Pro EZLC Chromatogram Modeler Help
Table of Contents
This help file explains the Pro EZLC chromatogram modeler’s interface and input fields. For further details on its use, see our technical article and introductory video.
What is Pro EZLC?
The Pro EZLC chromatogram modeler is an advanced tool for virtually modeling liquid chromatography (LC) separations. This free, online simulator allows users to simply input a compound list and instantly receive separation conditions that can be implemented directly in the lab. Results can also be further refined to meet specific analytical needs. No experimental input is needed because EZLC models are based on robust algorithms and experimental data already generated by Restek’s chemists.
The Pro EZLC Interface
The Compounds Tab
Work in the Pro EZLC modeler starts in the Compounds tab. Here, users select compounds to model on a column phase of their choosing. Note that the selected compounds are specific to the model being worked on. Exiting a work session, loading a saved model, or reloading the application will clear the selected compound list.
If any of the compounds chosen are isobaric, they will automatically be flagged for resolution by the modeler. Once final selections are made, the modeler will generate a model chromatogram displayed in the Model View area. Further optimization of this separation can be made in the Conditions tab.
To get started, first choose a compound class from the Compound Class drop-down menu. A list will appear showing all the compounds available for testing in that compound class.
To select a compound, click on its entry in the list. Clicking the entry again deselects the compound.
Click on a compound’s row to select that compound for modeling.
The Search field can be used to quickly filter the list for a compound by name or CAS number. Common synonyms for a compound name will also work. Click on the compound row to add it to the selected compound list. Once done, click the “X” in the Search field to clear it and return to the full list. Filtering in this way will not remove any existing selections.
Search for a compound by name or CAS # in the Search field.
Compounds in the library can be found by common synonyms.
Isobaric compounds are preselected to resolve (when the detector setting is MS), meaning the modeler will make an effort to separate any isobars in the selected compound list. A blue checkbox in a compound’s row indicates that the compound is isobaric with another compound in the selected list, and it is targeted to be resolved. When the detector setting is UV, the default is that no compounds are preselected to resolve. In this instance, targeted resolution of compounds is performed by either manual checkbox selection, or by clicking the "Target All" checkbox.
Isobaric compounds are automatically targeted for resolution.
If resolution of a particular isobar/compound is not needed, click on the compounds’ blue checkboxes to deselect them for resolving (they will still appear in the modeled chromatogram unless the compounds themselves are deselected).
Click the checkbox to toggle on/off targeting of a compound.
Note that run times can vary significantly when different compounds are targeted for resolution, and the resolution of other compounds may be sacrificed to gain resolution of the targeted compounds. Because of this, be sure to target all the isobaric compounds that should be resolved (when the detector method is MS).
To clear existing compound selections, click the Clear link in the Compounds Selected row of the legend below the compound list, or choose a new compound class from the Compound Class drop-down menu. All targeted compounds can be cleared via the Clear link in the Isobars/Compounds to Resolve row of the legend.
After compounds of interest are selected, choose a stationary phase from the Phase drop-down menu. Depending on the library selected, the Detector type will default to either MS or UV. (MS allows the user to target isobaric compounds for resolution [Rs] based on molecular weight/precursor ions. While UV focuses on the Rs of all compounds, it does allow for targeted Rs of any selected pairs/groups). Then, click the Generate Model button to send the selections for modeling.
The modeled result will appear in the Model View area of the app.
The Model View
The modeled results appear in the right panel of the interface. The view can be expanded (hiding the tabs) via the << arrows beside the My EZLC tab. To collapse the view and re-enable the tabs, click the >> arrows in the upper left corner of the interface.
The expanded view is great for printing a modeled result.
Zoom in on the graph by clicking and dragging across a region of the graph with the mouse (or by tapping and dragging with a finger if using a tablet). This method can be used again to zoom in tighter. To zoom out, double-click (or double tap) on the graph.
Clicking and dragging over a region of the chart will zoom that region.
Hovering over a peak’s number (desktop browser version only) will give its retention time (tR); resolution (to nearest peak, RS); and elution %B. For coeluting peaks, it will show those values for all coeluting compounds.
