Ransom Jones
At some point in every scalable synthesis, you need to develop a reliable method to purify the final compound. This step can feel overwhelming, often consuming both time and resources. However, you can reduce these challenges by building a transferable HPLC or UPLC method from the start. While moving from HPLC to flash chromatography is not always a direct one-to-one process, you can take advantage of the similarities between the two techniques. By designing with scalability in mind, purifying large-scale batches becomes much more manageable.
The first step is to focus on column chemistry. Choosing an HPLC column with a stationary phase that closely matches the chemistry of your larger flash column helps replicate the separation environment. This way, the results you see in HPLC are more likely to carry over when you scale up.
The second step is to develop gradients using column volume (CV) instead of time. Column volume refers to how much mobile phase (in milliliters) fits inside a column. By basing gradients on CV, you automatically account for differences in column size, geometry, and packing density. As a result, the method becomes transferable across multiple column formats, making scale-up far smoother and more reproducible.
There are two common ways to calculate column volume. The first method—and often the most consistent—is to calculate the volume of the cylindrical column itself and then adjust for packing density. To do this, you can apply one of two equations.
𝑪𝑽= 𝝅𝒓𝟐𝑳∗𝟎.𝟕
a. Where r is the radius, L is the length, and 0.7 assumes 70% packing density.
In the first equation, a packing density of 70% is assumed, since this is the standard for most modern columns. However, if the actual packing density is known or differs greatly from 70%, then the second equation provides a more accurate calculation.
𝑪𝑽= 𝝅𝒓𝟐𝑳∗𝒅
a. Where r is the radius, L is the length, and d is the packing density.
Although not every manufacturer includes packing density or pore volume on their COAs, some do. If they don’t, you can still calculate it from pore size, particle size, and other material details.
The second way to estimate column volume is much more hands-on: measure how long it takes for a non-retained compound to elute at a set flow rate. For example, on a 4.6 × 150 mm 5 μm C18 column, a highly polar compound elutes in 1.3 minutes at 1.5 mL/min. Multiply retention time by flow rate, and you get a column volume of 1.95 mL. This method isn’t exact, but it often proves more useful because it reflects real-world column variability. In practice, your choice comes down to preference and consistency.
Building a Transferable HPLC Method
Start by selecting an HPLC column that matches the chemistry of the flash column you’ll use later. Base your choice on the target molecule’s structure and chemical properties. Also decide what purity level you actually need. In process chemistry, some impurities are easier to remove in later steps. Don’t waste time chasing extreme purity if future chromatography will handle it anyway.
For this example, we use a C18 4.6 × 150 mm 5 μm column and calculate its column volume at 1.74 mL.
Designing the Gradient
Gradient design depends on the compound, but a good starting framework is simple:
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Start with an isocratic mix for 1–2 CVs.
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Shift to a ramping gradient over the next 5–10 CVs.
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Finish with a re-equilibration step for another 1–2 CVs.
For example, run 80:20 water/methanol for 1 CV, ramp to 80% methanol by CV 10, and hold until CV 12. By working in column volumes instead of time, you make the method fully scalable across different column sizes and flow rates.
Scaling to Flash Chromatography
Now transfer the method to a 25 g C18 21 × 117 mm flash cartridge, which has a column volume of 28 mL. Because we built the gradient in CVs, the HPLC method scales directly. Modern flash systems make this even easier since many come preprogrammed with CV values for their cartridges.
Final Tips
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Match your solid-phase column to a compatible flash cartridge.
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Stick with simple mobile phases and avoid buffers or ions unless required.
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Use UPLC to speed up development, as long as you continue to scale by CV.
In short, when you build methods with scalability in mind, you save time, lower costs, and make large-scale purification much easier.
About the Author – Ransom Jones
Analytical Scientist at Mikart LLC, focusing on product development for drug substances and drug products. MSc in Pharmaceutical and Biomedical Sciences from the University of Georgia. Background in nucleoside synthesis for antiviral purposes. Extensive knowledge in the drug development pipeline, including API synthesis, toxicology in-vitro testing, formulation development, FDA regulations (cGMP/21 CFR 210/211), pharmaceutical analysis, and more.
Graduate work focused on medicinal chemistry, specifically on antiviral nucleoside analogs. Included leading projects, complex synthetic organic chemistry, analytical development (NMR, HPLC, LC, Mass Spec), and more.
Experienced in solid dosage formulation development, including acetaminophen tablets and piroxicam capsules. Hands-on experience with rotary tablet press, dissolution, TGA/DSC, disintegration, HPLC, spray dryer, and more.