Capillary pH Gradient Fractionation for Protein Charge Variants
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Solution Overview
Problem
Existing methods for capillary isoelectric focusing (CIEF) fractionation, such as hyphenated cIEF-MS, face limitations in analyzing charge variants of proteins due to restricted downstream analysis methods, performance compromise with MS additives, and poor resolution, sensitivity, and operational complexity.
Innovation Solution
A novel icIEF fractionation system that integrates capillary electrophoresis with semi-automatic analyte separation, visualization, and fraction collection into wells, enabling further analysis without MS restrictions, using chemical mobilizers for selective elution based on isoelectric points.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If automated liquid handling and multi-dimensional chromatography are used, then analytical capability and productivity are improved, but system complexity and cost increase
Solution Approach 1:
The system is divided into multiple independent robotic arms (first robotic arm for sample injection, second robotic arm for fraction collection) that operate independently but coordinate through a centralized controller. Each robotic arm has its own end effector designed for specific tasks, allowing complex analytical operations to be broken down into manageable segments that can be automated separately.
Solution Approach 2:
The robotic arms are designed with universal end effectors that can perform multiple functions - the first robotic arm's end effector can both inject samples and manipulate HPLC components, while the second robotic arm's end effector can both monitor fractions and collect them. This multi-functionality reduces the need for specialized equipment for each task, managing system complexity while maintaining high analytical capability.
2Manufacturing precision
If HPLC fractionation with multiple columns is used, then separation precision is improved, but analysis time and system complexity increase
Solution Approach 1:
The system performs preliminary actions by automatically preparing and injecting samples before the actual HPLC separation process begins. The robotic arm pre-positioning and sample injection occur prior to column separation, allowing the multi-column HPLC system to operate at full precision without manual intervention delays during the separation process itself.
Solution Approach 2:
The system maintains continuous useful action through automated fraction monitoring and collection. While the HPLC system performs separation on multiple columns, the second robotic arm continuously monitors eluting fractions and automatically collects them in real-time, eliminating idle time between separation stages and ensuring that the precision gains from multi-column operation are fully realized without time loss.
3Device complexity
If manual HPLC operation is used, then system simplicity is maintained, but productivity and precision deteriorate
Solution Approach 1:
The system implements self-service automation where robotic arms independently perform sample injection, fraction monitoring, and collection tasks without requiring constant human intervention. The centralized controller coordinates these self-service operations, allowing the system to maintain simplicity from the user perspective while achieving high productivity through automated execution of precise analytical operations.
4Measurement precision
If automated robotic arms with end effectors are used, then operational precision is improved, but device complexity and cost increase
Solution Approach 1:
The robotic system implements local quality by designing specialized end effectors with specific properties for different tasks. The first robotic arm's end effector is optimized for precise sample injection with features suitable for HPLC port engagement, while the second robotic arm's end effector is designed for fraction monitoring and collection with appropriate sensors and container handling capabilities. This localized optimization achieves high operational precision for each specific function.
Solution Approach 2:
A centralized controller acts as an intermediary between the robotic arms and the HPLC system. It coordinates the complex interactions between sample injection timing, column separation processes, and fraction collection operations, translating high-level analytical requirements into precise robotic movements. This intermediary layer manages device complexity by providing a unified control interface while enabling precise coordination of multiple robotic components.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Provides streamlined, high-resolution, and sensitive fractionation of charge variants, allowing comprehensive characterization and isolation for downstream analysis like MS or ZipChip, with broad sample matrix compatibility and fast analysis times.
Implementation Method 1
a first robotic arm moves a high performance liquid chromatography (HPLC) syringe to inject a sample into an HPLC system
Implementation Method 2
fractionation and collection of analytes in a sample
Implementation Method 3
a second robotic arm moves collection vials to collect the fractions
Data Source
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AI summary
Embodiments include systems, apparatuses, and methods to efficiently separate analytes in a sample and elute fractions of the separated analytes. In some embodiments, a method includes introducing a sample in a capillary with a first end ionically coupled to a first running buffer and a second end ionically coupled to a second running buffer to form a pH gradient. The method includes applying a voltage between the first running buffer and the second running buffer, to separate a plurality of analytes in the sample. The method includes disposing the second end of the capillary in a collection well including a chemical mobilizer and applying a voltage to elute one or more analytes from the plurality of analytes in the sample, that have been separated, into the collection well. Embodiments include detection methods to monitor separation of analytes, mobilization of analytes, and/or elution of fractions containing analytes.