Pro EZLC Method Translator Help
The Pro EZLC Method Translator is a tool built for liquid chromatography (LC) method development. Generally, the goal of method translation is to allow alteration of LC column dimensions, flow rate, etc. while maintaining the same peak elution order and resolution, even though retention times will change. Note that method translation assumes that the LC stationary phase type, particle type (e.g., fully vs. superficially porous), and mobile phase solvents remain the same between original and translated methods.
Some of the most practical uses for Restek's Pro EZLC Method Translator are listed below:
- Increasing sample throughput by decreasing column length and/or inner diameter.
- Calculating time and solvent savings when switching to a shorter and/or smaller particle size column.
Basic Navigation in the Pro EZLC Method Translator
White cells represent user entry fields while blue cells are locked fields. Selecting the Gradient option under the Method Program section displays a Translation Validation Plot in the lower right corner of the tool (Windows version only). For more on this feature, see Translation Validation Plot.
Highlighting numerical values using the mouse allows easy user entry of new values. A double mouse click in any user entry cell highlights the value automatically for user entry. Hitting the Tab key (“Enter” will also work for Windows version) while in a cell updates the cell with the user-entered value and moves to the next cell for additional user entry.
Original / Translation
Original LC method parameters can be entered in the far left column. The Pro EZLC Method Translator will generate a new method in the translation column.
LC column length in millimeters (mm). This will be stated on the column box and the column label (e.g., 150 mm).
Inner diameter (ID) of the LC column in millimeters (mm). This will be stated on the column box and the column label (e.g., 2.1 mm).
The size of particles in the LC column, in micrometers (µm). This will be stated on the column box and the column label (e.g., 2.7 µm).
The amount of sample injected with the original method is called the “injection volume.” The Pro EZLC Method Translator uses a scaling factor based on the ratio of column IDs to calculate the recommended injection volume in the translated method. If the translated injection volume contains a decimal and your LC control software does not allow partial microliter (µL) injection volumes, round the translated injection volume to the nearest whole number when setting up your injection batch/sequence.
Changes in mobile phase composition during a gradient do not instantaneously arrive at the column. There is some system volume required for the new mobile phase composition to arrive at the column, called the instrument’s “dwell volume.” An instrument’s dwell volume affects retention times for gradient methods and may also affect selectivity for early-eluting compounds, so taking dwell volume into account can be very important for successful method translation. Dwell volumes are not necessarily the same from instrument to instrument (especially when moving between binary and quaternary pumps), so it is important that dwell volume differences be accounted for. Typically, an instrument’s dwell volume is determined to start at either the mixer (or the gradient proportioning valve for quaternary pumps) and includes tubing from the mixer to the injection valve, the sample loop, and tubing from the injection valve to the column. If you cut and install your own tubing, there are a number of online calculators that can easily determine the volume of various tubing lengths and IDs. For example:
To find your instrument’s dwell volume, consult instrument documentation or contact the instrument manufacturer. If instrument-specific dwell volume values are not available, you can use the default value. Translated retention time predictions may be affected, but they should still represent an approximation of the actual times observed under the new method conditions.
Extra Column Volume Effect
The following physical components of the LC instrument contribute to Extra Column Volume (ECV): needle seat, injection volume, injection valve, precolumn tubing, preheater, post-column tubing, and even detector volume in the case of flow cell-based detectors. ECV contributes to band broadening, or the increased width of the observed peaks, which, in turn, affects peak resolution. This widening of the observed peaks is called the “Extra Column Volume Effect” or “Extra Column Dispersion.” The ECV effect is typically reported in volume units because it represents the additional volume a band of analyte molecules occupies as a result of the instrument’s ECV. Accurately identifying the ECV effect allows the Pro EZLC Method Translator to calculate critical pair resolution in the translated method. Each LC instrument has its own value: if it’s not found in the documentation supplied with your instrument, contact the manufacturer.
