Unlocking Smoother HPLC Method Transfer
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High-performance liquid chromatography (HPLC) is one of the mainstay techniques in an analytical chemistry lab. However, a key challenge in highly regulated laboratories is when scientists must switch from one HPLC system to another, or exchange methods between different labs and sites. The process of transferring a method to the receiving HPLC system involves checking and adjusting multiple parameters such as instrument settings and configurations. If this is not carried out carefully, the receiving instrument and method might produce results inconsistent with the original system in terms of retention times and peak shapes for the analytes of interest, ultimately rendering analysis unreliable. Conversely, making method adjustments can lead to lack of compliance with regulator validated analytical protocols. In this article, we review the main challenges with method transfer and highlight how advanced HPLC technologies can help smooth this transition.
There are a few scenarios where analytical procedures might need to be transferred between HPLC instruments:
1) Method transfer to new equipment in the same lab, perhaps to a different vendor or a newer iteration of your existing instrument.
2) Method transfer to equipment in a different lab, for example from an R&D laboratory to a QC laboratory, or from a sponsor laboratory to a contract research organization.
This second scenario is becoming more common because of increased outsourcing to external laboratories, and because methods are more frequently shared across multiple international sites within an organization. In many such cases, chromatography instruments are not identical, and so difficulties arise when scientists attempt to reproduce their results.
All HPLC protocols must also comply with the standardized guidelines for analytical chromatography created by regulatory bodies such as the World Health Organization, the United States Pharmacopeia (USP621 regulations), and the EU Good Manufacturing Practice guidelines. This presents challenges for scientists trying to adjust variables to an extent permitted by regulators while ensuring accurate and reproducible analysis.
The extent to which a certain instrument parameter influences the success of a method transfer process will depend on the specific application. However, there are some factors that remain important across all HPLC methods.
With many liquid chromatography systems not set up to offer the flexibility to adjust these instrument inherent parameters, transfer can become time-consuming, complex and prone to failure. Thankfully, advanced HPLC technologies are now available to help address these challenges including:
- Column thermostatting: Mismatched temperature control can directly influence the selectivity of analyte separation. Some modern instruments enable you to choose the appropriate column thermostatting modes to correctly mimic the column thermostatting mode from the transferring instrument. This is of particular interest for ultra-high-performance liquid chromatography (UHPLC) methods creating frictional heating between the mobile phase and the small stationary phase particles at high pressures.
- Solvent temperature: In addition to column thermostatting, another variable that affects temperature is the use of a pre-heater for the mobile phase. To enable successful method transfer, the pre-heating capabilities of the original system should be transferred as accurately as possible. This should extend beyond simply deciding whether a pre-heater needs to be included, but also to consider differences in the design, function and volume of the pre-heater. There are two types of pre-heaters – passive and active. Conventional passive heaters work through contact with a temperature-controlled surface in the HPLC column, and heat is transferred over the pre-heater. By contrast, more advanced active pre-heaters work independently of the column temperature, allowing the user to fine-tune the temperature of the mobile phase.
- Extra-column volume (ECV): The ECV is defined as the volume from the injector to the detector excluding the volume in the column. If the receiving unit has a lower ECV compared with the original instrument (where switching from HPLC to UHPLC, for example) then adjustments will need to be made to avoid differences in analyte separation, especially for early eluting substances when strong solvents are used. Similarly, an extra-column volume that is too high can result in insufficient resolution of components. These potential issues can be resolved with advanced HPLC technologies that allow you to customize injection procedures.
- Sample mixing effects: Insufficient sample mixing can occur between the injector and the column entry and result in distorted peaks when strong sample solvents (stronger than the eluent itself) are used. Yet standard operating procedures might not permit modifying the chromatographic or sample preparation conditions to address this. An instrument that features a custom injection program or enhanced sample plug mixing can allow you to selectively adjust the effective injection solvent strength without changing the overall experimental settings, yielding consistent peak shapes.
- Flow cell volume: The detector flow cell volume is also crucial to consider during method transfer. Users need to ensure the volume is in accordance with the peak volume (no larger than 10% of the peak volume of the smallest peak) and the column diameter. Besides physical dimensions of the flow cell, the detector settings play an important role. HPLC software can help calculate the optimal settings for your flow cell volume, allowing you to obtain similar results between different instruments.
