High Performance Liquid Chromatography (HPLC), otherwise referred to “High Pressure Liquid Chromatography”, is a technique used to separate, identify, and quantify each component in a mixture.
The principle of chromatography is that, if a mixture of molecules is injected and percolated with an adequate solvent, (called eluent), through a chromatography column packed with beads of an adequate adsorbent, some molecules, having more affinity, will take longer than others to pass through a chromatographic column. More precisely, chromatography can best be described as a mass transfer process involving adsorption of analytes as they flow through a column filled with stationary phase, leading to the separation of the components according to their retention time.
In HPLC, the “High Performance” is related to the fact that very small beads (typically 5 to 50 µm of diameter) of adsorbent are used. There are two consequences…
Firstly, the band of distribution of the products in the eluent will be thinner (in other words, chromatographic peaks will be thinner), and secondly, the flow of eluent through this packed bed of small beads will generate high pressure. This is why HPLC is often called high pressure liquid chromatography. Pressure is the consequence, not the cause!
Preparative HPLC uses the principles of analytical HPLC, but it is used to produce purified molecules. Therefore, in preparative HPLC the goal is to inject and produce the maximum amount of targeted product per unit time. It is an important technique used for manufacturing of pharmaceutical and biopharmaceutical APIs & intermediates.
HPLC offers a higher separation performance (higher purity and higher yield), ease of scale up, and reduces the number of fractions collected, which need to be analyzed individually.
Flash chromatography is a separation technique like HPLC that is operated at low/medium pressure. The stationary phase used in flash chromatography is composed of a large particle size, inducing lower back pressure but with a lower efficiency compared with HPLC.
Batch chromatography is a sequential process taking place in one single column, composed of injection, elution and collecting steps. Continuous chromatography is based on continuous feed injection, and continuous collection, of target and unwanted streams, taking place in a series of columns connected in a loop.
In a batch process, you must wait for all products to be eluted at the outlet of the column before injecting for a second run. It is a time-sequenced collection of products. In continuous chromatography, the injection takes place while separation and collections are ongoing, at different places in the loop of columns.
With batch chromatography, the process is versatile, easy to implement and the initial investment is lower. Batch is particularly suitable for smaller quantities & complex mixtures when more than one purified product fraction is to be collected.
Continuous chromatography is more productive, optimizing the use of the stationary phase. Typically, the production gain can be up to 3 times compared to an equivalent batch process, and the consumption of eluent is proportionally lower. It is suitable for medium to large scale. Implementation of a continuous process requires more development & characterization work.
Continuous chromatography is a technique limited to binary separation.
MCC (Multi-Column Continuous) chromatography is a generic technique that notably includes SMB (Simulated Moving Bed) & Varicol®. All of them are continuous processes.
In an SMB, a true counter-current between the liquid and the solid phases is simulated by the periodic, synchronous shifting of inlet and outlet streams along the loop of columns.
In the case of Varicol®, Novasep’s optimized continuous chromatography process, the shifting of inlet and outlet streams isn’t synchronous, enabling a better utilization of the adsorbent, thus enabling the use of a lower number of columns and an improved productivity.
Cyclojet® is a process using a single column based on the well-known concept of Steady State Recycling (SSR). Like SMB or Varicol®, it is a binary separator, but it is a single-column process like HPLC.
In a Cyclojet®, purified fractions are collected while fresh eluent is injected, and the part of the outlet stream that contains both products is recycled into the column. This enables to increase the productivity and reduce eluent consumption compared with HPLC. Cyclojet® is particularly suitable for difficult separations.