Translations Between Differing Liquid Chromatography Formats: Advantages, Principles, and Possible Pitfalls - - Chromatography Online
Translations Between Differing Liquid Chromatography Formats: Advantages, Principles, and Possible Pitfalls


LCGC North America
pp. 558-567

The numerous advantages of translating gradient chromatographic methods between the differing formats of liquid chromatography (LC) have been explored and discussed. Although translations in principle obey well-defined chromatographic theories, the authors investigate a number of potential pitfalls that may result in poor translations as exhibited by selectivity differences, changes in efficiency, and hence failure to meet resolution system suitability criteria. The consequences of these pitfalls are examined and the regulatory implications of method translation are explored.



As a result of the introduction of commercially available sub-2-m porous particles (1), sub-2-m, 3-m, and 5-m superficially porous (2,3) particles, and ultrahigh-pressure liquid chromatography (UHPLC) instrumentation (4,5) from 2004 onward, there has been an increasing interest in the ability to perform accurate translations between different liquid chromatography (LC) formats. An example would be translating between 150 mm 4.6 mm, 5-m d p formats on standard high performance liquid chromatography (HPLC) systems and 50 mm 2.1 mm, sub-2-m formats on UHPLC systems while maintaining the same resolution. The findings of a recent survey of major chromatographic users predicted that the use of standard HPLC systems is expected to steadily decline from 2011 to 2015 with a concomitantly higher usage and purchase of UHPLC systems predicted over the same time frame (6).

There are a plethora of reasons for this shift in LC format usage and purchase, all of which are based on sound chromatographic theory (5,7,8). From the extensive experience of the authors within the pharmaceutical industry, the major drivers for this shift appear to be increased productivity (that is, reduced analysis time) coupled with minimal loss of information quality or an increased quality of data with no loss of productivity.

Advantages and Drivers

Increased Resolution

Reduction of the packing material particle size by a factor of two (that is, substitution of 3–3.5 m particles by 1.7-m particles), while keeping other operation factors constant, should result in an increase of resolution of approximately 30–40%.

Speed of Analysis

A reduction in column length (L) and particle size (d p) while keeping the L/d p ratio constant (for example, substitution of a 150-mm column with 3–3.5 m particles for a 75-mm column with 1.7-m particles) should maintain the same chromatographic efficiency and hence resolution, while reducing the gradient analysis time by 50% (same velocity typically used for large molecules) to 70% (higher velocity typically used for small molecules) and substantially increasing productivity. This approach is vitally important for the analysis of increasingly larger numbers of samples (that is, to better describe a process or formulation performance), increased utilization of instruments, the analysis of labile samples, and rapid at-line analysis (that is, process analytical technology).

A compromise between the approaches of increased resolution and speed of analysis has been the use of 100-mm columns with sub-2-m particles, which results in a 60% reduction in gradient time and an approximate 10% increase in resolution for small molecules.

Reduced Solvent Consumption

Converting a standard HPLC method that uses a 150 mm 4.6 mm, 3–3.5 m column to a 100 mm 2.1 mm, 1.7-m or 100 mm 1.0 mm, 1.7-m column in theory offers a possible reduction in solvent consumption of approximately 86% to 97%. In practice, it is less often because of the necessity to prime the LC lines. During a global implementation of UHPLC within AstraZeneca (during the period of 2007–2010) involving 41 UHPLC systems, a reduction in solvent consumption of 63% was realized compared to the theoretical reduction of 77% (9).

Ease of Method Transfer

Within many industries it is often standard practice to transfer the chromatographic testing from the research and development (R&D) laboratory to a contract research organization (CRO), operation, or quality control (QC) sites. This method transfer exercise can be made even more problematic because it is now quite common for many R&D departments to develop only UHPLC methods. However, not all receiving laboratories have sufficient UHPLC capacity or experience and, thus, method translations become necessary. The reverse of this is becoming true in that QC laboratories, which have moved predominantly to UHPLC, may have to use UHPLC methods for the analysis of legacy products or methods that use HPLC columns.

Increased Instrument Utilization

The drive for increased productivity and efficiency has necessitated an increased flexibility and utilization of available instrumentation. Many companies have a rolling program to replace their worn out HPLC systems with a reduced number of UHPLC systems capable of running LC methods based on both 5-m and sub-2-m particles. In addition, valve arrangements are used that allow queuing of both HPLC and UHPLC methods on the same LC system, hence a reduced number of LC systems allow continuous operation.


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