Also, for a separation containing isobaric compounds, the resolution of those isobars is easily visualized by using the Available Isobars menu below the modeled chromatogram. The Critical Pair (see note below) for each isobar will be listed in order of resolution from least to most resolved. Any isobaric critical pair with resolution values at or above Rs = 1.5 (baseline resolved) will appear as a green trace. If the resolution between isobaric critical pairs drops below Rs = 1.5, the trace will appear either orange or red based on the degree of coelution.
Available Isobars can easily be visualized in the chart.
NOTE: If three or more isobaric compounds are present for an isobar, as shown in colored traces in the figure above, only the two most closely eluting compounds will be displayed in the Available Isobars drop-down list. These two are defined as the “critical pair” for that m/z ion.
The peak list shows retention time (tR in minutes); resolution (Rs-to nearest peak); peak width (minutes); Exit %B; ESI ionization mode; and Precursor and Quantifier Product Ions (m/z). An isobar selected from the Available Isobars list will be highlighted in the peak list.
The Conditions Tab
Once the modeler has returned an initial result, the model’s conditions can be tailored further in the Conditions tab. It is important to note that the modeler is designed to provide conservative results that underpredict resolution. This means that if coelutions remain in the model even after optimization in the Conditions tab, separation may still be possible in the lab under the modeled conditions or by testing additional variables (e.g., mobile phase additives). For further discussion about separating unresolved isobars in the lab, please review our technical article.
The Conditions tab allows users to optimize their starting separation while maintaining critical pair separations by doing the following:
- Changing column dimension.
- Adjusting dwell and extra column volume to account for user-specific instrument/system effects.
- Changing mobile phase and run temperature.
- The ability to alter the Mobile Phase additive will be available for specific libraries (e.g., Cannabinoids library "Eluent A" buffer strength, mM).
- Creating a multistep gradient program.
Both the chromatogram and displayed results update with each optimization, allowing the user to track how each change affects the overall separation.
Alter the conditions of the model in the Conditions tab.
Column
Length
LC column length in millimeters (mm). This will be stated on the column box and the column label (e.g., 150 mm).
Inner Diameter
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).
Particle Size
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).
Available Columns
To pick a Restek-supplied column dimension, choose an alternative from the Available Columns list. Note that not every column format is presented in the list; a curated list of only the columns best suited to the analysis are presented for each compound library.
Volume Effects
Entering an instrument’s Dwell Volume and Extra-Column Volume will return a model more tuned to that specific instrument.
Dwell Volume
Changes in the mobile phase gradient do not reach the column instantly; time is required for the new composition to travel through the system. The system volume required for this change to occur is called the instrument’s “dwell volume,” and it is defined as the volume contained in the length and diameter of the flow path between the point at which the solvents are mixed and the column head. 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 development. Typically, an instrument’s dwell volume is determined to start at either the mixer or the gradient proportioning valve (for quaternary pumps), and it includes tubing from the mixer to the injection valve, the sample loop, and tubing from the injection valve to the column. If customizations have been made to the tubing, there are a number of online calculators that can easily determine the volume of various tubing lengths and IDs. Here is one:
https://www.omnicalculator.com/construction/pipe-volume
To find an instrument’s dwell volume, consult instrument documentation or contact the instrument manufacturer. If instrument-specific dwell volume values are not available, the default values applied by the software will provide a conservative estimation. Retention time predictions may be affected, but they should still represent an approximation of the actual times observed.
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, in the case of flow cell-based detectors, even detector volume. 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 chromatogram modeler to better calculate critical pair resolution in the model. Each LC instrument has its own value: if it’s not found in the documentation supplied with the instrument, contact the manufacturer.
If the 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, and mass on-column scaling errors). However, the calculated resolution values should be representative of observed resolution values within +/- 25%. If an instrument-specific ECV effect value is not available, the modeler’s default value will provide a conservative estimation. The model 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.
PFAS Delay Column Effects on tR and Pressure
Effects on tR: A PFAS delay column, correctly installed after the mobile phase mixer and before the sample injector, delays the system-related interference and allows accurate measurement of PFAS in your sample. Delay column effects on analyte retention time (tR) can be simulated by increasing the Dwell Volume parameter in the Volume Effects field in the Conditions tab. For example, the effects from using a 50 mm x 2.1 mm delay column can be accounted for by increasing the Dwell Volume parameter by ~0.09 mL (see calculation below).
