This almost sounds like a line from Shakespeare, but HPLC or high-performance liquid chromatography and ultra-high-performance liquid chromatography (UHPLC) came a little after the ‘Bard of Avon.’ Both HPLC and UHPLC are common techniques in the analytical laboratory, but recent years have seen a shift from HPLC to UHPLC. Why is this? What are the differences between the two and how do you transfer a method from HPLC to UHPLC? These are topics I will briefly discuss in this article.
Differences between HPLC and UHPLC
There are five main differences between HPLC and UHPLC:
Particle sizes – In HPLC particle sizes of the stationary phase are typically in the order of 3-5 µm, whilst UHPLC is characterised by particles of 2 µm or less.
Column dimensions – As with particle sizes there is a corresponding reduction in column dimensions with UHPLC. A typical HPLC column has an internal diameter of 4.6 mm and a length of 250 mm, whilst a UHPLC column has internal diameters of 2.1 mm or less and is much shorter, 100 mm for example.
Flow rates – UHPLC runs at much lower flow rates than HPLC, for example 0.2 – 0.7 ml/min against 1-2 ml/min respectively.
Backpressure – With the smaller particles and reduced column diameter then this manifests itself in to higher backpressures in UHPLC compared to HPLC. HPLC instruments typically operate at maximum pressures of 400-600 bar, whilst UHPLC instruments can operate at up to 1500 bar in the case of the Thermo Scientific™ Vanquish™ Horizon UHPLC System.
Detection parameters – Narrow peaks are produced with UHPLC, requiring a detector that can keep pace and provide the required number of data points per peak for detection. Most modern detectors, though, are capable of detection speeds of up to 250 Hz, which is sufficient for both HPLC and UHPLC.
These are the common differences between HPLC and UHPLC, but they can also be broadly separated by application area. HPLC is commonly used in routine environments, whilst UHPLC is more common in research and development, but this is not exclusive.
Why use UHPLC? (And why not?)
As alluded to earlier in the article, there has been a gradual shift to UHPLC form HPLC over the last decade. So why is this? There are a number of reasons:
Speed and throughput – Smaller columns and particle sizes, low system dispersion, with a pump pressure to match, means that separations can be achieved in a fraction of the time compared to HPLC thus offering a higher sample throughput. This application note provides a nice example of this: the separation took 2.5 minutes using a column with 4.0 µm particles, but when reducing this to 1.5 µm particles the separation was achieved in 1.5 minutes.
Better resolution – The use of smaller columns and particles also results in better resolved and sharper peaks to give better resolution and peak capacity.
Lower costs – As the flow rates are smaller and the separations fast, then solvent usage is reduced along with the associated disposal costs. The speed also offers the higher throughput so the instrument investment is fully utilised.
If UHPLC offers lower costs, increased speed and throughput and better results then why hasn’t everyone switched over from HPLC? Possibly because of two reasons: firstly, to achieve the higher backpressures and maintain accuracy and precision at lower flow rates then higher performance pumps and components are required which add cost to the instrument. However, even with a relatively low sample volume the higher throughput and reduced solvent costs will soon compensate for the initial higher investment in a UHPLC instrument. Secondly, as HPLC preceded UHPLC, most methods have been developed on HPLC and to transfer these over to UHPLC takes time and resources to optimise.
Transferring Methods to UHPLC
Transferring a method from HPLC to UHPLC is not as daunting as many fear if a few considerations are taken. Many methods can be quickly and easily transferred, and as shown above, the benefits of moving to UHPLC can be considerable. The topic of method transfer is an article in itself (and more), so here I would like to direct you to two on-demand webinars covering this topic entitled ‘From HPLC to UHPLC: How Fast Can I Be, and Is the Fastest Always the Best’ and ‘From HPLC to UHPLC: What are the Instrument Requirements and Pitfalls?’ These on-demand webinars are available in both English and German languages.
Can I Have Both?
This is a question that gets asked frequently: If I have a UHPLC instrument, can I also still use it for HPLC methods? The answer is simply ‘yes’ as there should be no technical limitations to prevent this. One consideration though, in a small number of cases, is the tubing as this is likely to be narrow UHPLC tubing that could need to be exchanged for standard HPLC tubing depending on the sample solvent used. One very interesting application note caught my attention recently that showed it was possible to do both HPLC and UHPLC on the same instrument in parallel. Using the Dual LC workflow on the Vanquish Duo UHPLC System means you can have both, and at the same time.
In summary, there are many advantages to switching from HPLC to UHPLC, but frequently the barriers are the initial investment and the transfer of methods. However, the lifetime cost of UHPLC is typically lower and there are many sources of help and advice to help transfer methods from HPLC to UHPLC. Finally, a UHPLC system does offer some flexibility to perform both HPLC and UHPLC applications and with the Vanquish Duo you can run both in parallel.
LC Method Transfer Calculator: Use this online calculator to transfer methods from HPLC to UHPLC conditions. Calculate the new gradient table, adjust sample volumes and run times if applicable.
Method Transfer Whitepaper: This whitepaper looks in detail at the instrumental parameters HPLC users need to consider during transfer of an analytical HPLC method between different instruments.