Related: paper chromatography, hplc with mass spec is a game changer for protein identification
An HPLC identifies and quantifies chemical components in liquid solutions, and is used in protein chemistry.
The workflow of the HPLC can be separated into two large sections: flow phase and stationary phase.
- Flow phase: the machine pumps liquid(s) through tubes at a set flow rate, then the samples are injected into these tubes so they also travel through the tubes at the same rate
- Stationary phase: the sample (now molecules mixed with the solvents) goes through a how HPLC columns work, and are separated based on their physical properties
In order for this the work, the stuff you want to quantify needs to have different affinities in the stationary phase. When it passes through the column, they should “travel” through various lengths of the column.
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the five-ish parts to an HPLC workflow
Here’s the overall path of the HPLC
- Solvent: these are buffers/solutions that are needed for the mobile phase to get started. these should be compatible with the machine, and are probably either sold as consumables or can be made in the lab
- Pump: this actually gets the fluids moving
- Sample injector: downstream of the solvent and pump, connected to the sample or the sample is loaded inside this component. it travels downstream
- HPLC column: chromatography!
- detection/analysis/waste: the particles in the column are detected via the column’s tech itself, which can be further analyzed using software. The solvent/sample mixture that’s been moving through the column now exits into a waste container.
- treat the liquid waste with caution - especially if you’ve used potentially dangerous samples or treatments!
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