Poor Resolution on Agilent 6890N GC

[Jana P]

In our laboratory we have a 6890N GC system with a G1888 Headspace unit.

Currently I am trying to run a method we developed on the Agilent GC system. I am experiencing problems with resolution and peak shape. I can’t identify anything to indicate a system problem. Documented below is a summary of the system checks and investigation performed to date.

Acetonitirle and Dichloromethane are the peaks of interest. The resolution use to be satisfactory (approx. 1.2) and they are now co-eluting.

The following had occurred between analysis:

• The column was removed from the system.
  The inlet was moved from the back (Volatiles) and set up on the front inlet (Split/Splitless) from the back inlet.
  Another method was used on the front inlet. No problems were encountered.
  The inlet was moved from the front (Split/Splitless) set up to run on the back inlet (Volatiles).
  The column used for this method was re attached.
  Analysis performed and poor resolution and peak shape obtained.
  All GC and Headspace parameters were reviewed. No problems were found.
  Temperature programs were checked and satisfactory.
  The column was the correct one for analysis and previously gave good chromatography.
  A different column was connected (same length, diameter etc. different brand). The same poor chromatography was obtained.
  A brand new column was placed on the system and conditioned. The analysis was performed on the new column and the poor chromatography was still seen.

As there is no obvious error, different columns have been assessed (brand and age of column of equivalent dimensions), multiple samples have been analysed (freshly prepared and older).

Can anyone suggest anything which may assist in the resolution of the two peaks and result in the chromatography being as it was originally (i.e. good resolution and good/sharp peak shape)?

If more information is required i can provide.

If i can figure out how i will post up example chromatograms too.

Thanks Everyone

[Peter Apps]

Hi Jana

Can you clarify some points:

When you say that the inlet was moved do you mean that the hardware was physically taken out of the instrument and put in the other location, or only that the gas lines were connected to one inlet and then to the other ?, or something else entirely ?

Is the "other method" an ordinary split-splitless vaporizing liquid injection, or was it a headspace method ?

Have you checked for leaks ?

You do not tell us anything about the analysis except what the analytes are. Please post all the instrumental conditions - length, diameter and stationary phase in the columns, flow rates and pressures, times and temperatures, sample matrix, detector etc .

Instructions for posting chromatograms are in a sticky at the top of the LC page.

Peter

[krickos]

Hi

Could you please list heaspace parmeters, temperatures and GC parameters, split, column type and so on.

Moving from a volatiles inlet to a split/splittless one (even with a tight liner) will normally make the resolution worse.
As you surely know the volatile inlet has a very small volume.

[Jana P]

Hi

Below are the method parameters which I am using:


GC Operating Parameters

• Carrier Gas Helium
  Flow or Linear Velocity 2.2 mL/min or 36 cm/sec
  Split Ratio 1:5
  Initial Temperature (1) 40°C
  Initial Hold Time (1) 20 min
  Temperature Ramp Rate 10°C/min
  Maximum Temperature (2) 240°C
  Hold Time (2) 20 min
  Injection Temperature 140°C
  Detector Temperature 250°C
  Sampling Rate 5
  Data Sensitivity HIGH
  Pneumatics Mode Constant Flow
  Fuel Flow 30 mL/min
  Oxidizer Flow 450 mL/min
  Make-up Mode Constant Makeup + Column
  Make up/Combo Flow 30 mL/min
  Gas Saver Flow Rate 15.0
  Gas Saver On Time 1.50
  Inlet Back
  Detector FID

Headspace Parameters

• Equilibration Temperature 80°C
  Vial Equilibration Time 45 min
  Transfer Line Temperature 105°C
  Loop Temperature 105°C
  Carrier Gas Helium
  Pressurization Time 0.2 min
  Injection Time or Volume 1.00 min or 1.0 mL
  Loop Fill Time 0.2 min
  Loop Equilibration Time 0.2 min
  GC Cycle Time 60 min
  Shake HIGH

When I refer to moving the inlet I simply mean that the gas lines were shifted from the back inlet to the front inlet (where the other method was perfromed) then re-connected to the back inlet again.

The method which was run inbetween the running of my method was a split headspace method.

I have found no leaks in the system and no other obvious instrument errors.


I hope this additional information can help shed some light on the possible causes.

Thank you

[krickos]

Hi Jana

What I see straight away is that Loop Equilibration Time 0.2 min could be the issue. I usually keep this at a minimum ie 0,05min when using a 1ml loop. The equilibration is basicly instant as it is a gas sample. Prolonging it further increases the risk of interaction with the loop wall especially for ACN (polar and bp of ~82°C).

You do not state if you use water or DMF/DMSO etc as sample solvent, but high bp solvents vapour like DMF (~155°C) will also condense/evapourate from the loop wall if Loop Equilibration Time is set too long.
You could also consider raising loop/vlave temp to 155°C and Tline to 165°C

[Peter Apps]

Hi Jana

Do you see any other changes apart from the loss of resolution ? e.g. changed retention times. Are the peaks symmetrically broader, or tailing, or has one peak moved relative to the other ? Posting a before and after chromatogram would help a lot.

If you put the current column into the split inlet with a narrow bore liner and run the same headspace method it will tell you whether the problem is with the volatiles interface.

Peter

[Jana P]

Hi Peter and Krickos

Thank you for your input on my problem so far, I have not replied sooner as I have been trying the few things suggested on this forum and by Agilent.

After contacting Agilent and reading your suggestions I had a bit of a play with the Agilent GC System and the method I was using.

Agilent advised that I should remove and clean the volatiles inlet, as well as bake out the loop and transfer line in the Headspace.
I baked the loop and transfer line overnight, following that I remove the inlet and cleaned it with methanol and then sonicated in methanol a couple of times. I dried the inlet in the GC oven before reinstalling it.

The injection I obtained following the cleaning had significantly improved peak shape and the peaks were no longer rounded or broad and are much sharper as they should be. However the resolution between the Acetonitrile peak and the Dichloromethane peak is still not there. It has improved but is not as it was (i.e. >1.0).

I subsequently have made a few injections altering the loop equilibration time (initially 0.2mins). As you suggested, Krickos, I tried an equilibration time of 0.05 min and this appeared to have little to no effect on the resolution (i.e. peaks are still not resolved and improvement is negligible).

Today I will be able to gain access to a second Agilent system and will be assessing the method on it. I am starting to believe that the problem may be arising from a “dirtyâ€

[krickos]

Hi Jana

About the the loop equilibration time, did not expect you to see any difference after you cleaned the loop just a good thing to always keep it short.

Well great that you seemed to have managed to get some contamination out of your system.

A question, what column do you use?
I am guessing that it could be a DB-624 (0,32 mm ID)or similar based on the tight resolution. Based on known data on that column you should have a resolution of close to 3 or better even with peakwidths close to 0,1min at halfpeak hight. So something seems off.

Do not think we have covered the concentrations in the standards. Whats the total amount in the vials? The high oven temp might give you too big peaks.
Given the from begining tight resolution (1,2), very small variations or problems can cause you problems.

[Jana P]

Hi Everyone

I have managed to solve the mystery of my poor resolution!!

After numerous experiments and consultation with Agilent we have found that our poor resolution is due to the EPC not functioning correctly, resulting in my split ratio being incorrect and hence more of my sample being injected.

Replacing the EPC should do the trick and resolve my problem. Fingers crossed that it all goes well. I will post up the result of replacing the EPC to confirm it works.

Thanks you all for your input and assistance.

[krickos]

Hi Jana

Great to hear that you found the problem.

Hehe well kinda funny, although we have a lot of cool gadgets it is never wrong to have a decent flowmeter around to verify that flows are accurate.