Translate Liquid Chromatography Methods Quickly and Easily
ACD/Labs have teamed up with experienced chromatographers Patrik Petersson and Mel Euerby to provide an application for translating liquid chromatography methods.
We also offer a full suite of commercially available chromatography software.
Compare the functionality of the freeware and commercial products.
| Functionality in Translation Tool | Freeware | ACD/Labs Chrom Software |
| Scaling of gradient times and a geometric scaling of flow and injection volume | ||
| >9 time points | ||
| User input of exp dead and dwell volumes | ||
| Dwell volume compensation | ||
| Scaling of flow to hit optima in van Deemter | ||
| Pressure estimate and warning | ||
| Solvent consumption | ||
| Change in Rs | ||
| Re-equilibration estimate and warning | ||
| Estimate of dead volume based on particle and column dimensions | ||
| Estimate of dwell volume | ||
| Scaling of inj volume to compensate overload | ||
| Calculation of linear velocity | ||
| Calculation of L/dp |
| Typical Dwell Volume Estimates | |
| Alliance | 1.40 mL |
| Acquity UPLC "classic" with 50 µL mixer | 0.10 mL |
| Acquity UPLC "classic" with 385 µL mixer | 0.44 mL |
| Acquity H-class with 100 µL mixer | 0.38 mL |
| Acquity H-class with 250 µL mixer | 0.53 mL |
| NB check also H-class extension loop size, i.e., add 50, 100, 250 or 1000 µL to the volumes above | |
| Dionex RSLC | 0.56 mL |
| Agilent 1100 | 1.22 mL |
| Agilent 1260 in low dwell volume config. | 0.29 mL |
| Agilent 1290 binary in low dwell volume configuration | 0.20 mL |
| Agilent 1290 quaternary in low dwell volume configuration | 0.57 mL |
| For an accurate translation it is recommended to measure the dwell volume as described in the European pharmacopeia 7.0, 2.2.46. This is only necessary once for a certain instrument model and configuration. | |
| Gradient Time Table | |||||||
| Original | Number column volumes | New selected not compensated for dwell volume differences | New selected compensated for dwell volume differences | ||||
| Gradient steps | tG1 (min) | %B | tG1 F1/VM1 | tG2 (min) | %B | tG3 (min) | %B |
| 0 | 0.00 | 27.00 | |||||
| 1 | 0.00 | 0.00 | 27.00 | ||||
| 2 | 18.18 | 25.69 | 65.00 | 25.69 | 65.00 | ||
| 3 | 18.24 | 25.77 | 90.00 | 25.77 | 90.00 | ||
| 4 | 20.00 | 28.26 | 90.00 | 28.26 | 90.00 | ||
| 5 | 0.00 | 0.00 | 0.00 | 1.00 | |||
| 6 | 0.00 | 0.00 | 0.00 | 1.00 | |||
| 7 | 0.00 | 0.00 | 0.00 | 1.00 | |||
| 8 | 0.00 | 0.00 | 0.00 | 1.00 | |||
| 9 | 0.00 | 0.00 | 0.00 | 1.00 | |||
| 10 | 0.00 | 0.00 | 0.00 | 1.00 | |||
| Start re-equilibration | 27.00 | 20.06 | 28.34 | 27.00 | 28.34 | 27.00 | |
| Stop re-equilibration | 27.00 | 26.06 | 36.82 | 27.00 | 36.82 | 27.00 | |
| Dwell volume warning | Note: in order to compensate dwell or dead volume differences the injection of the sample must be made X min after the gradient starts. | X = 0.41min | |||||
| On Waters equipment use the Solvent Manager Misc Pre-Injector Volume command set to Y µL. | Y = 83µL | ||||||
Tips and hints for using Method Translator
Check list for potential problems.
Typically translations give acceptable deviations in relative retention times. There are, however, potential problems that may result in selectivity differences:
- Programming error—double check the input to this sheet and the LC instrument methods.
- Dwell volumes are wrong—check actual dwell volumes as described in the European pharmacopeia 7.0, 2.2.46. This is only necessary once for a certain instrument model and configuration.
- Due to differences in column back pressure there will be a difference in the heat of friction generated in the column. This can in many cases be compensated by evaluating an increase in temperature if translating from UHPLC to HPLC (e.g. +2, +4, +6, +8C) to mimic heat of friction (or vice versa).
- Pressure induced selectivity differences have been described for separations involving mixtures of charged and neutral analytes. This is difficult to compensate.
- When sub-2 µm columns where introduced certain columns displayed selectivity differences between different particle sizes. Today this is less of a problem. Difficult to compensate.
- Differences in the design of the instrument can also introduce selectivity differences.
- Especially the column thermostat is a potential problem since large differences from vendor to vendor and even model to model from the same vendor has been observed.
- This can in many cases be compensated by evaluating an increase or decrease in temperature (e.g., +2, +4, +6C).
- Different LC systems have different injection principles and materials in the injector. This may result in non-linearity related to adsorption.
- In the validation of the translated method it is necessary to confirm maintained/sufficient selectivity and linearity. It is also strongly recommended to introduce a system suitability test that allows the compensation of column thermostat differences.
- A more comprehensive description on potential pitfalls and how to avoid them can be found in "P. Petersson, M.R. Euerby and M.A: James, Translations between differing formats of liquid chromatography: Advantages, principles and possible pitfalls, LCGC, submitted March 2014"