Help needed in troubleshooting the tailing peak

[iuliachiciudean]

Hi!
I don’t have any experience with GC-MS and I need your help in understanding some problem.

Briefly, I am trying to measure the residual concentration of hexadecane after bacterial growth.

I am doing the microbiology part of the experiment and I employed the expertise of another laboratory for the GC-MS measurements.
They are using SPME sampling technique, and Agilent GC 6890N.

I am adding some info's for the method exemplification and for what I get as a result at the end of the text.

I was supposed to have only one alkane in the medium (i.e. hexadecane), but I have multiple pick and even the hexadecane pick is a tailing peak.
In my opinion those are very bad results and I should stop working with this partner laboratory, but they say that is ok and they just need to do some adjustments of the data they obtain from the GC-MS with some software (MassLab).

I really need to be sure if their opinion is valid or what is exactly (more or less) the problem here (the extraction method, the column ….).

Just to know what should I recommend them to change for data acquisition or if I just should find someone else to do my GC-MS measurements.

Thank you very much!!!!
Respectfully,
Iulia

INSTRUMENT CONTROL PARAMETERS: GC 6890N
------------------------------------------

C:\MSDCHEM\1\METHODS\SPME.M
Tue Oct 25 09:46:45 2016

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

Sample Inlet : GC
Injection Source : Manual
Mass Spectrometer : Enabled

=============================================================================
6890 GC METHOD
=============================================================================


OVEN
Initial temp: 40 'C (On) Maximum temp: 325 'C
Initial time: 2.00 min Equilibration time: 0.50 min
Ramps:
# Rate Final temp Final time
1 10.00 320 7.00
2 0.0(Off)
Post temp: 0 'C
Post time: 0.00 min
Run time: 37.00 min


FRONT INLET (SPLIT/SPLITLESS) BACK INLET (UNKNOWN)
Mode: Split
Initial temp: 250 'C (On)
Pressure: 6.88 psi (On)
Split ratio: 0.2:1
Split flow: 0.2 mL/min
Total flow: 3.9 mL/min
Gas saver: On
Saver flow: 20.0 mL/min
Saver time: 5.00 min
Gas type: Helium


COLUMN 1 COLUMN 2
Capillary Column (not installed)
Model Number: Agilent 19091S-433
HP-5MS 5% Phenyl Methyl Siloxane
Max temperature: 325 'C
Nominal length: 30.0 m
Nominal diameter: 250.00 um
Nominal film thickness: 0.25 um
Mode: constant flow
Initial flow: 1.0 mL/min
Nominal init pressure: 6.88 psi
Average velocity: 36 cm/sec
Inlet: Front Inlet
Outlet: MSD
Outlet pressure: vacuum


FRONT DETECTOR (NO DET) BACK DETECTOR (NO DET)


SIGNAL 1 SIGNAL 2
Data rate: 20 Hz Data rate: 20 Hz
Type: test plot Type: test plot
Save Data: Off Save Data: Off
Zero: 0.0 (Off) Zero: 0.0 (Off)
Range: 0 Range: 0
Fast Peaks: Off Fast Peaks: Off
Attenuation: 0 Attenuation: 0


COLUMN COMP 1 COLUMN COMP 2
(No Detectors Installed) (No Detectors Installed)


THERMAL AUX 2
Use: MSD Transfer Line Heater
Description:
Initial temp: 280 'C (On)
Initial time: 0.00 min
# Rate Final temp Final time
1 0.0(Off)


POST RUN
Post Time: 0.00 min


TIME TABLE
Time Specifier Parameter & Setpoint




GC Injector


Front Injector:
No parameters specified

Back Injector:
No parameters specified

Column 1 Inventory Number : AB001
Column 2 Inventory Number :

MS ACQUISITION PARAMETERS


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

Tune File : atune.u
Acquistion Mode : Scan


MS Information



Area Percent Report

Data Path : C:\MSDCHEM\1\DATA\
Data File : CN-I.D
Acq On : 25 Oct 2016 9:09
Operator :
Sample : IULIA
Misc :
ALS Vial : 1 Sample Multiplier: 1

