文档介绍：Lecture 9 Introduction to Chromatography “The Science of 9讲介绍色谱学”
Objectives
Define High Performance Liquid Chromatoug candidates in animal models and human clinical studies.
Quality control (QC): assessing the quality of the final manufactured products against published specification for product release.
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
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. HPLC versus classical LC
High resolution (. better separation).
Small diameter ( mm) stainless steel, glass or titanium columns.
Column packing of stationary phase with very small (3, 5 and 10 μm) particles.
Relatively high inlet pressures and controlled flow of the mobile phase.
Continuous flow detectors capable of handling small flow rates and detecting very small amounts.
Rapid analysis.
These characters refer of course to HPLC, can you compare these characters to the ones related to classic column chromatography that you performed in the lab.?
© Dr. Rasha Hanafi, GUC
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Lecture 7 – HPLC, 23-10-2019
2. Schematic Set-Up of HPLC
© Dr. Rasha Hanafi, GUC
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Lecture 7 – HPLC, 23-10-2019
. Pumps
The quality of a pump is measured by how steady and reproducible a flow it can produce. A fluctuating flow creates detector noise that obscures weak signals.
Gradient construction:
High-pressure gradient
Low-pressure gradient (more common) where pro-portioning of the solvents (up to 4) through a four-way valve at low pressure takes place, then pumping the mixture at high pressure into the column is performed.
Requirements of pumping system:
Pulse free output.
Flow rates from -10 mL/min.
Flow control and flow reproducibility of a maximum error of %.
Corrosion resistant (stainless steel & Teflon).
Generation of pressure up to 6000 psi (400 Bar).
Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use much smaller particle sizes in the columns (<2 μm). These "Ultra High Performance Liquid Chromatography UPLC" systems work at up to 100 MPa