FAQ - Frequently Asked Questions
(LC) Settings for Fast Analysis (UHPLC)
(LC) Settings for Fast Analysis (UHPLC)
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Answer
1. Setting the Fast LC Mode
Fast LC (Mode): Select this for fast gradient analysis, such as when the mobile phase ratio is changed within 3 minutes. This mode allows obtaining satisfactory retention time reproducibility for fast gradients by dividing changes in mobile phase flow rate into micro-steps.
Fast LC (Mode): Select this for fast gradient analysis, such as when the mobile phase ratio is changed within 3 minutes. This mode allows obtaining satisfactory retention time reproducibility for fast gradients by dividing changes in mobile phase flow rate into micro-steps.
« Restrictions in Fast LC Mode »
The following restrictions apply when the control mode is set to Fast LC mode instead of Normal mode.
• Valid Instrument Configurations for Fast LC Mode
Operation will be conducted in the Normal mode even if the quaternary low-pressure gradient mode (LP.GE mode) is selected.
[Control Mode] can be set(displayed) on LabSolutions whenSCL-40, CBM-40/40Lite/20A/20Alite(ROM version 1.2 or later) is used. When using CBM-20A/20Alite(ROM version 1.1), set the Control Mode on the CBM-20A/20Alite by logging on it with Internet Explorer.
• Number of steps in Time Program
In Normal mode, the time program can comprise up to 400 steps. However, in Fast LC mode, the number of steps that can be used for pump-related commands is 320 (although the total number of steps that can be used, including non-pump related commands, remains 400).
If more than 320 steps of pump-related commands are set in the time program, those pump-related commands in excess of the 320 steps will not be executed.
• Settings for Gradient Profile
The gradient profile can be set with the B.Curve parameter in Normal mode, but in Fast LC mode the profile always becomes linear regardless of the B.Curve setting. Note that only linear profile is available for the gradient operation in Fast LC mode. The gradient profile can be set with the B.Curve parameter in Normal mode, but the profile always becomes linear in Fast LC mode regardless of the setting of B.Curve.
The following restrictions apply when the control mode is set to Fast LC mode instead of Normal mode.
• Valid Instrument Configurations for Fast LC Mode
Operation will be conducted in the Normal mode even if the quaternary low-pressure gradient mode (LP.GE mode) is selected.
[Control Mode] can be set(displayed) on LabSolutions whenSCL-40, CBM-40/40Lite/20A/20Alite(ROM version 1.2 or later) is used. When using CBM-20A/20Alite(ROM version 1.1), set the Control Mode on the CBM-20A/20Alite by logging on it with Internet Explorer.
• Number of steps in Time Program
In Normal mode, the time program can comprise up to 400 steps. However, in Fast LC mode, the number of steps that can be used for pump-related commands is 320 (although the total number of steps that can be used, including non-pump related commands, remains 400).
If more than 320 steps of pump-related commands are set in the time program, those pump-related commands in excess of the 320 steps will not be executed.
• Settings for Gradient Profile
The gradient profile can be set with the B.Curve parameter in Normal mode, but in Fast LC mode the profile always becomes linear regardless of the B.Curve setting. Note that only linear profile is available for the gradient operation in Fast LC mode. The gradient profile can be set with the B.Curve parameter in Normal mode, but the profile always becomes linear in Fast LC mode regardless of the setting of B.Curve.
2. Setting for detector
Detector Response (Time Constant) and Sampling Cycle
In fast analysis (UHPLC), as compared to normal analysis, the peaks have a relatively sharper shape (narrow peak width). Therefore, it is necessary to set a fast response time. When analysis is conducted with a slow response time, peak widening occurs, and peaks that are successfully separated in the column may not be shown separated on the detector signal.
However, if a fast response time is set, the noise, which is inversely proportional to the square root, will increase accordingly. Therefore, it is necessary to set an appropriate response considering both peak separation and sensitivity.
During analytical method development, after verifying the separation at a faster response time, the response setting is then set to a lower value that still gives the required separation and reduced noise.
The relation between the response setting and peak width from the viewpoint of separation is as follows.
Detector Response (Time Constant) and Sampling Cycle
In fast analysis (UHPLC), as compared to normal analysis, the peaks have a relatively sharper shape (narrow peak width). Therefore, it is necessary to set a fast response time. When analysis is conducted with a slow response time, peak widening occurs, and peaks that are successfully separated in the column may not be shown separated on the detector signal.