- Column Volume: V = πr2L
- V = π (1.05 mm)2 × 50 mm ≈ 170 mm3
- 170 mm3 × (0.001 mL / 1 mm3) ≈ 0.17 mL × ~0.5* ≈ 0.09 mL
If using the default Dwell Volume of 0.25 mL, increase the value to 0.34 mL.
*Approximates the free space in a packed column at 50%.
For a 50 mm x 2.1 mm delay column, expect retention time (tR) increases of ~6 seconds for early eluting compounds and ~12 seconds for late eluting compounds.
Effects on Pressure: A delay column will INCREASE your instrument system pressure. This effect on pressure is NOT accounted for in the modeler by increasing the Dwell Volume (to simulate tR effects). The amount of increase is dependent on particle size, as show in the examples below:
- 5 µm (50 x 2.1 delay column): ~1500–2000 psi increase
- 3 µm (50 x 2.1 delay column): ~2000–3000 psi increase
- 2 µm (50 x 2.1 delay column): ~3500–4000 psi increase
Mobile Phase
Eluent A, Eluent B
Pro EZLC libraries are curated by Restek’s chemists using the most appropriate mobile phases, additives, and buffers for the compound set. From library to library, different eluents, additives, or buffers may be available, and, if so, will be selectable via a drop-down list. For example, the Drugs of Abuse (DOA) library allows for either Methanol or Acetonitrile as Eluent B.
To learn more about the effects of solvent choice on LC separations, review our technical article: https://www.restek.com/articles/effect-of-organic-solvent-on-selectivity-in-lc-separations
Temperature
The allowed temperature range is specific to the library in use. For the Drugs of Abuse library, for example, the allowed range is 30 °C to 60 °C.
Back Pressure
Back pressure, calculated by the modeler, is estimated based on the flow rate and selected column parameters. Choose pressure units from the drop-down menu. Options are psi, kPa, bar, and atm. Selected pressure units will persist between searches and between sessions of usage of the modeler.
Practical Flow Rate Ranges by Column ID
- 2.1 mm ID columns: 0.4–0.6 mL/min
- 3.0 mm ID columns: 0.7–1.0 mL/min
- 4.6 mm ID columns: 1.4–1.6 mL/min
Pressure Limitations for Different Systems
- UHPLC: 15,000 psi (~1000 bar)
- Intermediate HPLC: 8700 psi (~600 bar)
- HPLC: 5800 psi (~400 bar)
Gradient Program
Gradient programs can be changed, added, and removed in this section. To add gradients, change the value in the # of Gradient Steps field to a value from 1 to 3. The modeler will automatically insert the new steps, assigning values that maintain the original gradient program’s slope. The steps can then be adjusted to change the slope of the gradient profile.
In addition to the gradient steps, holds can be added at the beginning and end of the slope. A final re-equilibration can also be added to aid in resetting the system for the next run.
In most cases, acceptable resolution can be obtained using a simple gradient with one to three steps. However, if a complex gradient with more steps is desired, the holds and re-equilibration field can be used to create the functional equivalent of a six-step gradient program. Users should note that six is the maximum number of program steps, including all holds, gradient steps, and re-equilibration.
Add Start Isocratic Hold
An initial isocratic hold is added to the gradient program by selecting the Add Start Isocratic Hold box. The program will automatically increment the gradient program with a one-minute hold, which can then be adjusted.
Add Final Isocratic Hold
To add an isocratic hold at the end of the analysis, selecting the Add Final Isocratic Hold will automatically increment the gradient program with a one-minute hold, which can then be adjusted accordingly.
| Some methods will require a final isocratic hold to allow all compounds to exit the column. When a final isocratic hold is required, it will be automatically added and will have the minimum value needed. |
Add Re-Equilibration Time
The Add Re-Equilibration Time box adds a step returning the gradient to the initial %B value. The default number of rinses time is what is necessary to rinse 10 column volumes at the given flow rate. Adjusting the # of Column Rinses field will increase or decrease the length of this re-equilibration step.
| There is a minimum number of column rinses allowed by the software. In addition to column volume, the software takes into account the extra column volume that is also necessary to rinse to completely return the system to the original mobile phase composition. |
Optimize Gradient Slope
The Optimize Gradient Slope button is a quick way to refine a model’s oven program for specific compounds. The button examines a wide range of alternate %B ramps and returns a model that meets or bests the previous run time and compounds resolved if possible. If more refinements are possible, a circular arrow appears next to the button. The button will disable when no further refinements are available. Changes can be undone via the Undo button in the Results section (or by pressing Ctrl-Z). Note: optimization of %B gradients that are very slight (e.g.,1%) may produce results with a larger gradient.
