Dynamic pH Profile Isoelectric Focusing Separation
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Solution Overview
Problem
Current isoelectric focusing methods face limitations in efficiently separating proteins based on their isoelectric points, particularly in achieving high resolution and rapid purification times, often requiring gel and ampholyte-based solutions which can be cumbersome and time-consuming.
Innovation Solution
A method and device utilizing a dynamic pH profile with controlled ion sources to induce the migration of molecular analytes along a pH axis, allowing for spatial and temporal separation of proteins based on their isoelectric points, enabling high-resolution separation without the need for gels or ampholytes, and facilitating shorter purification times.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If gel and ampholyte-based solutions are used for isoelectric focusing, then separation resolution is improved, but purification time and device complexity increase
Solution Approach 1:
The patent removes gels and ampholytes from the isoelectric focusing system, replacing them with a buffer solution containing pH gradient-forming ions. This extraction of problematic components eliminates the need for time-consuming gel polymerization and ampholyte addition while maintaining the essential pH gradient function through alternative means (ion migration under electric field).
Solution Approach 2:
The patent changes the fundamental parameters of the separation medium from gel-based to buffer-based, and from ampholyte-generated pH gradient to ion-generated pH gradient. This parameter change enables faster separation kinetics while maintaining resolution, as the buffer solution allows freer molecular movement compared to gel matrices.
2Manufacturing precision
If gel-based isoelectric focusing is used, then separation resolution is improved, but device complexity and ease of operation worsen
Solution Approach 1:
The patent extracts the gel matrix from the system, replacing it with a liquid buffer solution. This eliminates the need for complex gel polymerization procedures, gel casting apparatus, and associated handling complexities, while maintaining the separation functionality through the buffer solution's pH gradient.
3Manufacturing precision
If traditional isoelectric focusing methods are used, then proteins can be separated by pI, but purification time is excessive
Solution Approach 1:
The patent enables continuous pH gradient formation through sustained ion migration under the electric field, rather than relying on pre-formed static gradients. This continuous action maintains optimal separation conditions throughout the process, enabling faster purification while maintaining resolution.
Solution Approach 2:
The patent changes the pH gradient formation mechanism from chemical (ampholyte equilibrium) to electrochemical (ion migration under field). This parameter change accelerates the gradient establishment and maintenance, reducing purification time while preserving separation quality.
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
The method achieves efficient separation and purification of proteins by dynamically adjusting the pH profile, allowing for high-resolution separation of proteins with small pI differences and reducing purification times, while eliminating the need for gels or ampholytes, thereby enhancing separation efficiency and yield.
Implementation Method 1
The ion sources are set to establish a pH profile in the solution by injecting ion flows which lower or raise the pH in certain zones of the separation volume
Implementation Method 2
An electric potential is applied parallel to the proton concentration gradient between an isoelectric focusing anode and isoelectric focusing cathode. Molecules having a net positive charge migrate through the electrolyte solution towards the cathode while molecules having a net negative charge migrate through the electrolyte solution towards the anode.
Implementation Method 3
Isoelectric focusing is an analytical technique for separating molecules in an analyte sample by taking advantage of the differing ionic properties of the molecules. As the molecules migrate, the ambient pH change to reduce the net charge on the molecule until the molecule reaches an isoelectric point (pI) where, due to the ambient pH, the net charge on the molecule is zero.
Data Source
Figure 1
Figure 2A~2E
Figure 3A
AI summary
A method of separating a mixture of a plurality of molecular analytes having different isoelectric points (pIs). The method comprises placing a solution containing a mixture of a plurality of molecular analytes in a separation volume, generating a pH profile having a plurality of pH zones across an axis of the separation volume, and adjusting a profile of the pH profile to induce a migration of a first molecular analyte along the axis apart from a second molecular analyte. The first and second molecular analytes having different pIs.