Peak splitting problem?

[cfspence]

Advance apologies for my ignorance in knowing how to troubleshoot what may be a routine issue. My problem: Peaks on my chromatogram that should be from the same component are being split. I've attached a copy of the full chromatogram and an expansion of the mid-range times below. Below the images I detail information about my system and the parameters as well as the sample. Any ideas what is causing this?




GC-MS system: Agilent 7820A GC with a 5975 series MSD and a 7693A autosampler (ALS). Our column is a HP-5MS 30m x 0.250 mm with 0.25 um stationary phase film.

Run-time parameters:
Injection: 1 ul splitless injection using ALS standard injection with 0.2 ul air gap. Injector at 230 C with a flow of 1.0 ml/min with He carrier gas.
Temperature program: 70 C for 5 min.; ramp at 5 C/min for 325 C; hold at 325 C for 5 min.; re-equilibrate at 70 c for 1 min. prior to next injection.
Mass Spec parameters: transfer line at 250 C; ion souce at 200 C; speed at better than 2 scans per sec; mass range 50 to 600 u; solvent delay of 9 min.

Sample: Alkane retention index mixture containing C15, C18, C19, C22, C28, C32, and C36 straight-chain alkanes dissolved in dry pryidine (from new sure seal bottle, the prepared standard mixture is stored over 4 A molecular sieves and I added some KOH for good measure).

Note, for example, the peak splitting at a retention time of 28 min for the octadecane (C18) component.

Please let me know if I can provide further information. Thanks, in advance, for any assistance that you can provide.

cfspence

[Peter Apps]

You have an alkane retention index mixture dissolved in pyridine - are there any other solvents in there - is it a pre-mixed alkane solution that you then dilute with pyridine ?

Peter

[chromatographer1]

The chromatogram indicates that you have an improperly installed column at the inlet. The more volatile components show a greater problem.

Rod

[cfspence]

You have an alkane retention index mixture dissolved in pyridine - are there any other solvents in there - is it a pre-mixed alkane solution that you then dilute with pyridine ?

Peter

Hi Peter,

It is not a premixed solution. I added pure samples of the separate alkane standards to the pyridine myself. There should not be another solvent. Thanks.

[tlahren]

I agree with Chromatographer1. Recut and re-install the inlet side of the column. Make sure there are no burs or breaks in the end of the column and that you get a clean square cut. Then try again.

[chromatographer1]

It does make things go well quickly when the poster takes the time to post a chromatogram.

Your kind consideration is appreciated. Many of us here wish to help. Your cooperation makes this endeavor so much easier.

I hope this suggestion fixes your splitting issue. There are other less likely but possible causes.

Rod

[AICMM]

cfspence,

An inlet at 230 for C36 does not seem like it is high enough, especially since you take the oven to 325. It would not surprise me that you are leaving compounds in the injector. The 70 C starting point for the pyridine is good so that should not be the issue. Do you really have to use pyridine? Yech from lots of perspectives, least of all the smell.

Best regards,

AICMM

[cfspence]

Thanks to all for your help so far.

As advised I did re-install the column. During that process I also changed the septum, the injector port liner, and the "gold seal" at the bottom of the standard Agilent split/splitless injector (the latter was necessitated because when I removed the column the ferrule was left behind and I could not think of another way to get it out).

I ran the same method on the same sample and here is the resulting chromatogram:


Here is an expanded view of the central section:


Here is an expansion of the later section:


As you can see the early peaks are still being split but perhaps it does show some improvement. When I reinstalled the column I tried to carefully measure the ferrule to column tip distance which Agilent instructs should be 4 to 6 mm - I tried to aim for around 5.

I also ran a blank with a 0.2 ul injection of an empty vial that I had first purged with argon. I had failed to turn off the syringe solvent washes and so, in addition to the argon/air peak, I saw a fair amount of ethyl acetate. Neither peak was split but both seemed to be fairly broadened. I could post that chromatogram as well if anyone requests it.

Regarding the use of pyridine. I know... yuck. However my samples are plant extracts that are derivatized with hydroxyamine HCl and "per-TMS-ylated" with MSTFA. The literature appears to suggest running that reaction in (dry) pyridine. I thought it best to run the retention index standards in a similar solvent matrix.

Any suggestions from here?

cfspence

[chromatographer1]

I would guess a contaminated column. Something is causing the plug to split at the beginning of the chromatography. This might be from the expansion of the solvent exceeding the internal volume of the inlet or a piece of 'dirt' at the beginning few feet of the column.

best wishes,

Rod

[Peter Apps]

There is an odd variability in the tails on the peaks. In general alkanes should give sharp symmetrical peaks, but some of these are tailed. If they were all tailed I would suspect a damaged column of some absorptive crud in the inlet.

What does an injection of blank pyridine look like ?

What do the peaks look like if you make up the standard in hexane, octane or ethyl acatate ?

How many tmes are you rinsing the syringe the sample before the injection ?

Peter

[dblux_]

...
Injection: 1 ul splitless injection using ALS standard injection with 0.2 ul air gap.
...

Could you explain what is this air gap for ?

How doe's your chromatogram looks like when injection is done without air gap ?

[Johnny Rod]

I presume it's the same reason as you used to use one for manual injection, to pop the liquid plug out of the needle, also maybe one before it to prevent premature evaporation. No-one likes that.

[Don_Hilton]

Do you have a delay before the plunger on the syringe is pushed?

I assume that the air gap is between the syringe plunger and the sample and that there is a bit of sample in the needle on injection. Is there, by chance any delay between they syringe pentrating the septum and the plunger beign pushed - as in a hot needle technique? If this is done with no air gap following the sample, there could be some sampel evaporated into the GC while the needle heats.

[chromatographer1]

Peter and I both believe there is a contamination issue in the inlet.

I would remove a portion of column from the head and replace your inlet liner. Also reduce your sample size by half and see if the splitting still occurs.

best wishes,

Rod