- Gradient delay volume (GDV): The GDV describes the volume of all interconnected components from the point of mobile phase mixing to the entry of the separation column. Different instruments have different GDVs, and this can result in a particular solvent arriving at different time points on the head of a column. Of all the parameters that can prevent successful method transfer, the variable gradient delay volume is the factor that most often impacts result reproducibility on a new instrument. There are different approaches to adapting GDV, but advanced HPLC instruments include built-in capabilities that make these adjustments more precise and straightforward to implement.
Gradient delay volume — a deeper dive
GDV is essentially the sum of the volume after the point of mixing in the pump, flushed autosampler volume and connecting capillaries to the column head. It causes the delay from the time when the HPLC or UHPLC pump is programmed to produce a specified solvent composition, to when the composition reaches the column.
As different instruments have different components, the GDV might need to be adjusted when methods are transferred. HPLC systems can also have different pumping technologies known as low- and high-pressure mixing pumps. A large GDV difference between the pump types, but also within one pump type, can cause a significant difference in GDV.
One common way to measure the GDV is to program the pump to deliver a linear gradient from 0% to 100% B, with channel B containing a UV-absorbent compound, such as caffeine. The GDV can then be calculated using the time it takes for the UV trace to reach 50% of the maximum (Figure 1).
Figure 1: Method for determination of an instrument gradient delay volume. Two different instrument behaviors are shown as well as two commonly used data evaluation procedures (blue and green arrows).
Once you know the GDV of your receiving instrument, you can determine if you need to change this to achieve the same retention times and peak profiles for your analyte generated on the original instrument. Making software changes to compensate for differences in GDV, such as by altering the time of injection or changing the gradient profile, can raise concerns of regulatory authorities. However, changes to the GDV are permitted under the standard guidelines for analytical chemistry and so these adaptations are popular tools for supporting more successful method transfer.
If you need to adjust the GDV, there are two approaches commonly used. The first option is to physically change the GDV of the receiving system to match the original system’s GDV. This might be achieved by placing mixers or large volume capillaries between the pump and autosampler. Although these changes usually help to mimic the parameters of the source instrument, the mixers and capillaries have fixed volumes, and do not allow for precise GDV adjustment. Moreover, in regulated environments, these hardware changes would require a (re)validation of the altered instrument.
The second option is to move the injection point relative to gradient start. In practice, this means that if the difference in GDV on the receiving instrument is +1 mL, and you have a flow rate of 1 mL/min, you could delay injection time by one minute after the programme has started. In this way, the slope and duration of the gradient would not be affected. Although this approach might be feasible if the difference in GDV is substantial, such adjustments might overcompensate where the difference between instruments is small.
To address this, some advanced HPLC systems now feature autosamplers that can freely fine-tune the GDV. Rather than manually adjusting the GDV in fairly large increments, as is required with most modern HPLC systems, a tunable GDV allows the end-user to gradually and subtly change the GDV. The resulting adjustments do not alter the gradient table and thus ensure optimal method transfer.
A common theme across all the parameters discussed is the requirement for end-users to make manual adjustments to reproduce settings from their original HPLC system, which can be time consuming, subjective and prone to errors. Advanced HPLC systems eliminate this need through sophisticated data analysis software.
For example, features such as gradient pre-start can support users in better adjusting the injection point to achieve the same GDV as their original instrument. Software can also be used to optimize other parameters such as flow cell detector response times.
Moreover, today’s advanced HPLC software can centrally control all instruments within a workflow, integrating different systems, regardless of vendor or location. Most advanced HPLC analysis software provides predefined workflows to support new or experienced users in setting up reproducible methods on different HPLC systems, as well as integrated data management processes that provide an instant audit trail, ensuring labs maintain the integrity of their data.
Increased outsourcing to contract research organisations and method-sharing between different labs within an organization means that scientists often need to transfer HPLC methods between different instruments. If careful consideration is not given to each instrument parameter in the HPLC system, this can lead to irreproducible results. At the same time, any adjustments to the system must adhere to regulator standards.
Many different variables influence the success of method transfer, and there are multiple adjustment approaches to choose from. Innovative HPLC technology can help make the method transfer process smoother and ensure compliance through solutions such as tunable GDV, customizable injections and column thermostatting modes. Moreover, today’s state-of-the-art HPLC software can not only calculate optimal parameters for the same method on a different instrument, but can also centrally control all instruments in a vendor-neutral way across different labs.
Together, these tools ensure users can confidently approach each method transfer whenever they need to move their analytical procedures between different equipment or labs.