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Errors in Method Transfer

What if Two Chromatograms do not Look Perfectly Alike?

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How to support successful method transfer

Table 1 lists the most common root causes of method transfer deviation between instruments with recommendations for addressing them. Three selected examples will be discussed here.

The first example is depicted in Figure 2, with a gradient-based acetaminophen impurity method transferred from a quaternary (LPG) Agilent system to a quaternary Thermo Scientific Vanquish Flex system. Due to the 120 µL larger GDV of the original instrument (note that this system has a strong GDV dependence on system pressure caused by a pulse damper in its pump), all peaks that eluted during the gradient where shifted to a later elution, while the retention of the first peak eluted during the isocratic step still closely matched (Figure 2A). The change to the next available gradient mixer on the Vanquish system from 200 µL to 400 µL would increase GDV by 200 µL, which was too high. Therefore, the sample loop was changed from the default 50 µL to the largest 130 µL version, and additionally the idle volume setting of the metering device was tuned to achieve a better match (Figure 2B). Changing autosampler GDV contribution with a metering device idle volume setting is explained in Figure 3.


Gallery with 6 images

The next example shows a loss of resolution due to thermal effects occurring in UHPLC columns (Figure 4). This isocratic method, which was designed for the analysis of preservatives, runs at a pressure close to 700 bar where frictional heating in the column matters, and the relative retention of the critical pair (methyl parabene/ dimethyl phthalate) strongly depends on column temperature (3). While the Thermo Scientific Ultimate 3000 system employs a forced air column thermostat, the Thermo Scientific Vanquish thermostated column compartment (V-TCC) allows the instrument to monitor and adjust fan speed to operate in still air or forced air modes. The still air mode minimizes the dissipation of frictional heat from the column and maintains a homogeneous temperature in the radial direction across the column intersection. While this provides the greatest levels of efficiency when frictional heat is present, the axial heat gradient from the column inlet to the outlet is greater (close to 10 °C here), resulting in a higher average column temperature than in the Ultimate 3000 system. In addition to the adjustable fan speed, the active mobile phase pre-heater in the V-TCC can be set to a lower temperature than the column compartment temperature. This enables to compensate for the friction-generated heat by lowering the temperature of the mobile when entering the column. The near-perfect match of the retention times with the method adapted on the active eluent pre-heater even improved peak
resolution, as illustrated in Figure

The last example (Figure 5) demonstrates a solvent mismatch by insufficient mixing in front of the column, which can occur with the 4-aminophenol peak in the impurity method illustrated in Figure 3, for example. According to the US Pharmacopeia-based method description, the sample is injected in a vehicle of pure methanol, while the mobile phase composition in the initial isocratic step is as weak as 1:99 (v/v) acetonitrile/water. If the mixing of the recommended 5 µL injection volume with the surrounding mobile phase is insufficient, this can result in severe peak distortion. Figure 5B shows the positive effect of a smaller injection volume on the peak shape. If the method fully passes the SST with the 1 µL injection volume and this change is clearly documented, the new method can be used with GLP compliance and would not require re-validation. The other possible solution is modifying system fluidics between the sample injector and column. This would however likely require a complete re-qualification of the system in a regulated environment, even if the SST passed on the modified system.


[1] Taylor, T., Dwell Volume – Still Relevant in our UHPLC World?, Link:

[2] Dolan, J. W., Gradient Elution, Part IV: Dwell-Volume Problems, LCGC Europe, Jun. 2013, 330-336 [Online], Link:

[3] Paul, C., Gruebner, M. Heidorn, M., Krajewski, M., Patzelt, S., Piecha, T., Steiner, F., An instrument parameter guide for successful (U)HPLC method transfer, Link:

* Dr. F. Steiner, Dr. C. Paul, Maria Gruebner Thermo Fisher Scientific, 82110 Germering/Germany