Find valuable information.
Optimize your experiments to get the best results. We’ve compiled a detailed knowledge base of the top tips and tricks to meet your analyses needs.
View the relevant questions below:
Mass deviation is usually defined in the relative means of ppm. We specify 3 ppm for 24h for external calibration and 1 ppm for internal calibration (working with lock mass).
Mass deviation (ppm) = (Theoretical monoisotopic mass –Observed mass) / Theoretical monoisotopic mass x 106.
This depends on the substances and the matrix you are working with. Theoretically, SIM would be the most sensitive mode. However, sometimes PRM turns out to be most sensitive due to interference of the matrix. Usually Full Scan provides sensitive measurements, too.
AGC targets of Full Scan: 1e6 (1e6= 1.000.000), SIM/PRM: 2e5 (2e5=200.00) are good starting points. The more similar the ions are which are collected in the C-trap, the lower the target should be.
Max IT: The following scan times are required for the different resolution settings below:
|Instrument resolution||Transient time|
|Exactive Plus (EMR)
Q Exactive Focus
Q Exactive Plus
|Q Exactive HF Q Exactive HF-X|
|a only Q Exactive Plus
b highest resolution for Q Exactive Focus
c only Q Exactive HF-X
In the simplest case, set the max IT to times just below these values – parallel acquisition.
In Full Scan mode, usually the defined max IT time is not used by the system as the ion flux is very high. In data dependent MS2 scans, the time can be extended to receive a better spectrum quality. However, this might generate less data points.
Suggested resolution settings are as follows:
|SIM||35,000 or 70,000
(30,000 or 60,000)
|*depending on instrument type|
In PRM you can choose a lower resolution because of the selectivity of MS2 ions.
If you need to do polarity switching in Full Scan mode, it is a good idea to reduce resolution to 35,000 to generate more data points. However, this might generate less data points.
If you want to work qualitatively you might want to consider to work with lock masses and the highest resolution (fine isotopic pattern).
Mass calibration should be performed daily by default. If the lab qualified the instrument in their controlled conditions, weekly calibration is fine. System calibration should be performed monthly. Generally, we specify less than 3 ppm mass deviation within 24 h.
Yes, it is possible to work in polarity switching mode. The switching time is about 250 ms. With a resolution of 35000 it takes about 1 s for a cycle of one positive and one negative scan (data point).
AGC stands for Automatic Gain Control.
The Automatic Gain Control alters the number of ions in the Orbitrap by filling the C-trap for a calculated period of time. This way, ions can enter the mass analyzer at specified times only.
The ions are gated in the C-Trap and compressed into a narrow cloud before being pushed out to the Orbitrap. The AGC is calculated and during the injection time is when the ions can pass into the C-Trap. Hence, Ion “gating” occurs by changing the polarity of the split lens after the first multipole.
Depending on the experiment and other instrument parameters, the AGC is calculated in three different ways:
AGC Calculated with a Prescan:
When the system is performing the first scan of the run, the instrument will perform a Prescan to calculate the number of ions at that time and to adjust the injection time for the analytical scan accordingly. This is achieved by opening the trap for 1 ms and by performing an acquisition in the Orbitrap (~200 ms)
AGC Calculate on a Scan-to-Scan basis:
When two consecutive full scans are acquired within 400 ms, the instrument will use the previous Full Scan TIC to adjust the injection time.
pAGC (predictive AGC):
When the method is setup to perform data dependent MS experiments, the dependent scans will use their master scan (Full Scan) as a reference for the AGC calculation.
The HCD Cell consists of a straight multipole (octapole) device mounted within a metal tube. The metal tube is in turn connected to the C-Trap in direct line of sight.
The voltage offset between the C-Trap and the HCD Cell is used to accelerate the precursor ions into the gas filled cell. Inside the HCD cell, ions collide with the neutral gas causing rotation, stretching and ultimately cleaving bonds to create fragment ions and neutral species, which are subsequently removed by the vacuum system.
The generated fragment or product ions are then pushed back toward the C-Trap and focused before being pushed toward the Orbitrap analyzer.