Prep LC 101: Scaling up with Preparative HPLC

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Prep LC 101: Scaling up with Preparative HPLC

Team TFS
Team TFS

preparative-hplc-column-brochure-cover-imageEver heard the term “prep LC” and have not been entirely sure what it meant? You’re not alone! We’re here to help you understand what preparative liquid chromatography is and why it’s important.

Let’s start with the basics and work our way up to things you should consider when choosing the right column:


What is preparative HPLC and where is it used?

Preparative HPLC, also known as prep LC, is used for the purification and isolation of molecules. Common industries which use prep LC are the pharmaceutical, biopharma and associated chemical industries.


How is preparative HPLC different to analytical HPLC?

As stated above, preparative HPLC is concerned with the purification and isolation of molecules. The goal of analytical HPLC is different in that it is predominantly used for the identification and gaining quantitative and qualitative information about the compounds in your sample. For a more detailed review of the differences between these two techniques, please see this informative article.


Why is prep LC important?

In these industries where prep LC is used, the production of highly pure compounds are extremely important as the production of side products or impurities can have a huge effect on the intended chemical activity and toxicity. They also want these pure compounds in large quantities for a commercially viable final product.


What does the general prep LC workflow look like?

Typically, scientist begin their work at the analytical scale to synthesize new compounds in small test tubes, refining their reaction conditions producing only a few micrograms of material, e.g. for enzymes in biopharmaceutical applications, or a few milligrams, e.g. for structural or identification of synthesized or natural products. At this stage, the scientist can use an analytical column to purify the small amount of crude material they need at this stage to confirm the target molecule’s characteristics are what they desire. The analytical scale also means this can be done quickly and without much waste. They can use these screening results to go back to the drawing board and tweak their synthesis.

The scientist continues to refine their reaction to hone in on the perfect recipe for their target compound, with the goal that their compounds may be the winning compound they’ve been searching for! The analytical purification also provides them with vital information about how to separate their target compound from all the other components in the sample.



Why would you want to scale up?

In pharmaceutical environments, as a drug target compound moves further in the drug development pipeline, there is a need to “scale up” the reaction to create more volume of the targeted compound – think gram quantities, which may be required for standard and reference compounds for pharmacology and toxicology testing procedures and also the final product itself.



What needs to be considered from analytical to prep LC?

One of the biggest things to remember about going from analytical to preparative scale LC is that at the prep LC stage, it’s no longer about efficiency, it’s about yield.

    • How much of the purified product did you make?
    • Is it enough to run all of your analysis to confirm that you actually have the product you intended without impurities or side products?
    • Can I purify enough of my compound (and quickly enough) for it to be commercially viable, for example, as a monoclonal antibody therapeutic?

As you can imagine the stakes can be high and you need to have high yields to be considered successful. In order to do that you really do need to consider all aspects of your workflow and see how you can optimize each area to achieve the highest yield.

One way to try to maximize your yield (and sanity) is to use the same media in your analytical and prep LC column. By using the same media, you keep the consistency of chemistry, particle size and quality. Prep LC columns are larger, so there are some differences in fluidics in the column that need to be considered, but by minimizing the other variables like the chemistry and quality of your media, it makes the transition from analytical to preparative LC easier.

Ideally you would also want to perform both your analytical and preparative LC on the same instrument and software. This is not always possible depending on the yield required and the sample size; in analytical chromatography think 1-2 µl sample injections, but preparative LC can sometimes be hundreds of liters! If possible to run on the same instrument, this has advantages as again this reduces additional variables such as different pump operating parameters, miss-matched gradient-delay volumes, etc. You can then also use the same Chromatography Data System (CDS) software which means not having to learn and train on two different packages.



What does Thermo Fisher Scientific offer to scientists who are ready to scale up?

We’ve recently expanded our preparative HPLC column portfolio to offer prep LC columns in more than 40 stationary phases in an assortment of column dimensions. This allows you to utilize the same chemistries between your analytical and preparative separations, which reduces a variable in your scale up from analytical to prep.

By offering high quality and cost-effective columns, with so many chemistries to choose from you can bet you’ll be able to find a prep column to suit your needs. What’s even better is that if you don’t find the column you’re looking for, just reach out to your local sales rep as there is a good possibility we will be able to get you what you need!

While using a guard column can help extended the life of your preparative HPLC column, we are also offering some chemistries to be packed in our XtendedLife hardware which uses Dynamic Axial Compression (DAC) to keep the bed packed and uniform, improving lifetime and robustness.

Check out our full offering online at 

Now that you’re ready, use our free online tool to determine best starting conditions for scaling methods between analytical to preparative HPLC columns. The tool will calculate the new injection volume, flow rate, gradient table and expected eluent usage so you can get your new method up and running faster.

Get started at