Preparative high performance liquid chromatography (HPLC), like its analytical counterpart, provides fast and sensitive separations of mixtures using high pressures and flow rates. Preparative HPLC is a valuable technique for purifying components from even complex mixtures, such as drug ingredients, food additives and other valuable industrial compounds. While utilizing the same selective separation methodology and much of the same instrumentation as analytical HPLC, preparative HPLC allows purified target compounds to be collected separately after elution and also requires different column dimensions and run parameters to achieve the desired result. The following tips can help improve your purification HPLC workflows, from strategies in method development and scale up to general best practices.
1. Think Beyond %B
It’s important not to underestimate the significance of mobile phase composition on separation resolution and purity. Developing a method for prep HPLC involves both selecting a stationary phase and determining a suitable mobile phase through trial runs and scouting gradients at the analytical scale. Both of these components will have a major impact on selectivity, which in turn dramatically influences resolution, more so than other factors such as retention and efficiency.1 While the stationary phase selection is key to this resolution equation, there is less flexibility to test a wide range of different stationary phases than there is to tune and optimize the solvent system, so understanding the different factors involved in mobile phase selectivity is especially important, and should go beyond just looking at strong solvent ratio (%B).
Methanol and acetonitrile are the most common strong solvents used in reverse phase HPLC, and the chemical differences between them can cause selectivity and resolution to differ depending on which one is used. For example, methanol is polar-protic while acetonitrile is polar-aprotic with a stronger dipole moment.2 Due to these differences, if you’re having trouble resolving peaks with one solvent, switching to a different strong solvent could do the trick. Just keep in mind that acetonitrile has a greater elution strength than methanol, meaning greater %B of methanol will be needed to elute compounds in the same amount of time as acetonitrile.3 Additionally, methanol and acetonitrile can be mixed together to further fine-tune the selectivity of the strong solvent.
Another tool you have to work with when designing your solvent system is pH, which is controlled with the addition of buffers like trifluoroacetic acid (TFA) or ammonium salts. Acidic compounds are deionized in a mobile phase about 2 pH units below their pKa, while basic compounds are deionized at about 2 pH units above their pKa.4 Deionized compounds will be more strongly retained, which can result in better separation of coeluting analytes.5 Thus, adjusting the pH based on the pKa of the target compound or compounds is another strategy you should take advantage of when optimizing your method. Ensure that the buffers used are volatile enough to be easy removal after fraction collection, and that the pH of the mobile phase falls within the acceptable range for the column you are using. Typical silica-based compounds will usually tolerate a pH range around 2-8, while non-silica columns or silica columns with specially designed ligands will provide more flexibility in terms of buffer use.4
2. Plan for Scale-up Ahead of Time
Once you have perfected your mobile phase, tuned parameters like flow rate, temperature and gradient, performed loading studies for your sample and determined the dwell volumes of your prep and analytical systems, you will now need to scale your method up from an analytical column to a preparative column using a series of well-established equations. The availability of scale up calculators can greatly simplify this process, but planning in advance can make scale up even easier and ensure a smooth transition to the preparative scale.
The path to easy scale-up begins with column selection, as there is a lower chance of unexpected results when the analytical and prep columns are as similar as possible. For any analytical column you use for method development, you should seek to ensure there are preparative columns available with the same stationary phase chemistry, particle size and column length.6 When these elements are kept the same at both the analytical and preparative scale, scaling up is much more straightforward with less “moving parts,” reducing the chances for error and unexpected hurdles. It is possible to accommodate for a difference in particle size by maintaining the ratio between column length and particle size (L/dp) between methods, but identical column chemistry is especially key for maintaining separation beformance between the different columns.
It is also important to consider the capabilities of your preparative system hardware while developing your method at the analytical scale. You do not want to set up the perfect analytical-scale method only to hit a wall when attempting to scale-up, due the pressure and injection needs of the new method exceeding system capabilities. It can be useful to consider what your goals will be at the preparative scale (such as yield, purity and throughput), take into account the capabilities of your pump, autoinjector and other hardware, and work backward to tailor your method within parameters that will suit system capacities. If you find that your goals can not be realistically met within these limitations, it may be time to consider some system upgrades.
3. Take Special Care When Working with Large Solvent Volumes
By its nature, preparative HPLC involves a much higher volume of solvent being used, increasing the safety risks associated with organic solvents like acetonitrile and methanol.7 Therefore, extra precautions must be taken to prevent and manage spills and leaks. It is highly recommended to use an HPLC system with built-in leak detection, and fortunately most modern systems are equipped with leak sensors. However, it is still important to remain vigilant and regularly inspect the system for possible leaks, as even miniscule leaks can become a larger problem if the issue is not resolved. Regular maintenance should be performed to mitigate issues that can lead to leaks, such as loose fittings or build-ups and blockages. If you notice problems during a run, such as a drop in pressure or distorted peak shape, check to make sure that leakage is not the culprit.
