passing through the uv tube was determined for Irradiances (W/m^2) over the range of potential sources in consideration (9-50 uW/cm^2) in relation to the maximum distance a bacteria in the quartz tube could pass from these sources. The Intensities were calculated conservatively, factoring the “end of life” intensity of 80% of the starting intensity. The transmissibility through air was neglected, but the transmissibility of the quartz tube was factored for a 10mm thickness with reflectivity accounted for. This is also conservative, since the actual thickness of the tube wall is approximately 1.5 mm.
Adjust for:
Intensity reduction over life of lamp
Transmissibility of UV light through quartz tube wall dA A

21. UV Block Design

22. 13 loops necessary with an 18W UV bulb and thin wall FEP tubing
UV Block Design
The Dose for any particle is a function of the Irradiance and the time exposed. The residence time, or time of exposure, required to obtain a Dose of at least 40mJ/cm^2 for any particle passing through the uv tube was determined for Irradiances (W/m^2) over the range of potential sources in consideration (9-50 uW/cm^2) in relation to the maximum distance a bacteria in the quartz tube could pass from these sources. The Intensities were calculated conservatively, factoring the “end of life” intensity of 80% of the starting intensity. The transmissibility through air was neglected, but the transmissibility of the quartz tube was factored for a 10mm thickness with reflectivity accounted for. This is also conservative, since the actual thickness of the tube wall is approximately 1.5 mm.
Adjust for:
Intensity reduction over life of lamp
Transmissibility of UV light through quartz tube wall
13 loops necessary with an 18W UV bulb and thin wall FEP tubing

23. Test Planning Verification Test
Does the Device Meet Design Requirement?
Pump Particle-Laden Water from Bottles With and Without Device
Compare Backpressure and/or Flow Rate
Pump
Device
Sensor
Column

24. Backpressure was reduced by 28% when our device was used
Test Results
Backpressure was reduced by 28% when our device was used

25. Cost Analysis Developed target costs by estimating:
Annual costs without the assistance of our device (excluding operational costs)
Savings in material costs by implementing our device
Potential savings for high-end users = $44,000
Minimum estimated annual savings = $600
Target production cost = $500
Target prototyping cost = <$1500
This cost analysis was conducted to determine a baseline prototyping cost that would be acceptable considering all of Waters’ customers.
Therefore an estimate of the current annual cost for all levels of customers was calculated. The cost analysis displays the maximum and minimum number of columns that any customer could purchase each year.
The cost analysis also displays the savings for all levels of customers after implementing the filtration system. This is mainly from reducing the number of columns required but also from reducing the time required to maintain the UPLC systems’ functionality.
Ultimately, a device cost limit was determined by considering the range of UPLC customers.

26. Recommendations for Further Development
Improve manufacturability of the design
Simplify tubing system
Smaller pump
Custom filter size
Analyze effectiveness of UV with microbiological testing

27. Summary Introduction to liquid chromatography
The problem and its source
Requirements of a good solution
Design considerations
Prototype design and analysis
Recommendations

28. Questions???