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

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

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.

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 http://www.waters.com/WatersDivision

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 http://www.waters.com/WatersDivision

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.

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

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

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

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

Preliminary Design – UV Probe Inexpensive Simple Design

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.

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

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

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

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

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

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

UV Block Design-Initial Concept

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

UV Block Design

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

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

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

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.

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

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

Questions???