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How U is Your UHPLC?

Ultra High Performance Liquid Chromatography (UHPLC) is a powerful tool for increasing HPLC sample throughput, chromatographic efficiency, and sensitivity. However, how simple is it to transfer between HPLC and UHPLC applications?

There are several parameters associated with both the chromatographic method and the system hardware that need to be considered when switching to UHPLC. This article will give you some practical tips to make sure your UHPLC is as U as possible and that you are getting the best possible chromatography.

Tips for Reducing System Extra Column Volume

Extra column volume is the total volume contributed by all system components and capillary tubing from sample injection to detection, which are not directly involved with the separation process. Extra column volume is the predominant factor in loss of efficiency when using narrow i.d. columns due to the contribution of peak dispersion (Figure 1); therefore, it is very important to minimize extra column volume throughout the system.

 

Figure 1: Effect of extra column peak dispersion on peak efficiency and resolution.

 

This can be done by using short lengths of tubing with a reasonable internal diameter.  Peak dispersion is related by the Aris-Taylor equation (Eqn 1) to flow rate (F), tubing length (L) and internal diameter (i.d.), and the molecular diffusion coefficient of the analyte in the mobile phase (DM); with tubing i.d. having the greatest impact. 

Therefore, it would seem that to reduce peak dispersion we should be using the narrowest possible tubing.  However, using very narrow tubing comes with a compromise. 

When tubing i.d. is decreased the pressure required to move mobile phase through the tubing increases - for example, plumbing a system with 0.002” tubing and running a flow rate of 3 mL/min requires a pressure input of 1500 bar (and this is only to move the mobile phase through the tubing) leaving no pressure capability in the system to be able to install a column and run a separation (Figure 2). 

A good compromise between dispersion and pressure is to use 0.005” tubing, which at the same flow rate would only require 200 bar of pressure. 

 

Figure 2: Pressure/diameter relationship for tubing.  Assume: Viscosity of water at room temperature and L = 50 cm.



It is also essential to use the correct column end fittings - ideally zero dead volume fittings should be used, with many manufacturers providing specialist UHPLC fittings.  Incorrect fittings can lead to leaks or an increase in extra column volumes resulting from voids (Figure 3). 

Figure 3: Differences in fit of column end fittings.

If you are using an in-line filter (and they are important and can be considered one of the most high value pieces of the system, as they can prevent blockages in guard columns, the analytical column, tubing etc.) make sure it has a low dead volume. 

Look carefully at the injection system, for example, for a flow through system look at the apparently small contribution from the needle seat capillary.  It is also worth considering the injection volume, which should be matched to the column geometry. 

A good rule of thumb is to maintain the injected volume between 1-5% of the column dead volume.  Most UHPLC experiments are performed with a 50 x 2.1 mm column
(V0 = 120 μL), the injected volume should, therefore, be between 1 and 5 μL, to limit peak dispersion.


Tips for Minimizing Frictional Heating

Frictional heating is the viscous heating of the mobile phase as it passes through very small diameter particles; causing a rise in temperature over the length of the column (Figure 4).

 
Figure 4: Oversimplified temperature profile in an HPLC column with no temperature control.  The temperature increases in the flow direction (left to right); each temperature zone is identified with a different colour.

Temperature impacts separation efficiency, retention, and selectivity, therefore, it is imperative to accurately and reproducibly control the column temperature. 

Frictional heating effects can be minimized through the use of smaller i.d. columns (2 mm is the sweet spot) which are less susceptible to heating and heat dissipation is more effective.  The use of a column oven will ensure that the column is accurately temperature controlled. 

Pre-heating mobile phases reduces differences in temperature between the inlet and outlet which can cause diffusion of the sample plug which leads to peak dispersion. 

Conversely, post column cooling can be applied to mitigate any peak dispersion (Figure 5).  Superficially porous particles also have improved heat dissipation.

Figure 5: Effect of uneven column heating: (a) column and mobile phase fully equilibrated, (b) cold incoming mobile phase, (c) frictional heating within the column.


Tips for Detecting UHPLC Peaks

Smaller peak volumes and narrower peaks in UHPLC have an impact on both detector hardware and settings.
Detector flow cells are often the main source of extra column volumes; therefore, in order to minimize this, and in conjunction with the reduction in injection volumes, reduced flow cell volumes are commonly employed (≤ 2 μL, Figure 6).

One compromise of reducing cell volume is the need for a reduction in path length which can have an impact on sensitivity; therefore, if greater sensitivity is required then selecting a larger cell volume and losing some efficiency may be necessary. It should be noted that if injection volumes are not correctly scaled then overloading of the detector can be encountered (flat topped peaks).

Figure 6: Effect of detector flow cell volume on peak efficiency and resolution.


There is also the issue of how the detector acquires the data and reports to the data system (this can differ between manufacturers).  Detector sampling rates (measured in Hz) must be high enough that enough points are detected across the peak to allow proper quantitation. 

At low sampling rates, peak apices can be missed and numerical integration will be inaccurate.  Low data sampling rates alone do not lead to peak broadening (lower resolution), rather a combination of sampling rate and filtering electronics. 

For example, in one system, if the detector is set to acquire data at 40 Hz lots of data points will be detected across the peak and the data will exhibit fine structure (Figure 7).  Reducing the sampling rate to 1.5 Hz results in one out of every 15 points, relative to the first case, being detected resulting in a loss of some of the fine structure, for example, the peak apex.  In this case, however, the baseline is less noisy. 

An alternative system may exhibit peak broadening when the detector sampling rate is reduced - this can probably be attributed to how the data is handled in the analogue or digital domain inside the instrument module or software.

Figure 7: Effect of sampling rate and data reporting on peak identification and quantitation.

In conclusion, it can be seen that getting the most out of your system is often a balancing act and may require some amount of compromise. 

Watch the related webcast here


Dr. Dawn Watson
 

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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