If your instrument has been modified such that it is no longer representative of a stock configuration from the manufacturer, the Pro EZLC Method Translator can be used to calculate the ECV effect. Simply run an isocratic analysis using two different column dimensions while keeping the stationary phase and particle type (SPP or FPP) the same for both columns, and then adjust the ECV effect until calculated resolution values are comparable to observed values. Typical values for ECV range from 2–50 µL. Proper determination of ECV effect may still result in biased calculated resolution values due to a number of factors (e.g., non-Gaussian peak shapes, slight shifts in elution %B, mass on-column scaling errors). However, the calculated resolution values should be representative of observed resolution values within +/- 25%. If you are not able to obtain an instrument-specific ECV effect value, you can use the default value. The method translation and the predicted retention times will still be accurate, but the predicted critical pair resolution values will not be as accurate as they would be with an instrument-specific value.
Isocratic, Gradient, Number of Gradients
The Method Program can be toggled between Isocratic and Gradient. Isocratic means that the %B will not change during the analysis. Selecting Gradient and then entering the number of steps and original program values for Time and %B allows translation of LC method programs. Additionally, selecting the Gradient option displays a Translation Validation Plot in the lower right corner of the tool (Windows Version Only). For more on this feature, see Translation Validation Plot.
Note on Gradient Methods: There are special cases where you will notice a mismatch between the number of steps in your original and translation gradient methods. If your translation results in one of these cases, one of the following messages will appear in the Method Program Header:
- “Isocratic hold is required” If your translated method would benefit from the introduction of an initial isocratic hold period, the method translator will add one to the translated method conditions, resulting in a translated mobile phase program with one extra step compared to the original conditions.
- “Isocratic hold not required” If your original method contained an initial isocratic hold time, but the translated column does not require one, the method translator will automatically eliminate the initial isocratic hold step, resulting in a translated mobile phase program with one fewer step compared to the original conditions.
For more information on why and when these scenarios occur, see the FAQ page.
Time (min), %B (min), Flow (mL/min)
Enter the original starting %B and flow rate in Line 1 of the method program. You can have up to 8 steps for %B changes at different times for linear or step gradients. The last line should be the “Stop” time for the method.
You can override the recommended flow rate in the translation column to evaluate the results (Speed Gain, Total Time, etc.) at different flow rates. Changing any other conditions (column dimensions, dwell, or extra-column volume) and then changing back to the initial value will return the original translation value for flow rate.
Note: Pro EZLC Method Translator does not allow changes in flow rate during a method program.
Note on Translated Flow Rates: In an effort to provide the most practical translated flow rate for a given column dimension, we have applied a simple “dampening term” to our general flow rate conversion equation. This dampening term will restrict the translator from initially providing flow rates that fall outside of typical practical flow ranges. It does assume, however, that the original method conditions fall within the ranges listed below for the original column dimensions. If the original method conditions fall outside the listed ranges, the translator may return a translated value that also falls outside the ranges. The translated flow rate can still be adjusted by the user after the initial translation.
Practical Flow Rate Ranges by Column I.D.
- For 2.1 mm I.D. columns, 0.4 – 0.6 mL/min
- For 3.0 mm I.D. columns, 0.7 – 1.0 mL/min
- For 4.6 mm I.D. columns, 1.4 – 1.6 mL/min
To the best of our knowledge, no other method translator incorporates this dampening term into the flow rate conversion equation. Keep this in mind if you try another translator and the initially returned value for the translated flow rate falls outside of the practical ranges listed above.
Note on Translation Quality: A short statement commenting on the quality of the translation will display to the right of the Results header. The message reflects the degree to which compounds from the original and translated methods elute at the same %B. In general, a mismatch occurs when there is a difference in the original and translated instrument dwell volumes when using a gradient method. Five possible messages can appear:
- “Excellent Translation”
- “Peak order may change for very early peaks”
- “Peak order may change for early peaks”
- “Peak order may change in the first half of the gradient”
- “Peak order may change throughout gradient”
These messages will correlate with the information displayed in the Translation Validation Plot (Windows version only).
