Transitioning From HPLC to UHPLC

Justin Ringling, senior scientist in Analytical R&D at Pfizer Consumer Healthcare, talks to contributing editor Tanuja Koppal, PhD, about his work, which involves developing and validating analytical methods using HPLC, UHPLC, ICP, and MS. He explains why he is choosing to transition some of his older methods from traditional HPLC to UHPLC and what factors need to be considered to ensure a successful transition. He also elaborates on the pros and cons of moving from HPLC to UHPLC, which he thinks are important to be aware of before the transition is made.

Q: What is your current role and how does UHPLC (ultra high performance liquid chromatography) play a part?

A: Our group develops and validates methods to support Pfizer’s Consumer Healthcare, which produces, among other things, over-the-counter medications and dietary supplements. I have been in this role for nearly two years and earlier I spent almost twenty years on the pharmaceutical side, working on small-molecule drugs across all phases of development. There is not much UHPLC being used on the consumer side when compared to pharmaceuticals, and we are hoping to change that by bringing in what I have learnt over the years. Currently we either co-validate or validate and then transfer methods to the commercial sites of the organization. One of the biggest hurdles has been getting the buy-in from commercial sites for using UHPLC, since all their methods have been set up for HPLC and the personnel have been trained on them. There would be some disruption in transitioning to UHPLC methods and some investment in capital for buying the UHPLC systems. However, today UHPLC systems are not that much more expensive than HPLC and I think the benefits far outweigh the additional costs.

Q: When were you first introduced to UHPLC?

A: My introduction to UHPLC was back in 2004 when Waters first came out with their UPLC system. I was then in a non-GMP (good manufacturing practices) environment, supporting discovery chemists who were synthesizing a lot of compounds. The HPLC methods that we used for screening those compounds took too long, about 45 to 60 minutes, on a 4.6 mm internal diameter (i.d.) column. The flow rates were high, the run times were long, we used up a lot of solvents, and it took a long time to analyze the samples. So when we heard about the Waters UPLC system that could reduce run time and improve resolution with smaller-particle columns, we went ahead and bought a couple of those systems. However, today I would recommend that if you have a good relationship with a vendor, to go ahead and first demo a system before buying one.

Q: Did you face any challenges transitioning from HPLC to UHPLC?

A: At that time the biggest problem was that UHPLC columns were very different from HPLC columns. The stationary phases that we used on HPLC were not available in the smaller i.d. columns, so we had to make some conversions in our methods. There would be a big problem if you had a validated method in HPLC and had to convert it to UHPLC and not have the same stationary phase to work with. However, that is not the case today. Vendors are now manufacturing columns with stationary phases in a variety of lengths, dimensions, and particle sizes. Hence, transferring from HPLC to UHPLC is much simpler today than it used to be in the past. Being in the non-GMP technology evaluation group, we tested and evaluated the new UPLC systems and methods for parameters such as linearity, precision, and reproducibility to make sure they could do what an HPLC system could. We then transitioned from 150 or 250 mm columns to 100 x 2.1 mm columns with sub 2μm packing. We wanted to make sure that we reduced our run times while retaining the peak capacity and ability to resolve all the impurities that may be in a sample. That’s where I saw the great benefit of UHPLC, a good compromise between run time and resolution, so the throughput goes up automatically.

Q: What are the challenges you face today?

A: On the consumer side, UHPLC has not been totally integrated into the various units. However, in the divisions that do have UHPLC systems, we need to show that we can successfully transfer methods while saving time and using less solvent. In some locations it is more expensive to use and to dispose of solvents due to environmental concerns, and in those cases the smaller i.d. columns will have a big advantage.

The vendors have done a great job in offering the same stationary phase in different particle sizes, and that gives you lot of different options to go back and forth between UHPLC and HPLC. In the past we have sometimes screened compounds in UHPLC and then back-calculated to HPLC methods too. I don’t recommend this, because it doesn’t always work, but today there is a better chance that it will work and you’ll get the same type of specificity and resolution for the peak you are trying to resolve.

