Where do these peaks come from?

We are running a method to analyze assay and some impurities in a pharmaceutical material. We are running under the following conditions:

Agilent HP-1 30m x 0.53mm x 3.0 um column

200 C inlet

1:1 split

5 mg/mL (high concentration to detect impurities)

2.5 mL/min flow with temp gradient with 250C bakeoff for 20min

2 uL inj

Diluent: ACN 

Liquid inject

Liner: 4mm deactivated straight through with wool (Restek cat # 20782)

Septum: Bleed/Temp Opt. non-stick 11mm (Agilent PN 5182-3413)

 

We have peaks that look like "impurities" peaks but do not increase and decrease with same magnitude as the sample concentration and are inconsistent between injections of the same sample. They are not in the ACN blank. They increase with inlet temperature increase to 250C.

I also see them (with lesser magnitude) with an injection of just hydrogen peroxide in ACN only.

These peaks are seen at 7.8  min and 8.2 min in the attached chroms.

 

Any clues where these are coming from? Interaction with injection and something in the inlet? Is there a more inert liner or septum I should be using?

attachments.zip
  • So I see you say your method is a split method 1:1 (so 50% of sample goes through). Is there a reason for you to not use splitless and do injections of 1uL instead?

    Also, your liner looks to me like a liner I would use in a splitless method and not splitless unless you have the cross gold plate in your inlet instead of regular gold plate. Also, what is the pressure of your inlet with that flow?

     

    So you say it is not part of your ACN injections but it is part of your sample, and you say you are looking for impurities in your sample. So is the issue that it does not seem to be linear with the concentration of your samples? If so, if it is not part of your ACN Blank injections, are you sure it is impurities from the sample and not something like sample degradation from high temperature of the GC?

  • Thanks for taking the time to reply. I am not sure why the injection is split and the liner was chosen. Is there a liner that you would recommend? But when using a splitless injection and 1 uL injection, those peaks are still present.

    The peaks are not linear with sample concentration (i.e. 10x sample concentration results in 3x bigger unknown peaks). All other impurities present behave as expected with varying sample concentrations.

    The peaks may be from sample degradation in the inlet but the real mystery is when I injected just hydrogen peroxide and ACN (as part of a degradation study) and saw these same peaks. This made me think that there is some kind of reaction with an inlet material in the presence of a sample. GCMS data was inconclusive.

  • If you want to try Agilent's deactivated liners, this is a part number for it 5183-4693. These are for splitless injections. Also we have deactivated split/splitless inlet that you might want to look into if that could interest you if these are reactions with something in the inlet, if it is strictly liner then just a change of liner should fix it, but I cannot say what is creating that issue. I am not aware.

     

    If the peak increase with temperature, I would suspect there is a degradation or a reaction of some kind in the inlet that is happening for sure.

  • If the GC is used for more years, possibility of inlet contamination. As Gary has mentioned cleaning with Methylene chloride is good and maintaining total flow 20ml/min by rising split flow. However many laboratories, methods are developed during 5890 and 6890 GC period and following it and not ready to revise their methods. Some Non-Agilent software are not functioning for splitless mode and users need to use split mode with very low split ratio.

     

    I have observed in some old GC inlets are having lot of deposit.

    It has to be cleaned with scrapper or inlet cleaning metal wire brushes. Split vent port is also very important, it gets choked and affect the split ratio. Refer attached picture below.

    Next glass wool may be also have impact on unknown peaks or low response. Use Agilent Glass wool. I suggest even new liner remove the glass wool, sonicate it for 3 minutes in IPA to remove the powders and insert new glass wool. It will improve the performance.

    Use this brush for cleaning inlet

  • With that low a split what is total flow? It is recommended at least 20 ml/ min total flow I think. Try cleaning off the gold seal and cleaning the inlet with cotton swabs wet with methylene chloride. We use a tapered liner at the bottom.

  • Hi Nila,

    Any updates to share? I will mark this as assumed answered for now.

    Regards

    James

  • Hi all,

    I'll contribute to this topic with a few comments.

    I'm in no way a GC expert but I have been working with a GC-MS, basically on my own, for about a year now and I have been trying to gain knowledge on how to best operate the machine.

    Please correct me if I'm wrong in any way.

     

    The first thing that caught my attention was the injection volume (2µL).

    According to a solvent expansion calculator, with the injector temp @ 200ºC and ACN as solvent, backflash happens during the injection.

     

    I used another calculator to find the head pressure according to the column dimensions and other parameters.

     

     

    So, according to this document, backflash "leads to poor reproducibility, sample loss, ghost peaks, carry-over, split peaks, tailing peaks and loss of resolution. Without doubt, backflash is the primary cause for the largest number of reported gas chromatographic anomalies."

     

    With such a low head pressure (dictated by the type of column used and method parameters), there is not much to do to lower the expansion volume of the solvent: decrease inlet temp (if possible), decrease injection volume (best option for backflash, worst for contaminant analysis), change solvent.

    There is the option of using Pulsed Pressure Splitless GC Injection if your instrument has that possibility. I'll just leave a link to a document explaining better this feature.

    The liner you are using is fine for split/splitless injection. Just don't use split liners for splitless injections.

     

    About injecting hydrogen peroxide.

    I would not be amazed if H2O2 would decompose in the injector to give water and oxygen. Two compounds not typically detected by FID (if it is the detector used here).

    As well, GC columns do not usually like oxygen when hot.

     

    So, what I would do first (here is the part that might need more tuning by experts...):

    - clean the injector,

    - change septum and liner,

    - bake injector @ 300ºC (good practice when putting a new liner) without the column attached if possible because max temp of the column is 260-280ºC,

    - bake column @ max temp,

    - if working splitless, choose starting oven temp below solvent boiling point (solvent effect),

    - choose correct linear velocity for carrier gas, @ starting oven temp, for my column (Van Deemter), this will give me the head pressure of the carrier,

    - determine the volume of the injection to avoid backflash,

    - determine column holdup time and calibrate column,

    - inject standard(s) (not H2O2) indicated in the column documentation.

  • This question has been marked as assumed answered.

Was this helpful?