However, if a fast response time is set, the noise, which is inversely proportional to the square root, will increase accordingly. Therefore, it is necessary to set an appropriate response considering both peak separation and sensitivity.
During analytical method development, after verifying the separation at a faster response time, the response setting is then set to a lower value that still gives the required separation and reduced noise.
The relation between the response setting and peak width from the viewpoint of separation is as follows.
As an example of fast analysis (UHPLC), if the first eluting peak has a half height width of 1 second, then Response should be set to 0.1 sec.
The S/N ratio improves as the response increases, but on the other hand, the sharper the peak, the worse the resolution.
Peak S/N ratio when analyzed for each response (The values in parentheses are the ratio when the S/N ratio of 20 msec is 1)
Peak resolution when analyzed in each response (The values in parentheses are the ratio when the resolution of 20msec is 100%)
The sampling period is the time required for detector signal processing, and it affects the reproducibility of peak areas and peak heights. Although, a shorter sampling period is necessary in the fast analysis (UHPLC), for reasons same as response settings, it increases the size of analysis data. Generally, the sampling period will be the same value as the response time.
* If you want to set the sampling rate to less than 100 ms (initial value) in LCsolution/LabSolutions, you must also change the detector base rate in the configuration settings (excluding PDA detectors).
* If you want to set the sampling rate to less than 100 ms (initial value) in LCsolution/LabSolutions, you must also change the detector base rate in the configuration settings (excluding PDA detectors).
3. Method setting for LCsolution/LabSolution
• The Minimum Half-Height Width of Peaks: Width
The [Width] is the most fundamental parameter among all peak integration parameters. The software detects peaks according to the [Width] value, and performs peak integration under the conditions most suitable for the peaks.
In [Width], specify the minimum half-height width (width at 50% of peak height) of peaks detected during analysis. Specifically, set the value equal to or slightly smaller than the half-height width of the sharpest peak in obtained chromatogram.
The unit of the [Width] is sec.
• The Minimum Half-Height Width of Peaks: Width
The [Width] is the most fundamental parameter among all peak integration parameters. The software detects peaks according to the [Width] value, and performs peak integration under the conditions most suitable for the peaks.
In [Width], specify the minimum half-height width (width at 50% of peak height) of peaks detected during analysis. Specifically, set the value equal to or slightly smaller than the half-height width of the sharpest peak in obtained chromatogram.
The unit of the [Width] is sec.
Appropriate Width example
The default value for Width in the LCsolution and chromate-pack series is 5s. It is appropriate to set the Width to about 4 to 5s for an ODS column with a particle size of about 5μm, and to about 0.4 to 1s for an fast LC (UHPLC) with a particle size of about 2μm.
• Peak Detection Sensitivity Determining Peak Start/End: Slope
When q is set to [Slope] as shown in Fig., the peak start is detected when the graph slope exceeds the angle. Conversely, the peak end is detected when the negative graph slope falls under the angle.
If a large value is set to [Slope], the software detects only sharp peaks with a large graph inclination. When a smaller value is set, the software detects broader peaks, as well as a lot of noises in some case.
When q is set to [Slope] as shown in Fig., the peak start is detected when the graph slope exceeds the angle. Conversely, the peak end is detected when the negative graph slope falls under the angle.
If a large value is set to [Slope], the software detects only sharp peaks with a large graph inclination. When a smaller value is set, the software detects broader peaks, as well as a lot of noises in some case.
The Slope test is the feature to automatically calculate the [Slope] value from baseline noise and drift on chromatograms.
There are two methods for obtaining the slope value: (1) using the baseline before analysis when analytical equipment signals are displayed, and (2) using the baseline on chromatograms already obtained.
The result of the Slope test depends on the value of Width, so let's perform the Slope test after confirming the Width.
There are two methods for obtaining the slope value: (1) using the baseline before analysis when analytical equipment signals are displayed, and (2) using the baseline on chromatograms already obtained.
The result of the Slope test depends on the value of Width, so let's perform the Slope test after confirming the Width.
The higher the noise level, the higher the slope of the individual noise, and the higher the appropriate Slope value. In fast LC (UHPLC), the response speed of the detector is set low, so noise is often louder than in normal HPLC, and the Slope value must also be set higher.
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