Q: Are there any improvements you would like to see in UHPLC?

A: Not really, and we would like to bring in UHPLC wherever we can. In my previous group at Pfizer we had switched 80 to 90 percent of our systems to UHPLC. There will always be a special circumstance, like a size exclusion or chiral chromatography, that you cannot do on a UHPLC system. But today a lot of the chiral work has moved over to SFC (super critical fluid chromatography) or UPCC (ultra performance conversion chromatography), so HPLCs are not really needed for those applications either.

Q: Are there any issues that people should be aware of before transitioning to a UHPLC?

A: There are some questions I would ask before moving to UHPLC. One is on compatibility. What is the data acquisition system you are using currently, and is UHPLC compatible with the system? Sometimes if the system is not compatible you have limited functionality or access. If you are going to have to build a standalone acquisition system, then what is the cost of the software?

The cost of the service plan is another important factor, as the preventive maintenance and qualification for UHPLCs are going to cost a little more. When we transitioned to UHPLC we were able to drive more throughput from one system and hence ended up reducing the number of systems and our service costs. Theoretically you can replace two to three HPLC systems with one UHPLC and so reduce equipment space and save on utilities and service costs. The downside to that is if that UHPLC system goes down you may now be impacting two or three users.

You also need to compare all the UHPLC systems that are out there and the ability to transfer methods across those systems. If you are using contract labs for your work, make sure that the methods developed in your lab are transferrable to their systems. We did compare systems and methods side-by-side and made sure that we got the same results. This is important even if you are transferring across different sites within the company.

Q: What about the costs of columns and consumables?

A: You definitely save on the cost of solvents like methanol and acetonitrile, and you use less buffer too. But on the other hand, you require high-quality solvents and buffers to keep the UHPLC systems clean. UHPLC columns are slightly more expensive but if your throughput is higher, the costs do even out. However, you need to use guard or pre-filter columns to extend the column lifetime. You also have to filter or centrifuge the samples to remove particulates, as smaller diameter columns and tubings are more susceptible to clogging. UHPLCs are also less forgiving to tubing size and connections and the smaller tubings can increase the back pressures. So it’s best to keep the system operating at less than the maximum recommended pressure in order to minimize the wear and tear.

Overall, UHPLCs require more attention to buffers and solvents, the use of guard columns and tubing, and careful following of vendor recommendations for maintenance. In HPLCs we could swap tubing and other accessories but the UHPLC systems are more sensitive and may not support those changes. Finally, it’s important to choose a vendor that has a good history of customer support. Equipment downtime is not good, so customer support becomes very important.

Tips for Successfully Moving from HPLC to UHPLC
Source: Justin Ringling

Pros

Methods will have shorter run times, with equal or greater peak resolution
Higher signal to noise, due to peaks with higher plate count
Can use smaller internal diameter (i.d.) columns, with lower flow rates
Use less solvent and buffers
Generate less waste
Lower flow rates are ideal for the interface with Mass Spectrometry
Shorter run times equal more sample throughput or the reduction of systems needed
Less preventive maintenance (PM), qualification and maintenance costs
Less systems translates to less lab space needed for instruments
Ability to convert HPLC method to UHPLC easier today due to the wide variety of columns available with the same stationary phase in different particle sizes

Cons

UHPLC systems more expensive than HPLC systems
PMs and qualification are more expensive
Systems more susceptible to problems with particulates in the mobile phase or sample solutions
Need to use higher grade buffers and solvents to minimize possible interferences from contaminants

Areas to be aware of

UHPLC’s have different dwell volumes and should be considered when transferring methods across different UHPLC types
Ability to integrate the UHPLC system into the current data acquisition system should be considered
Consider the vendor’s ability and reputation in regards to providing system service and support
UHPLC vendors are continually expanding their instruments and columns to cover other specialized areas of HPLC, such as GPC/SEC and 2D