Mannose-6-Phosphate Detection Using Tris Buffer and Hydroxyapatite Chromatography
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Solution Overview
Problem
Conventional post-column fluorescence detection methods for analyzing reducing sugars, particularly for detecting mannose-6-phosphate, face issues with noise interference and reduced sensitivity due to thermal expansion and convection at high temperatures, and pose environmental and health risks from boric acid usage.
Innovation Solution
The method employs Tris buffer with phosphate in the mobile phase and hydroxyapatite column chromatography instead of anionic exchanger columns, along with a back-pressure generator to reduce noise and eliminate boric acid, enabling sensitive detection of mannose-6-phosphate without boric acid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If boric acid is used in the mobile phase for post-column fluorescence detection of reducing sugars, then the detection can be performed, but thermal expansion and convection occur at high temperatures causing noise and reducing sensitivity
Solution Approach 1:
The patent changes the chemical composition parameters of the mobile phase by replacing boric acid with Tris buffer containing phosphate, thereby eliminating the harmful thermal expansion and convection effects while maintaining detection capability
Solution Approach 2:
The patent extracts and removes boric acid from the mobile phase composition, eliminating the source of thermal expansion and convection noise while preserving the essential detection function through alternative reagents
2Reliability
If boric acid is used in the mobile phase, then reducing sugars can be detected, but environmental and health risks are posed
Solution Approach 1:
The patent removes boric acid from the mobile phase composition and replaces it with Tris buffer containing phosphate, thereby eliminating environmental and health risks while maintaining the detection capability for reducing sugars
Solution Approach 2:
The patent changes the chemical composition parameters by substituting boric acid with Tris buffer containing phosphate, achieving both elimination of harmful effects and preservation of detection function
3Measurement precision
If high temperature is used for heat reaction of reducing sugars with basic amino acid, then fluorescence detection is enabled, but thermal expansion and convection cause noise
Solution Approach 1:
The patent changes the mobile phase composition from boric acid-based to Tris buffer containing phosphate, which allows high-temperature heat reaction to proceed without causing thermal expansion and convection noise, thereby maintaining measurement precision
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
This approach enhances detection sensitivity and eliminates the environmental and health hazards associated with boric acid, providing a more efficient and environmentally friendly method for analyzing mannose-6-phosphate.
Implementation Method 1
a column with a solid phase having affinity for phosphate
Implementation Method 2
a heater for heating the eluate in the flow path to cause mannose-6-phosphate and a basic amino acid contained in the eluate to react
Implementation Method 3
The fluorogenic derivatives are melanoidins, brown compounds which are produced by a heat-reaction between reducing sugars and a basic amino acid, which is an amino compound (Maillard reaction)
Implementation Method 4
a fluorescence detector installed in the flow path downstream of the heater for continuously irradiating the eluate with excitation light and measuring the intensity of fluorescence emitted from the eluate
Data Source
AI summary
Disclosed are an apparatus and method for separation analysis of mannose-6-phosphate (M6P) by post-column fluorescence detection method. The apparatus is based on chromatography, and includes a column with a solid phase having affinity for phosphate, a flow path for the eluate, a heater installed on the flow path for M6P and a basic amino acid to react by heating the eluate in the flow path, and a fluorescence detector installed downstream of the heater for continuously irradiating the eluate with excitation light and measuring the intensity of the emission, and may include in the flow path a supply channel for addition of a basic amino acid between the column and the heater. The method is characterized in that it uses the apparatus and a second mobile phase consisting of a second buffer containing phosphate of predetermined concentration and adjusted to a predetermined pH.