Frequently Asked Questions about High Performance Liquid Chromatography - HPLC

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.

To learn more about the purity level Novasep can achieve:

A batch may be defined either by the batch size of the crude material to be purified or by the capacity of the pure product collection tank/receiver. It is applicable to both batch and continuous processes.  

In batch processes, a batch typically contains several successive injections performed under automatic and controlled conditions.

 In preparative chromatography, the productivity of a process is often expressed in KKD, which is defined as kg of purified compound (or sometimes kg of crude compound to purify) per kg of stationary phase per day. 

Productivity is related to the complexity of the crude mixture to purify, such as selectivity between target compound and impurities to remove, retention times of the components, loadability, the specification required, and type of process (SMB/Varicol^®, Cyclojet® or HPLC).

The first step of a chromatography project is to select the chromatographic phases (stationary and mobile) to ensure that the target product can be separated from the impurities. This is called the screening/scouting step and is performed using analytical scale columns.

The next step, still at laboratory/analytical scale, consists of developing & optimizing the process in terms of loadability and productivity.

Finally, a robustness study, Proof of Concept batch (POC), stationary phase aging study and a stability study would also be performed.

Although at small scale, these studies are operated in similar conditions as in production, using industrial-grade solvents and stationary phases. This scale-down of operating conditions enables the predictability of the future scale-up.

To learn more about the development of chromatographic purification:

To learn more about the quantity required for the development of a chromatographic project:

The key factor is the ratio between the cross-sections of the columns. Injected quantity, flow rate & stationary phase quantity are all multiplied by this factor to do the scale-up. Column length, stationary phase characteristics, temperature and eluent composition is kept the same for each scale.

To discover a case study presenting which column size is needed for purifying 100kg within one to three months:

Novasep operates 50 mm up to 1200 mm id columns (from hundreds of grams batches to 100’s tons/year production).

As implied in the thermodynamic equilibriums between solute, stationary, and mobile phases, the temperature modifies the selectivity between the peaks. A higher temperature results in thinner peaks (more theoretical plates on the column), but generally the selectivity (distance between peaks) is reduced, so there is generally an optimum to find. Maintaining a stable temperature can be critical in some separations.

Novasep has more than 30 years of experience in contract development and services using industrial chromatography. In many cases, this involved the successful transfer of a process from a third party to our sites, and between our sites.

We can offer development on our Pompey (near Nancy), Chasse-sur-Rhône (near Lyon) & Le Mans sites (HPAPIs) in Europe, on Boothwyn (near Philadelphia) site in the US.

To learn more about the stationary phase we use for High Performance Liquid Chromatography (HPLC): 

For a bed length of 25 cm, and taking into consideration a silica packed stationary phase density of 0.6, amounts of stationary phase are...

To learn more:

To discover how we store the stationary phase:

The lifetime of the stationary phase really depends on the process and the quality of the crude material but usually, several years lifetime can be expected for commercial productions. Lifetime studies would usually be carried out at the development stage.

To learn more about the reutilization of the stationary phase:

To learn more about the use of the stationary phase:

Discover how SMB works in this video:

To get more information about Varicol^®, visit our dedicated webpage.

Novasep designs & manufactures chromatography equipment and is the recognized leader in preparative HPLC, SMB and Cyclojet^® technologies.

The equipment design is continuously improved, customized and adapted based on both customer's feedback and Novasep's experience acquired through more than 30 years of Contract Development and Manufacturing Organization (CDMO) activity. We have acquired extensive experience in efficiently validating equipment.

Cyclojet^TM is a steady-state recycling process which has characteristics that are intermediate between batch HPLC and SMB, and using a single column.

To discover how we pack columns:

To discover how we clean columns and equipment between projects:

To learn more about the maximum pressure to be applied on Novasep systems:

Discover what is the maximum column diameter and the maximum flow rate for HPLC :

A longer and thinner column will certainly show a better efficiency. However, it will have very high pressure drop as the pressure is proportional to the column length while it is inversely proportional to the column diameter. In HPLC, small silica beads will compensate the loss of efficiency due to a shorter bed height compared to a flash chromatography with a bigger silica.

Novasep has fully integrated evaporation and solvent recycling systems in the global chromatography unit operation. We are typically using fully automated falling film evaporators and in some plants, rectification, distillation or solvent stripping technologies.

Novasep has designed proprietary control systems integrating full solvent recycling. This enables to operate large scale systems with minimum amounts of solvent storage, generally corresponding to only a couple of hours of autonomy.

Very often, binary mixtures are used in chromatography processes. When these mixtures are not 100% recycled, the recycled eluent generally gets enriched in the solvent having the lower boiling point.

Novasep has developed proprietary technologies to control and readjust the composition of recycled solvents to a very high degree of precision (down to less than +/-0.05%), to ensure perfect reproducibility of the automated chromatography process.

Yes, Novasep has multiple cGMP facilities operating industrial chromatography.

Our custom purification facilities of Mourenx, Chasse-sur-Rhône and Pompey have been successfully inspected by the FDA and the European authorities (ANSM) recently. Some facilities are also approved by the Japanese PMDA.

Learn more about our quality assurance track record in our brochure.

Find out more about the solvent we can work with:

Find out more about the level of solvent we can recycle:

HPAPIs, and, in particular, ADC toxins, are often very complex molecules. Hence chemical processes are delicate and often generate closely related impurities that can only be eliminated by HPLC.

Novasep’s Le Mans site has more than 30 years’ experience in industrial chromatography for HPAPIs & cytotoxic compounds (including development, scale-up and production).

As we do for all HPAPI operations, we have implemented all appropriate monitoring and safeguards to ensure that we can safely operate HPLC to purify products classified up to and including Safebridge band 4.

HPLC is a liquid-solid separation process. All components must remain in solution to avoid precipitation or settlement inside the column, which can generate pressure drops or separation failure. Non-soluble material must be removed beforehand. 

High solubility brings high performance.

Discover how we test our column after packing :

Discover how we define fraction collection: