Peak saturation with calibration curve

Hello,

I am trying to increase the range of my calibration curve but I have problems with saturation. First, if the integration list says the peak is saturated, does that mean the peak area is unreliable to use in the calibration curve? In the attached image, the red peak is the standard extract with 10:1 split ratio, the blue peak is the same extract with 50:1 split ratio, and the black peak is 5x diluted extract with 50:1 split ratio. According to the integration list only the black peak is saturated even though we know less analyte has been injected. How does this saturation relate to concentration? I am also assuming the saturation has to do with the number of data points at the peak. Are there certain MS parameters I should adjust to eliminate the saturation? I am also interested in advice to reduce the tailing as concentration decreases.

0 paulsalverda 2 months ago

The maximum abundance per ion on Agilent Single Quadrupole GCMS instruments is approximately 8.4 × 10⁶, To stay within the linear range of the electron multiplier it is recommended to stay at or below 2.0 × 10⁶ for SQ.

This large peak is 54,000,000 counts tall. You need to further dilute your samples, dramatically increase your split ratio, inject less sample, or do all three. You want your smallest calibration peak to be maybe 10,000 counts or less tall. The smallest peak shown is 2,000,000. The smallest peak of interest only needs to be about three to five times taller than the background noise to be valid.

The less you inject of the lowest concentration and cleanest samples possible will require less inlet, column, and ion source maintenance.

What is the sample? What are the GC parameters? Please upload a copy of the acqmeth.txt file that is located underneath your method's ****.M subdirectory.

Also older versions of MassHunter Acquisition and old MSD Chemstation sometimes had trouble with data files names longer than 40 characters and this one is already 42 characters long.
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0 kayleemorton 2 months ago in reply to paulsalverda

Thanks for the response.

The sample I am measuring is methyl methacrylate. In the attached image, the black peak (measured in scan mode) is still saturated even though the counts are relatively lower. The red peak is the EIC for 100.0 ion, which we use for quantification, and it is not saturated. If the peak is saturated in scan, but the EIC for quant ion is not saturated is it okay to use the peak areas for calibration curve? Is any "saturated" peak reliable for calibration?

We also noticed that smoothing the chromatogram eliminates the saturation issue, but I am not familiar with smoothing parameters. Some of the saturated peaks have flat tops prior to smoothing, which is why I thought increasing the MS sampling may help.

Fullscreen 4657.acqmeth.txt Download

                       INSTRUMENT CONTROL PARAMETERS:    1
                       -----------------------------------

      Wed Feb 14 11:37:32 2024

Control Information
------- -----------

Sample Inlet             : GC
Injection Source         : External Device
Mass Spectrometer        : Enabled

Injection Location:  Front


 No Sample Prep method has been assigned to this method.


GC
GC Summary
Run Time                                     22.133 min
Post Run Time                                0 min

Oven
Temperature
Setpoint                                     On
(Initial)                                    32 °C
Hold Time                                    7 min
Post Run                                     70 °C
Program
#1 Rate                                      10 °C/min
#1 Value                                     70 °C
#1 Hold Time                                 0 min
#2 Rate                                      30 °C/min
#2 Value                                     320 °C
#2 Hold Time                                 3 min


Equilibration Time                           0.25 min
Max Temperature                              320 °C
Maximum Temperature Override                 Disabled
Slow Fan                                     Disabled

Front SS Inlet He
Excluded from Readiness                      ***Excluded from Affecting GC's Readiness State***
Mode                                         Split
Heater                                       On    250 °C
Pressure                                     On    17.915 psi
Total Flow                                   On    91.23 mL/min
Septum Purge Flow                            On    3 mL/min
Pre-Run Flow Test                            Off
Gas Saver                                    On    20 After 3 min mL/min
Split Ratio                                  50 :1
Split Flow                                   86.5 mL/min
Liner                                        Agilent 5190-2293: 900 μL (Splitless, single taper, wool, Ultra )

Back PTV Inlet He
Mode                                         Sleep
Pressure                                     Off
Septum Purge Flow                            Off
Pre-Run Flow Test                            Off
Liner                                        Agilent G1544-80730: 675 μL (Direct connect, single taper, bottom )

Column
Column #1
Flow
Setpoint                                     On
(Initial)                                    1.73 mL/min
Post Run                                     0.57353 mL/min

Column Information                           Agilent
Restek-Rxi5SilMS                             
Temperature Range                            -60 °C—350 °C (350 °C)
Dimensions                                   30 m x 250 μm x 0.25 μm
Column lock                                  Unlocked
In                                           Front SS Inlet He
Out                                          PSD 2 
(Initial)                                    32 °C
Pressure                                     17.915 psi
Flow                                         1.73 mL/min
Average Velocity                             34.773 cm/sec
Holdup Time                                  1.4379 min
Control Mode                                 Constant Flow

Column #2
Pressure
Setpoint                                     On
(Initial)                                    2 psi
Post Run                                     10 psi

Temperature Range                            -1 °C—325 °C (325 °C)
Dimensions                                   5 m x 200 μm x 0 μm
Column lock                                  Unlocked
In                                           PSD 2 He
Out                                          MSD 
(Initial)                                    32 °C
Pressure                                     2 psi
Flow                                         1.5224 mL/min
Average Velocity                             109.14 cm/sec
Holdup Time                                  0.076352 min
Control Mode                                 Constant Pressure

Column Outlet Pressure                       0 psi

Front Detector FID
Excluded from Readiness                      ***Excluded from Affecting GC's Readiness State***
Makeup                                       He
Heater                                       On    300 °C
H2 Flow                                      Off
Air Flow                                     Off
Makeup Flow                                  Off
Carrier Gas Flow Correction                  Constant Makeup and Fuel Flow
Flame                                        Off

Detector Evaluation
Perform Detector Evaluation Test             Off
Signal Selected                              Signal #1: None
Checkout Sample                              None


MSD Transfer Line
Temperature
Setpoint                                     On
(Initial)                                    320 °C



Aux EPC 1,2,3
Aux EPC 1 N2
Pressure
Setpoint                                     Off
(Initial)                                    10 psi
Post Run                                     10 psi

Excluded from Readiness                      ***Excluded from Affecting GC's Readiness State***

Aux EPC 2 N2
Pressure
Setpoint                                     Off
(Initial)                                    10 psi
Post Run                                     10 psi

Excluded from Readiness                      ***Excluded from Affecting GC's Readiness State***

Aux EPC 3 N2
Pressure
Setpoint                                     Off
(Initial)                                    10 psi
Post Run                                     10 psi

Excluded from Readiness                      ***Excluded from Affecting GC's Readiness State***


PSD 2
Excluded from Readiness                      ***Excluded from Affecting GC's Readiness State***
PSD Purge                                    On    0 mL/min
PSD 2 He                                     Supplies Column 2

Signals
Signal #1: 
Description                                  None

Signal #2: 
Description                                  None

Signal #3: 
Description                                  None

Signal #4: 
Description                                  None

Signal #5: 
Description                                  None

Signal #6: 
Description                                  None

Signal #7: 
Description                                  None

Signal #8: 
Description                                  None






                            GERSTEL MAESTRO


SYSTEM SETTINGS
   Maestro Runtime         : 22.13 min
   GC Cool Down Time       : 5.00 min
   Cryo Timeout            : 15.00 min



                               GERSTEL CIS


   CIS                     : not used  -  use these parameters if it becomes 'used'

CRYO COOLING
   Cryo Cooling            : used

TEMPERATURE PROGRAM
   Heater Mode             : Standard

   Liner Type              : 013882-010-00 glass liner CIS4, baffled, non deactivated
   Liner Max Temperature   : 400 'C

   Initial Temperature     : -10 'C
   Equilibration Time      : 0.50 min
   Initial Time            : 0.50 min
   Ramp 1                 
       Rate                : 12.00 'C/s
       End Temp            : 325 'C
       Hold Time           : 5.00 min
   Ramp 2                 
       Rate                : 0.0 'C/s

   Low Split               : not used



                               GERSTEL MPS PREP


   Sample Prep             : used

PREP ACTION LIST

     Action          Method/Value          Source                Destination
     =============   ===================   ===================   ===================






                        GERSTEL MPS Liquid Injection

   Syringe                 : 10ul 65mm USM

SAMPLE PARAMETERS
   Sandwich                : not used

   Inj. Volume             : 2.0 uL
   Air Volume below        : 0.0 uL

   Inj. Speed              : 50.00 uL/s
   Fill Volume             : 5.0 uL
   Fill Strokes            : 5

   Fill Speed              : 2.00 uL/s
   Viscositiy Delay        : 0 s
   Eject Speed             : 50.00 uL/s

   Pre Inj. Delay          : 0 s
   Post Inj. Delay         : 0 s

   Inj. Penetration        : 40.00 mm
   Sample Tray Type        : VT54/2-CVM
   Bottom Sense            : used
   Fast Injection          : used

CLEANING PARAMETERS
   Preclean Sample         : 2

   Wash Station 1          : Pos1@Standard Wash 1
   Preclean Solv.1         : 2
   Postclean Solv.1        : 6
   Clean Volume Solv.1     : 90 %
   Fill Speed Solv.1       : 5.00 uL/s
   Viscosity Delay Solv.1  : 0 s
   Eject Speed Solv.1      : 50.00 uL/s
   Information Solv.1      : Hexane

   Wash Station 2          : Pos2@Standard Wash 1
   Preclean Solv.2         : 0
   Postclean Solv.2        : 0
   Clean Volume Solv.2     : 70 %
   Fill Speed Solv.2       : 5.00 uL/s
   Viscosity Delay Solv.2  : 0 s
   Eject Speed Solv.2      : 50.00 uL/s

MS Information
-- -----------


General Information
------- -----------


Acquisition Mode         : Scan
Solvent Delay (minutes)  : 3.3
Tune file                : D:\MassHunter\GCMS\1\5977\etune.u
EM Setting mode Gain     : 5.000000

Normal or Fast Scanning  : Normal Scanning
Trace Ion Detection      : Off
Run Time (if MS only)    : 23.67 minutes 

[Scan Parameters]
Start Time               : 3.3
Low Mass                 : 40
High Mass                : 300
Threshold                : 300
A/D Samples:             : 4


[MSZones]

MS Source                   : 230 C   maximum 250 C
MS Quad                     : 150 C   maximum 200 C

Timed Events
----- ------
Number Events= 1


[Timed MS Detector Table Entries]

Time (min)                 State (MS On/off)
10.00                  off





                     END OF MS ACQUISITION PARAMETERS


                        TUNE PARAMETERS for SN: US2018M021
                        ---------------------------------

 Trace Ion Detection is OFF.

    34.593   :   EMISSION     
    70.007   :   ENERGY       
     3.790   :   REPELLER     
    89.822   :   IONFOCUS     
    20.181   :   ENTRANCE_LENS
  1669.658   :   EMVOLTS      
                              2061.7  : Actual EMV 
                               5.00   : GAIN FACTOR 
  1872.000   :   AMUGAIN      
   133.875   :   AMUOFFSET    
     1.000   :   FILAMENT     
     0.000   :   DCPOLARITY   
     6.990   :   ENTLENSOFFSET
     9.502   :   Ion_Body     
     4.006   :   EXTLENS      
  -250.000   :  MASSGAIN        
   -31.000   :  MASSOFFSET      

                        END OF TUNE PARAMETERS
                        ----------------------



                      END OF INSTRUMENT CONTROL PARAMETERS
                      ------------------------------------

0 paulsalverda 2 months ago in reply to kayleemorton

There's a lot going on. You have Column 1 in Constant Flow to a PSD in Constant Pressure with PSD Purge at 0 ml/min. Is there a purged splitter plate installed or a Purged Ultimate Union? The flow into the MS changes throughout the run due to the PSD Constant Pressure setpoint, so at the beginning of the run when the Oven Temp is 32°, the flow into the MS is a bit high at 1.5524 ml/min but at the end of the run the flow is only 0.498 ml/min. The analytical column constant flow is 1.4379 - so if there is no other place for it to go like to an FID through a splitter, at the end of the run the excess 0.94 ml/min has to go somewhere, so it is going out the PSD purge line and contaminating the PSD flowpath. This needs some investigation.

Here are some observations and recommendations.
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0 kayleemorton 2 months ago in reply to paulsalverda

We have the front inlet connected to column> CFT Device> MS transfer line. Our CFT has a purge/vent valve. We have not noticed any carryover or contamination between runs so we are not immediately worried about the purge line, but would you recommend connecting the 2nd column to FID in the future?

Referring to the figures-

The red peak was injected under the previous method. Saturation did not occur. Then we changed to the splitless liner and re-injected (black peak), and the signal increased with saturation. Then we applied some of your recommendations to the method (eqb time, injection temp, and A/D samples) and re-injected (green peak) and again we see saturation. Why is the green peak saturated when it is the lowest abundance? Is there another parameter besides abundance which determines saturation, like the peak width?

Red peak- injected using the previously described method

Black peak- Changed from splitless liner to split liner (5190-2295), injected with the same method

Green peak- 2295 split liner and some recommendations (equilibration time, injection temp, and A/D sampling)

Fullscreen 7851.acqmeth.txt Download

                      INSTRUMENT CONTROL PARAMETERS:    1
                      -----------------------------------

      Thu Feb 29 11:53:58 2024

Control Information
------- -----------

Sample Inlet             : GC
Injection Source         : External Device
Mass Spectrometer        : Enabled

Injection Location:  Front


 No Sample Prep method has been assigned to this method.


GC
GC Summary
Run Time                                     18.8 min
Post Run Time                                0 min

Oven
Temperature
Setpoint                                     On
(Initial)                                    32 °C
Hold Time                                    7 min
Post Run                                     70 °C
Program
#1 Rate                                      10 °C/min
#1 Value                                     70 °C
#1 Hold Time                                 0 min
#2 Rate                                      30 °C/min
#2 Value                                     280 °C
#2 Hold Time                                 1 min


Equilibration Time                           2 min
Max Temperature                              320 °C
Maximum Temperature Override                 Disabled
Slow Fan                                     Disabled

Front SS Inlet He
Excluded from Readiness                      ***Excluded from Affecting GC's Readiness State***
Mode                                         Split
Heater                                       On    250 °C
Pressure                                     On    17.915 psi
Total Flow                                   On    91.23 mL/min
Septum Purge Flow                            On    3 mL/min
Pre-Run Flow Test                            Off
Gas Saver                                    On    20 After 3 min mL/min
Split Ratio                                  50 :1
Split Flow                                   86.5 mL/min
Liner                                        Agilent 5190-2293: 900 μL (Splitless, single taper, wool, Ultra )

Back PTV Inlet He
Mode                                         Sleep
Pressure                                     Off
Septum Purge Flow                            Off
Pre-Run Flow Test                            Off
Liner                                        Agilent G1544-80730: 675 μL (Direct connect, single taper, bottom )

Column
Column #1
Flow
Setpoint                                     On
(Initial)                                    1.73 mL/min
Post Run                                     0.57353 mL/min

Column Information                           Agilent
Restek-Rxi5SilMS                             
Temperature Range                            -60 °C—350 °C (350 °C)
Dimensions                                   30 m x 250 μm x 0.25 μm
Column lock                                  Unlocked
In                                           Front SS Inlet He
Out                                          PSD 2 
(Initial)                                    32 °C
Pressure                                     17.915 psi
Flow                                         1.73 mL/min
Average Velocity                             34.773 cm/sec
Holdup Time                                  1.4379 min
Control Mode                                 Constant Flow

Column #2
Pressure
Setpoint                                     On
(Initial)                                    2 psi
Post Run                                     10 psi

Temperature Range                            -1 °C—325 °C (325 °C)
Dimensions                                   5 m x 200 μm x 0 μm
Column lock                                  Unlocked
In                                           PSD 2 He
Out                                          MSD 
(Initial)                                    32 °C
Pressure                                     2 psi
Flow                                         1.5224 mL/min
Average Velocity                             109.14 cm/sec
Holdup Time                                  0.076352 min
Control Mode                                 Constant Pressure

Column Outlet Pressure                       0 psi

Front Detector FID
Excluded from Readiness                      ***Excluded from Affecting GC's Readiness State***
Makeup                                       He
Heater                                       On    300 °C
H2 Flow                                      Off
Air Flow                                     Off
Makeup Flow                                  Off
Carrier Gas Flow Correction                  Constant Makeup and Fuel Flow
Flame                                        Off

Detector Evaluation
Perform Detector Evaluation Test             Off
Signal Selected                              No Signal Selected
Checkout Sample                              None


MSD Transfer Line
Temperature
Setpoint                                     On
(Initial)                                    320 °C



Aux EPC 1,2,3
Aux EPC 1 N2
Pressure
Setpoint                                     Off
(Initial)                                    10 psi
Post Run                                     10 psi

Excluded from Readiness                      ***Excluded from Affecting GC's Readiness State***

Aux EPC 2 N2
Pressure
Setpoint                                     Off
(Initial)                                    10 psi
Post Run                                     10 psi

Excluded from Readiness                      ***Excluded from Affecting GC's Readiness State***

Aux EPC 3 N2
Pressure
Setpoint                                     Off
(Initial)                                    10 psi
Post Run                                     10 psi

Excluded from Readiness                      ***Excluded from Affecting GC's Readiness State***


PSD 2
Excluded from Readiness                      ***Excluded from Affecting GC's Readiness State***
PSD Purge                                    On    0 mL/min
PSD 2 He                                     Supplies Column 2

Signals
Signal #1: 
Description                                  None

Signal #2: 
Description                                  None

Signal #3: 
Description                                  None

Signal #4: 
Description                                  None

Signal #5: 
Description                                  None

Signal #6: 
Description                                  None

Signal #7: 
Description                                  None

Signal #8: 
Description                                  None






                            GERSTEL MAESTRO


SYSTEM SETTINGS
   Maestro Runtime         : 22.13 min
   GC Cool Down Time       : 5.00 min
   Cryo Timeout            : 15.00 min



                               GERSTEL CIS


   CIS                     : not used  -  use these parameters if it becomes 'used'

CRYO COOLING
   Cryo Cooling            : used

TEMPERATURE PROGRAM
   Heater Mode             : Standard

   Liner Type              : 013882-010-00 glass liner CIS4, baffled, non deactivated
   Liner Max Temperature   : 400 'C

   Initial Temperature     : -10 'C
   Equilibration Time      : 0.50 min
   Initial Time            : 0.50 min
   Ramp 1                 
       Rate                : 12.00 'C/s
       End Temp            : 325 'C
       Hold Time           : 5.00 min
   Ramp 2                 
       Rate                : 0.0 'C/s

   Low Split               : not used



                               GERSTEL MPS PREP


   Sample Prep             : used

PREP ACTION LIST

     Action          Method/Value          Source                Destination
     =============   ===================   ===================   ===================






                        GERSTEL MPS Liquid Injection

   Syringe                 : 10ul 65mm USM

SAMPLE PARAMETERS
   Sandwich                : not used

   Inj. Volume             : 2.0 uL
   Air Volume below        : 0.0 uL

   Inj. Speed              : 50.00 uL/s
   Fill Volume             : 5.0 uL
   Fill Strokes            : 5

   Fill Speed              : 2.00 uL/s
   Viscositiy Delay        : 0 s
   Eject Speed             : 50.00 uL/s

   Pre Inj. Delay          : 0 s
   Post Inj. Delay         : 0 s

   Inj. Penetration        : 40.00 mm
   Sample Tray Type        : VT54/2-CVM
   Bottom Sense            : used
   Fast Injection          : used

CLEANING PARAMETERS
   Preclean Sample         : 2

   Wash Station 1          : Pos1@Standard Wash 1
   Preclean Solv.1         : 2
   Postclean Solv.1        : 6
   Clean Volume Solv.1     : 90 %
   Fill Speed Solv.1       : 5.00 uL/s
   Viscosity Delay Solv.1  : 0 s
   Eject Speed Solv.1      : 50.00 uL/s
   Information Solv.1      : Hexane

   Wash Station 2          : Pos2@Standard Wash 1
   Preclean Solv.2         : 0
   Postclean Solv.2        : 0
   Clean Volume Solv.2     : 70 %
   Fill Speed Solv.2       : 5.00 uL/s
   Viscosity Delay Solv.2  : 0 s
   Eject Speed Solv.2      : 50.00 uL/s

MS Information
-- -----------


General Information
------- -----------


Acquisition Mode         : Scan
Solvent Delay (minutes)  : 3.3
Tune file                : D:\MassHunter\GCMS\1\5977\etune.u
EM Setting mode Gain     : 5.000000

Normal or Fast Scanning  : Normal Scanning
Trace Ion Detection      : Off
Run Time (if MS only)    : 23.67 minutes 

[Scan Parameters]
Start Time               : 3.3
Low Mass                 : 40
High Mass                : 250
Threshold                : 300
A/D Samples:             : 2


[MSZones]

MS Source                   : 230 C   maximum 250 C
MS Quad                     : 150 C   maximum 200 C

Timed Events
----- ------
Number Events= 1


[Timed MS Detector Table Entries]

Time (min)                 State (MS On/off)
10.00                  off





                     END OF MS ACQUISITION PARAMETERS


                        TUNE PARAMETERS for SN: US2018M021
                        ---------------------------------

 Trace Ion Detection is OFF.

    34.593   :   EMISSION     
    70.007   :   ENERGY       
     3.989   :   REPELLER     
    89.822   :   IONFOCUS     
    20.181   :   ENTRANCE_LENS
  1668.925   :   EMVOLTS      
                              2059.7  : Actual EMV 
                               5.00   : GAIN FACTOR 
  1875.000   :   AMUGAIN      
   133.563   :   AMUOFFSET    
     1.000   :   FILAMENT     
     0.000   :   DCPOLARITY   
     6.990   :   ENTLENSOFFSET
     9.502   :   Ion_Body     
     4.106   :   EXTLENS      
  -251.000   :  MASSGAIN        
   -31.000   :  MASSOFFSET      

                        END OF TUNE PARAMETERS
                        ----------------------



                      END OF INSTRUMENT CONTROL PARAMETERS
                      ------------------------------------

0 paulsalverda 2 months ago in reply to kayleemorton

Saturation is reported by ion, not by TIC abundance.

The maximum possible abundance per ion on SQ instruments is approximately 8.4 × 10^6.
To stay within the linear range of the electron multiplier it is recommended to be <= 2.0 × 10^6 per ion.

Please inject less sample, maybe 0.3µL, and/or increase the split ratio, like up to 250:1 or 500:1.
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