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Translate My Method (HPLC↔UHPLC)

Instructions

ACD/Labs have teamed up with experienced chromatographers Patrik Petersson and Mel Euerby to provide an application for translating liquid chromatography methods. Simply fill in your method information below and translate HPLC methods to UHPLC and vice versa (applicable for any type of analytical gradient chromatography).

Compare Functionality of ACD/Labs' Method Translation Tools in ACD/Chrom Workbook and the Freeware Application

Functionality in Translation Tool Freeware ACD/Chrom Workbook
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
Instrumental and Column Parameters -
Original New Scaled New Selected
Flow (mL/min)
Flow scaled against column dimensions (mL/min). Typical for large molecules 0.20
Column i.d. (mm) 2.1
Particle diameter (um) 3.4
Column length (mm) 283
Injection volume (uL) 9.4 5.0
Dead volume (mL)
Dwell volume (mL)
Dwell time (min) 2.88 2.88
L/dp 83,333.3 83,333.3 44,117.6
In order to get accurate translations it is recommended to use experimentally determined dead and dwell volumes. A 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".
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 0.00
6 0.00 0.00 0.00 0.00
7 0.00 0.00 0.00 0.00
8 0.00 0.00 0.00 0.00
9 0.00 0.00 0.00 0.00
10 0.00 0.00 0.00 0.00
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 warningNote: 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"

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