Speed is normalized to "1" for the original method, and Speed Gain is the ratio of the Original Method Total Time divided by the Translation Method Total Time. Depending on the translation results, speed will either be gained (a number greater than 1), or lost (a number less than 1).
Back pressure is normalized to "1" for the original method, and the Results pressure is estimated based on the translated flow rate and column parameters. Smaller column ID and particle size in translated methods produce higher back pressure, and the pressure increase will be even greater if the translated flow rate is changed to a higher than recommended value.
Critical Pair Resolution (Rs)
A critical pair is a group of two closely eluting compounds whose resolution is desired to be monitored in a translated method. The original Rs value is calculated by your data acquisition software; refer to the vendor for instructions. The Pro EZLC Method Translator uses the extra column volume as part of the calculation of critical pair resolution in the translated method, so it is important to enter an accurate value in this field.
Compound Retention Time
Retention Time is the time between sample injection and analyte detection. The Compound Retention Time field allows the user to determine when an analyte of known retention time (on the original column) will elute on the new column.
Enter the number of injections in your batch/sequence to calculate the total time and solvent usage with the translated method compared to the original.
The Total Time (min) is calculated for both original and translation methods from the method program values and number of injections.
Solvent usage (mL) is calculated for both original and translation methods from the method program values of time and flow rate and the number of injections.
Save as Program Default Settings/Restore Program Default Settings (Windows Version Only)
If you have entered original column and method information that you intend on using for multiple translations, you may save those entries as your default settings for the translator by simply clicking the “Save as Program Default Settings” button below the results section of the method translator and selecting “Yes.” These default settings will be retained regardless of the translation you are currently working on. If you wish to change the translator’s inputs, at any time, back to the default settings you chose, simply click the “Restore Program Default Settings” button.
Translation Validation (Windows Version Only)
This plot can be used to verify how accurately the translation maintained the same separation under the new method conditions.
The translator scales gradient conditions—maintaining the relative relationship between %B composition and time—with the intention of having compounds elute at retention times that correspond to the same %B composition in the translated method as they did in the original method. It also attempts to scale flow rates—optimizing mobile phase linear velocity and column efficiency—with the goal of maintaining the original method’s separations within practical flow rate restrictions (see the Time section above for more on practical flow rate ranges).
The plot has lines that represent hypothetical retention times for compounds that elute at different points on the gradient profile. They are not representations of your actual chromatography. The space between the lines represents the time difference between the apexes of the hypothetical peaks (note that the plot does not attempt to portray resolution since no peak width information is provided). A translated method will have the lines on the lower original method trace align vertically with the lines on the upper translated method trace, and the method translator will report “Excellent Translation,” indicating that the translated method conditions can be used without further adjustment needed to other system parameters, most notably the dwell volume.
There are circumstances where the translation validation plot will display a misalignment between the retention times, indicating that the Pro EZLC Method Translator is predicting a problem. This result typically occurs when the translation column dimensions are significantly smaller than the original column dimensions. In these instances, HPLC system volume may be too large to allow for the proper method translation because of unaccounted for band broadening outside the column. If this occurs, the translation validation plot will provide a message in red text indicating the region of the gradient program that will be most affected, and the affected retention times will also be displayed in red text. To proceed with the translated method without making the appropriate system adjustments (typically, a reduction in dwell volume to be compatible with the increased efficiency of the translation column) may result in differences in the separations, especially at the earlier part of the gradient program.
It is important to note that this alert is not a failure of the method translation; it is a notice that the system set up as defined in the translation LC parameters is not fully compatible with the translation column dimensions, and adjustments should be made to avoid instrument-related deviations from the translation.
The Pro EZLC Method Translator itself is built on the seminal work of Csaba Horváth [http://molnar-institute.com/fileadmin/user_upload/Literature/_1976_Horvath_Solvo.pdf] that describes the fundamentals of reversed-phase separations.