UHPLC Method Transfer Basics

Introduction

High performance liquid chromatography (HPLC) has been utilized in laboratories for decades , and its robust nature makes it ideal for a wide range of applications. As the demand for faster separations, better peak resolution and lower detection limits has increased in recent years, many labs have begun to transition their HPLC methods to ultra high-performance liquid chromatography (UHPLC) techniques. The first article in our series on HPLC to UHPLC method transfer offered a general discussion on HPLC and UHPLC methodologies. Now, we will turn our attention to converting methods from an HPLC technique to UHPLC, exploring the benefits of method transfer, as well as a discussion on whether transferring a method from HPLC to UHPLC is right for your lab.

The trend of transferring methods from HPLC to UHPLC techniques was first seen on a large scale in the pharmaceutical industry, which has significant demands for fast method development. Many other industries, especially those that are highly regulated such as the food, industrial and environmental sectors, have recently begun to embrace UHPLC methods. The primary benefits of transferring a method from HPLC to UHPLC include:

• Faster analytical separations.
  
  
• As we detailed in the first article in this series, transferring a method from HPLC to UHPLC can result in substantial productivity gains, as separations are achieved much faster with smaller columns and higher-pressure solvent delivery. Faster separations can allow for the analysis of more samples in a day, which can improve the productivity of high throughput laboratories. This is likely a major contributing factor in the shift towards UHPLC witnessed in the food, industrial and environmental industries.
• 
  To illustrate this benefit, consider the chromatograms in Figures 1 and 2. Figure 1 displays the chromatogram from an HPLC analysis of seven anxiolytic agents. The seven target compounds eluted in approximately 18 minutes. Figure 2 displays the chromatogram from the same analysis utilizing a UHPLC instrument. All compounds eluted in approximately 3 minutes utilizing the UHPLC method. Utilizing this example, if a lab were analyzing a batch of 20 samples, analysts could save 5 hours utilizing UHPLC (60 minutes versus 360 minutes).

Figure 1¹: Analysis of seven anxiolytic agents compounds an HPLC method – 1) bromazepam 2) nitrazepam 3) oxazepam 4) clonazepam 5) alprazolam 6) flunitrazepam 7) diazepam

Figure 2¹: Analysis of seven anxiolytic agents compounds a UHPLC method – 1) bromazepam 2) nitrazepam 3) oxazepam 4) clonazepam 5) alprazolam 6) flunitrazepam 7) diazepam

• Better peak resolution. 
  
  Peak resolution is a measurement of how well one chromatographic peak can be differentiated from another. Apart from the need for faster separations, the demand for higher resolution separations was one of the driving forces behind the development of UHPLC methods. Owing to the smaller particles in the column, as well as the smaller column internal diameter, UHPLC methods routinely offer taller, more narrow peak shapes, which can result in more confident identification and quantification of analytes and improve sensitivity

• Lower long-term costs.
  
  It is important to consider cost when evaluating whether method transfer is right for your lab, as cost is rarely not a factor in these decisions. UHPLC instrumentation does tend to have a higher initial price point than that of HPLC instruments, owing to the more powerful pump and associated components. However, these costs can often be offset in the longer term, as you begin to realize a reduction in per-sample analysis operating costs. The lower flow rates associated with UHPLC analyses result in less solvent consumption. This reduction in solvent consumption also results in reduced waste disposal and management costs.

Is UHPLC Right for Your Analysis?

The benefits of UHPLC are clear. However, not every laboratory operation can realize these benefits, and transferring an existing method from HPLC to UHPLC may not be right for every lab.

As you consider transferring your method, we suggest you reflect on a few points. First, at the most basic level, it’s important to determine whether the analytical method and sample matrix you are analyzing is compatible with UHPLC principles. Methods and applications that require separation and quantification of a large number of analytes are ideal candidates for UHPLC method conversion, as the improved resolution and separation efficiency will reduce analysis time and data review complexity. Further, filtered or relatively clean samples are preferred for UHPLC techniques. As UHPLC columns contain smaller, more tightly packed particles, unfiltered samples could cause clogs within the column.

Next, consider whether the improved efficiency associated with UHPLC techniques will offset the increased initial cost. If your lab only analyzes a few samples each day, and your HPLC technique provides sufficient resolution for your application, UHPLC may not be the right choice. On the other hand, looking back at the previous example where UHPLC saved the analyst 5 hours of time in a day, one can see how the improved efficiency could result in higher laboratory throughput and increased revenue.

Also consider how you will utilize your instrument long term. Although you may only analyze 5 samples today, are there any trends or regulations on the horizon that may increase your workload? Investing in a UHPLC instrument today may future-proof your investment and allow your operation to remain agile in its reaction to changing customer demand. As an example, consider the buying decision of a US-based cannabis lab director in 2011. Medicinal cannabis use began to rise in the United States at the beginning of the 21st century as states legalized the use of cannabis in the treatment of a number of conditions. When the lab director was determining the needs of the lab in terms of throughput and projected sample load, she should be considering the several states that were set to vote on the legalization of recreational cannabis use, and the subsequent increase in laboratory demand this would spur. Although her lab may only be analyzing 100 samples per week in 2011 for cannabis label-claim accuracy, these sample loads could be increased drastically as regulations change, necessitating a more powerful and efficient instrument.

After reading this article, we hope that you have a better understanding of why UHPLC may or may not be the right choice for your laboratory and application. In the next article in our series, we will explore best practices associated with transferring a method from HPLC to UHPLC, to ensure that you maintain data quality, while achieving optimal efficiency.

Sources

1. Guillarme, D., & Veuthey, P. (2009). UHPLC Instrumentation and Method Transfer Guidelines [Scholarly project]. In PerkinElmer White Paper.