Target Resolution
The Target Resolution field is defaulted to 1.5, which is full baseline separation. If a resolution lower than baseline is acceptable, it can be changed here. Always ensure minimum Target Resolution values meet your lab's analytical or quality requirements.
Raising this value may result in longer run times, and lowering this value may decrease the run times (sometimes significantly).
| Warning! When optimizing the gradient program, compounds not already meeting the Target Resolution may be sacrificed to achieve better run times for the compounds that are adequately separated. If the optimization does not resolve all the targeted compounds to the Target Resolution after refinement, undo to the initial starting position, enter a lower value for the Target Resolution, and try again. To maintain the resolution of particular compounds, lower the Target Resolution to match the resolution of those compounds before refining. |
To maintain the resolution of peaks 160 and 165 above, change the target resolution to 0.60 before refining.
Isocratic Program
An isocratic program option is available for specific libraries (e.g., Cannabinoids). Flowrate and %B, with range max and min respectively, are the sole adjustment parameters. The total run time is based on the Rt of the latest eluting compound, whereby ~1 min of additional time is added to account for remaining system volume.
Results
To undo any changes made in the Conditions tab, click the Undo button in the Results section, or press Ctrl-Z on the keyboard. To redo a change, hit the Redo button or press Ctrl-Y on the keyboard.
| Rerunning a compound list in the Compounds tab will reset the Conditions tab, clearing any changes. |
Gradient Time + Delay/Run Time
“Gradient Time + Delay” is the total time necessary for the programmed gradient to reach the end of the column. It takes column volume, extra column volume, and flow rate into account. Note that this time may be longer than the mobile phase program provided by the modeler because it may not be necessary to achieve the final gradient condition throughout the column to elute the last compound being modeled. To see the mobile phase composition at the time of a given compound’s elution, check the peak list table provided. The “Exit %B” column reports the actual mobile phase condition present in the column at the time of that compound’s elution.
“Run Time” adds the time of any steps added to the mobile phase program, such as additional isocratic holds or column rinses to the Gradient Time + Delay. Note that this may not correspond exactly to the actual analysis time for the reasons noted above.
The Void Time (T0)
The void time (T0) is the time it would take for an unretained compound to travel from the injection port, through the column, to the detector.
Isobaric Compounds Separated
The Isobaric Compounds Separated value shows the number of targeted compounds that meet or exceed the Target Resolution. (See more on Target Resolution in the Optimize Gradient Program section.) This value does not reflect the actual total number of compounds separated–only the isobaric compounds.
Critical Pair
Reports the peak numbers of the least resolved isobaric compounds in the entire analysis.
Critical Pair Resolution
Reports the resolution of the least resolved isobaric compounds in the entire analysis.
Compound Structure and Properties View
To view more information on a particular compound, click on the magnifying glass beside the compound name. The pop-up that appears will contain compound details, as available.
The My EZLC Tab
The My EZLC tab houses saved models.
Saving a Model
To save a model, simply do the following:
- Give it a title by clicking on “Untitled. Click here to edit.” at the top of the modeled result. A title textbox field will become visible. Enter a title and click OK or hit the Enter key on the keyboard to lock in the new title.
- Hit the Save button in the top bar of the Pro EZLC chromatogram modeler.
| Untitled saves are possible, but not recommended. A title can be added, and the model resaved, even after loading the model on a subsequent visit. |
To save a variation on a model, make changes in the Conditions tab, change the model’s title as needed, then click the Save A Copy button. A new entry will appear in the My EZLC list of saved models. Models can be compared by clicking on their entries in the My EZLC list.
A model from the My EZLC list can be resaved by clicking the Save button.
To delete a model, click the X in the upper right corner of a model’s listing, and click the OK button in the confirmation alert that pops up.
Note that saved models can be further optimized using the Conditions tab; however, compounds cannot be added to or subtracted from the Compound tab during subsequent work sessions.
Printing a Model
Models can be printed by selecting the print button in the toolbar, which will bring up the browser’s print dialog.