Column Bleed & Septa Bleed – Same Old Thing!

On a seminar trip I was confronted with describing why septa bleed and column bleed are different. My canned answer is that septa bleed has a base peak of m/z 73 and column bleed has a base peak of m/z 207 and we can prove this with a GC-MS. Keep in mind that both column bleed and septa bleed are made up of cylic siloxanes which begs the question of what makes them different.

First let’s review what we already know about septa bleed from work that has been done by Amanda Rigdon, Alan Sensue, Jaap de Zeeuw, Jack Cochran and a variety of other sources. One overlooked area for septa bleed is the vial septa, and while less common, it is characterized as sharp repetitive peaks even after several blanks have been run. Preparing separate vials that are PTFE lined can minimize or eliminate septa bleed from the vial (1). With vial inserts the issues can be greatly exaggerated and can also degrade target compounds; for example Endrin & DDT (2). Evaporative loss following puncturing results in high bias in addition to concentrating contaminants in the sample. Coring, whether from the sample vial or the injection port septum, will result in an increased amount of siloxanes that will manifest themselves as “ghost” peaks.  Tapered cone style 23s-26s needles minimize septa particles in the inlet liner. The use of a Merlin Microseal can reduce bleed and eliminate the potential for coring and can reduce activity in the inlet (3,4,5,6).

Figure 1: Total Ion Chromatogram (TIC) overlayed with Extracted Ion Chromatogram m/z 73 and m/z 207.

Figure 1: Total Ion Chromatogram (TIC) overlayed with Extracted Ion Chromatogram (EIC) m/z 73 and m/z 207.

Now that we know how to minimize septum bleed let’s take a look at the difference between column bleed and septa bleed by examining a chromatogram where we can see both. The chromatogram in figure 1 is the first run after the instrument sat idle over the weekend. The oven temperature was set at 35°C which means that any contamination in the carrier gas (septa bleed) is concentrated at the head of the column. The black trace is the total ion chromatogram (TIC) whereas the red trace represents m/z 73 and blue trace m/z 207. First notice the m/z 73 are discrete peaks, that is analytes have concentrated at the head of the column and partitioned in and out of the stationary phase over the course of the temperature programmed analysis. Column bleed on the other hand is shown as a rise in the baseline and is a mix of siloxanes.

Figure 2: Mass Spectrum of D3 & D4 cyclosiloxanes with EIC representing column bleed (blue trace m/z 207) and septum bleed (red trace m/z 73).

Figure 2: The degradation of polydimethylsiloxane (PDMS) stationary phases forms predominately two cyclic siloxanes; hexamethyl-cylcotrisiloxane (D3) and octamethyl-cyclotetrasiloxane (D4). The combination of both of these siloxanes produces the distinctive m/z 207 and the m/z 281. Notice that the fragmentation results in a low abundance of m/z 73 compared to larger cyclic siloxanes which all share the m/z 73 as the base peak (figure 3). The septum bleed represented as the EIC of m/z 73 (red trace) has a series of higher molecular weight cyclic siloxanes, D7 through D10 (and beyond).

Figure 3: Typical spectrum of septa bleed where the base peak is m/z 73

Figure 3: Typical spectrum of septa bleed where the base peak is m/z 73

Septa bleed will generally be identified as one of the above spectra especially when the scan range is limited to m/z 550. Higher molecular weight siloxanes are present but will not be correctly identified since the target spectrum exceeds 550 atomic mass units (AMU).

Identifying the source of siloxanes makes troubleshooting much easier. While it’s true that both septa bleed and column bleed are the same; that is, they are both made up of cyclic siloxanes, the molecular weights are quite different, resulting in different spectra.

 

1.) The Forgotten Septum

2.) Silicone Autosampler Vial Septa Cause Endrin Breakdown and Sample Contamination

3.) Troubleshooting Injection Port Septa

4.) Septum particles in my insert.. Good,  Bad or Ugly?

5.) Using a Merlin MicroSeal Septum to Reduce Endrin and DDT Breakdown for EPA Method 8081b – Organochlorine Pesticides by Gas Chromatography

6.) Unraveling the Mysteries of Ghost Peaks: It’s Time to Pull the Sheet Off

Other links:

Reduce impact of septum “sweat”   by using septum purge..

Merlin microseal or Septa… should they really  “SEAL” ?..

An Evaluation of the Performance of GC Septa in Different Commonly Used Procedures

 

 

 

 

 

 

 

5 Responses to “Column Bleed & Septa Bleed – Same Old Thing!”

  1. Paul says:

    What about siloxanes from an SPME-Fiber?

  2. Paul:
    That’s a great question. We have performed phase digestions with PDMS, taken those samples and injected them into a GC with a thin film column. We can clearly see mostly D3 & D4 but there are larger cyclics in the mix. These all show up as distinct peaks. I would expect to see the same with a PDMS SPME fiber, although I have not done SPME. Keep in mind whatever off-gasses from the fiber will concentrate at the head of the column and elute as peaks.
    Thanks for reading the blog,

    Chris.

  3. Marc says:

    Do you have a mass for the peak next to 221 in figure 3 (lowest two spectra), is this 235?
    (Looks like the 221 is a bit before the marker on the x-axis)

  4. Marc:
    The spectral peaks are shown to the right of the blue m/z values. The confusion here is that x-axis is off by 10amu to the left. So the value that looks like 235 is actually 221 and the spectra directly below 221 is 207. I will get these figures cleaned up an place a comment that the figures have been updated to let everyone know. Thanks for your help with the figure and let me know if you need anything and I’ll be happy to help,
    Chris.

  5. Yuki says:

    Hi Chris,

    D3 producing fragment 96 is a real mystery to me..133, 177, 191, 207 are explainable, but 96 must be from some sort of rearrangement, but do you have any idea what this 96 might be??

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