Frequently Asked Questions (FAQs): Pro EZLC Method Translator
- Can you translate between one column that uses superficially porous particles (SPP) and another column that uses fully porous particles (FPP) or vice versa?
- What is the meaning of the messages to the right of the Results header?
- The gradient for my translated method goes to 7 minutes, but I’m seeing a retention time marker appear at 8.3 minutes in the translation validation plot (Windows version only). Why is it coming out after my run is complete?
- Can you enter a step in the original gradient program that is a lower %B that the initial value set for %B?
- Sometimes when my original method contains a gradient there is a mismatch between the number of steps in my “Original” and “Translation” methods. Why does this happen and when should I expect it to happen?
- The translated Compound Retention Time is later than the end time of the method program. Why is this the case?
1. Can you translate between one column that uses superficially porous particles (SPP) and another column that uses fully porous particles (FPP) or vice versa?
No, at this time you can only translate between columns that use the same particle type (e.g., from SPP to SPP, or from FPP to FPP).
2. What is the meaning of the messages to the right of the Results header?
The meaning of each message is as follows:
- “Excellent Translation”—this message is displayed when the compounds in the original and translated methods elute at the same %B. Translations for isocratic methods will always be “Excellent.”
- “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 the gradient”*
*These messages are displayed when not all of the compounds in the original and translated methods elute at the same %B during a gradient run. The severity of the discrepancy is reflected in each message. This mismatch occurs when the translated column and method conditions may require adjustments to the instrument’s dwell volume. See the glossary within the software itself for more information about dwell volume.
3. The gradient for my translated method goes to 7 minutes, but I’m seeing a retention time marker appear at 8.3 minutes in the translation validation plot (Windows version only). Why is it coming out after my run is complete?
The plot has lines that represent hypothetical retention times for compounds that elute at different points in the gradient profile. They are not representations of your actual chromatography. In addition, the time you enter as the end of the gradient (7 minutes in this example) is the time at which the pumps switch over to the final %B. Extra time is required for that final mobile phase concentration to travel through the system to the detector.
4. Can you enter a step in the original gradient program that is a lower %B that the initial value set for %B?
No. The initial %B entered at Time 0 min in the original gradient program table is the lowest %B the method translator will allow for that mobile phase program.
5. Sometimes when my original method contains a gradient there is a mismatch between the number of steps in my “Original” and “Translation” methods. Why does this happen and when should I expect it to happen?
This occurs because of a significant change to either column diameter or dwell volume (e.g., when transferring a method from one LC system to another LC system). In some instances, these differences can effectively act as a hold time at the initial %B. Depending on the scenario, an isocratic hold may be needed or not needed in the translated method for equivalence.
- Scenarios when an initial isocratic hold will be added to the translated method:
- The dwell volume of the original method is greater than the dwell volume of the translated method.
- Going from a narrow bore (ID) column to a wide bore (ID) column.
- Going from a shorter (length) column to a longer (length) column with the ID of the columns changing.
- Any combination of the above.
- Scenarios when an initial isocratic hold is not needed in the translated method:
- The dwell volume of the translated method is greater than the dwell volume of the original method.
- Going from a wide bore (ID) column to a narrow bore (ID) column.
- Going from a long (length) column to a shorter (length) column with the ID of the columns changing.
- Any combination of the above.
6. The translated Compound Retention Time is later than the end time of the method program. Why is this the case?
If you have a late-eluting peak that elutes very close to the end of your analysis under your original run conditions, it is possible that under the translated run conditions the peak will not elute without adding an isocratic hold. Alternatively, if you are able to reduce your instrument’s dwell volume that can also affect compound retention time, allowing the late-eluting peak to elute within the translated method conditions.