Additional precautions include not leaving the preparative HPLC system running unattended, placing the fraction collector under a fume hood and drying down fractions quickly to prevent evaporation of solvent vapors into laboratory air,7 and placing trays underneath areas where leaks are most likely to occur, such as the pump plunger head, to facilitate easy clean up in case of a spill.8 It is also recommended to position equipment away from potential ignition sources and keep waste containers grounded to prevent potential ignition from static electricity generated by the HPLC system.9
4. Avoid Common Mistakes When Evaluating Collected Fragments
Once your purified fractions are eluted and collected, you will want to evaluate them to determine their purity, yield and activity. There are several common mistakes that can occur between collection and analysis that may lead to unexpected or undesirable results. Ensure you avoid these errors in order to accurately determine the quality of your purified sample and whether steps need to be taken such as repurification or further method optimization.
One issue that can distort results is the testing of an unrepresentative sample from the fraction container. This can occur if the sample in the container is not well-mixed, due to the formation of concentration gradients across the fraction.10 Without mixing, a small sample taken from one part of the container could yield different results from that taken from a different part of the container, and will not be representative of the complete fraction. The solution to this problem is simple: mix the fraction prior to analysis.
Another problem is the crystallization of samples that are more soluble in a strong solvent, like DMSO, but not in the mobile phase used for separation. If you draw a liquid sample from the collection vessel for purity testing after crystallization has occurred, it will not be representative of the fraction; for example, if an impurity in the sample crystallizes, the sample will appear much more pure than it really is, or vice versa if an impurity is soluble and the target analyte crystallizes. Therefore, you want to ensure that crystals have not formed in the liquid phase you are using for purity testing, and may need to redissolve the sample in a stronger solvent before purity testing is performed.
Lastly, the active compound in the sample could decompose between collection and testing, including during the dry-down process. This can lead to discrepancies between testing performed directly from the fraction container and tests performed after dry-down. Thus, it is advisable to evaluate the fractions for purity and activity both before and after dry-down to ensure nothing is missed, and that the results are truly representative of the purified product.10,11
References
- "Improving Prep HPLC," Webinar Presented by Josh Lovell, Teledyne ISCO (2020). https://www.youtube.com/watch?v=FbyeASJP3ck
- "The Role of Methanol and Acetonitrile as Organic Modifiers in Reversed-phase Liquid Chromatography," Article by Trevor Hopkins on behalf of Advanced Chromatography Technologies, Chromatography Today Knowledebase (2019). https://www.chromatographytoday.com/article/hplc-uhplc/31/advanced-chromatography-technologies/the-role-of-methanol-and-acetonitrile-as-organic-modifiers-in-reversed-phase-liquid-chromatography/2507
- "7 Key Differences in the Use of Methanol and Acetonitrile," Shimadzu. https://www.shimadzu.com/an/service-support/technical-support/lib/methanol-acetonitrile.html
- "Preparative Liquid Chromatography Primer: pH," Waters. https://www.waters.com/waters/en_US/Preparative-Liquid-Chromatography-Primer/nav.htm?cid=134929152
- "Utilizing Mobile Phase pH to Maximize Preparative Purification Efficacy," Blog Post, Phenomenex. https://phenomenex.blog/2017/08/22/mobile-phase-ph-improve-preparative-purification/
- "Preparative Liquid Chromatoraphy Primer: Method Scale-Up," Waters. https://www.waters.com/waters/en_US/Preparative-Liquid-Chromatography-Primer/nav.htm?cid=134929155
- Schulenberg-Schell, Helmut; Tei, Andreas; Rieck, Florian; Guilliet, Ronald. (2019). Principles and Practical Aspects of Preparative Liquid Chromatography: A Primer. Agilent Pub No. 5994-1016EN. https://www.agilent.com/cs/library/primers/public/primer-preparative-liquid-chromatography-5994-1016EN-agilent.pdf
- "About Preparative HPLC," Shimadzu. https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/hplc/prep1.html
- "Beware Static Electricity Generated by Flowing Liquids," Shimadzu. https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/lib/lctalk/14/14lab.html
- Huber, Udo. (2003). Automated fraction re-analysis — does it really make sense? Agilent Pub No. 5988-8653EN. https://www.agilent.com/cs/library/applications/5988-8653EN.pdf
- "Preparative Liquid Chromatography Primer: Fraction Analysis," Waters. https://www.waters.com/waters/en_US/Preparative-Liquid-Chromatography-Primer/nav.htm?cid=134929161