Preparative Chromatography Instrumentation

Preparative Chromatography Course

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1 - Fundamentals of Preparative Chromatography 

2 - Preparative Workflow 

3 - Preparative Chromatography Instrumentation 

4 - Key Considerations: Part 1 

5 - Key Considerations: Part 2 

6 - Column Selection 

7 - Automated Preparative Purification Instrumentation - UFPLC  

8 - Automatic Analytical to Preparative Scale Up 

9 - Triggering Fractionation 

10 - Preparative SFC 

11 - Course Summary & Test 

preparative chromatography online course

Preparative Chromatography Instrumentation 

Welcome back to our Preparative Chromatography Online Course, where in this unit, we will be looking at the different parts that make up a prep instrument.  

If you have used HPLC’s before, you will be comfortable with most of the modules that comprise a prep LC system. We need to have: 

  • A pumping unit capable of pushing the mobile phase around the instrument 
  • A means of introducing the sample into the mobile phase 
  • A mode of separating the compound of interest from impurities, side reactions or starting material 
  • A suitable technique for detecting the compounds based on the physicochemical properties of the analyte 
  • A method of collecting the fraction of interest 
 
Preparative chromatography workflow showing pump, sample injection, detector options (UV/PDA, fluorescence, ELSD, MS), and fraction collection modules selected according to sample and fraction requirements

Figure 1. Preparative Chromatography System Components

Click on each of the module images below to find out more information. 

Loading Amount
The pump needs to be capable of reliably delivering the prescribed flowrates to accommodate the different scales (i.e. semi micro 0.001-10 mL/min such as the LC-40D, through to large scale 150 mL/min like the LC-20AP pump).  Sometimes, pumps can be adapted such as by adding multiple solvent lines to enable gradient analysis, and recycling kits can be used to recycle isocratic solvents. 

 

Injection Volume Sample Amount
An autosampler or liquid handler is required to automatically inject the sample into the flow path. These modules must have low carryover, reliably inject specific volumes and manage highly repetitive tasks. Automated systems are required to maximise throughput in laboratories and are superior to manual injectors. An autosampler can simply inject the mixture into the prep system, whereas a liquid handler has dual purpose with sample injection and fraction collection functions, thereby saving space.
Character of Compound
Detection mode is selected based on the physicochemical properties of the compound of interest. The most used detectors are mass spectrometry and photodiode array (PDA) based on the chromophore of the compound. Alternatives include fluorescence and ELSD. It is possible with a single quadrupole MS detector to use this as a trigger to collect a specific compound, known as mass-directed prep, rather than collecting a fraction based on time. Mass directed prep will be discussed in greater detail later in the course.
Fraction Amount
The aim of prep HPLC is to collect a pure fraction, which therefore means the system needs to either have a fraction collector or a liquid handler. Criteria for selecting one is usually based on throughput requirements. For example, the FRC-40 can accommodate up to 540 10 mm test tubes, which can be expanded to 3,240 test tubes via six units which can be controlled in one system.  

 

Additional modules can be included, such as column hubs, recycling valves, solvent selection valves and degassers. These can enhance the functionality of the system, but the main units are all described above.  

In the next unit, we will continue our journey to learn more about the critical questions to understand such as impact of solvent and temperature on chromatography, as well as loading studies.  

Best regards, 
 
Your Shimadzu Chromatography Team 

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