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Chromatographic Resolution

Chromatographic Resolution

In chromatography, a solute moves through a chromatographic column and tends to spread, forming a Gaussian-shaped band. The longer the solute spends in the column, the broader the band becomes. The broadening can lead to overlaps within the column, affecting separation effectiveness.
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In chromatography,...

Ion-Exchange Chromatography

Ion-Exchange Chromatography

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High-Resolution Mass Spectrometry (HRMS)

High-Resolution Mass Spectrometry (HRMS)

The resolution of a mass spectrometer depends on the efficiency of separating ions with different ion masses. The mass of an atom is approximated to the sum of the masses of protons and neutrons inside, considering the masses of protons and neutrons as equal. However, the masses of the proton (1.6726 × 10−24 g) and neutron (1.6749 × 10−24 g) are not truly equal. There is a minor error in the expression of atomic masses relative to the simplest atom of hydrogen. For...

Size-Exclusion Chromatography

Size-Exclusion Chromatography

In size-exclusion chromatography (SEC), also known as molecular-exclusion or gel-permeation chromatography, molecules are separated based on their sizes. This technique is important for separating large molecules such as polymers and biomolecules. The two classes of micron-sized stationary phases encountered in SEC are silica particles and cross-linked polymer resin beads. Both materials are porous, but their pore sizes vary significantly.
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Related Experiment Video

Updated: May 30, 2025

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Published on: July 31, 2011

sgRNA Single-Nucleotide Resolution by Ion-Pairing Reversed-Phase Chromatography.

Joshua D Jones¹, Todd D Maloney¹

¹Synthetic Molecule Design and Development, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States.

Analytical Chemistry

|January 30, 2025

Summary

This summary is machine-generated.

Researchers developed a novel chromatography method for analyzing single-stranded guide RNAs (sgRNAs), essential for CRISPR/Cas9 genome editing. This breakthrough offers single-nucleotide resolution, improving the characterization of these vital genetic medicines.

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On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Published on: March 2, 2012

Area of Science:

Biochemistry
Analytical Chemistry
Molecular Biology

Background:

Single-stranded guide RNAs (sgRNAs) are crucial for CRISPR/Cas9 genome editing therapies.
Current analytical methods for sgRNAs, including chromatography, lack sufficient selectivity and mass spectrometry (MS) compatibility.
The complex structure of sgRNAs complicates their characterization, hindering therapeutic development.

Purpose of the Study:

To develop a highly selective ion-pairing reversed-phase chromatography (IP-RPC) method for sgRNA analysis.
To overcome limitations in existing chromatographic techniques for sgRNA characterization.
To enable high-resolution analysis of sgRNA therapeutics for improved process development and toxicological studies.

Main Methods:

Systematic evaluation of chromatographic parameters for sgRNA separation.
Optimization of ion-pairing reagents, flow rates, and temperatures for IP-RPC.
Development of an MS-compatible mobile phase for enhanced analysis.

Main Results:

Achieved the first single-nucleotide resolution chromatography method for sgRNAs.
Demonstrated that stronger, more hydrophobic ion-pairing reagents enhance selectivity for truncation impurities.
Identified that reduced flow rates and temperatures improve selectivity near the FLP (presumably Full-Length Product).

Conclusions:

The developed IP-RPC method provides high-resolution characterization of sgRNA therapeutics.
This methodology facilitates insights into sgRNA impurity profiles, crucial for toxicological assessments.
The findings support the advancement of sgRNA-based genetic medicines through improved process development and quality control.