Integration Parameters: events.e
Integrator: ChemStation

Method : C:\MSDCHEM\1\METHODS\SPME.M
Title :

Signal : TIC: CN-I.D\data.ms

peak R.T. first max last PK peak corr. corr. % of
# min scan scan scan TY height area % max. total
--- ----- ----- ---- ---- --- ------- ------- ------ -------
1 1.513 231 238 257 BB 5389284 85330000 2.41% 0.611%
2 1.908 268 304 319 BV 649381 88019386 2.48% 0.630%
3 2.010 319 321 486 VB 2 571589 149750691 4.23% 1.072%
4 12.468 2056 2067 2069 BV 2 6097807 147013068 4.15% 1.052%
5 12.510 2069 2074 2076 VV 3 6210374 156096876 4.40% 1.118%

6 12.538 2076 2079 2080 VV 3 6197368 90336038 2.55% 0.647%
7 12.587 2080 2087 2089 VV 3 6453707 196938283 5.56% 1.410%
8 12.620 2089 2093 2210 VB 4 6533185 533381807 15.05% 3.819%
9 15.440 2547 2564 2569 BV 2 827557 33559434 0.95% 0.240%
10 15.506 2569 2575 2580 VV 6 723879 28458657 0.80% 0.204%

11 15.555 2580 2583 2587 VV 2 702124 15278297 0.43% 0.109%
12 15.605 2587 2591 2597 VV 2 654922 22252325 0.63% 0.159%
13 15.652 2597 2599 2601 VV 2 584894 8530029 0.24% 0.061%
14 15.689 2601 2605 2610 VV 3 546885 16962155 0.48% 0.121%
15 15.746 2610 2615 2620 VV 2 519484 17158853 0.48% 0.123%

16 15.787 2620 2622 2623 VV 2 422826 4684691 0.13% 0.034%
17 15.815 2623 2626 2631 VV 4 429639 10780053 0.30% 0.077%
18 15.858 2631 2633 2636 VV 3 373729 6721507 0.19% 0.048%
19 15.899 2636 2640 2642 VV 3 333298 7667751 0.22% 0.055%
20 16.199 2679 2690 2692 VV 3 515621 17094049 0.48% 0.122%

21 16.274 2692 2703 2706 VV 4 614284 26652459 0.75% 0.191%
22 16.331 2706 2712 2715 VV 8 554277 18850711 0.53% 0.135%
23 16.360 2715 2717 2741 VV 3 530040 32695171 0.92% 0.234%
24 17.097 2747 2840 2842 PV 6 9357398 3544119290 100.00% 25.373%
25 17.151 2842 2849 2853 VV 4 7961674 322656208 9.10% 2.310%

26 17.185 2853 2855 2857 VV 3 7034564 88271615 2.49% 0.632%
27 17.221 2857 2861 2864 VV 5 6123803 153461577 4.33% 1.099%
28 17.253 2864 2866 2869 VV 3 5063819 89824779 2.53% 0.643%
29 17.279 2869 2871 2878 VV 7 4255177 125826818 3.55% 0.901%
30 17.331 2878 2879 2881 VV 2 2703891 31417896 0.89% 0.225%

31 17.353 2881 2883 2894 VB 6 2114858 55155489 1.56% 0.395%
32 17.526 2908 2912 2918 VV 7 2242505 79294665 2.24% 0.568%
33 17.585 2918 2922 2930 VV 6 3905362 186825147 5.27% 1.338%
34 17.646 2930 2932 2939 VV 2 5722912 189594656 5.35% 1.357%
35 17.703 2939 2942 2945 VV 7369981 157900616 4.46% 1.130%

36 17.748 2945 2949 2952 VV 3 8711778 215630530 6.08% 1.544%
37 17.793 2952 2957 2962 VV 5 10006325 344321486 9.72% 2.465%
38 17.843 2962 2965 2973 VV 4 11453502 470565051 13.28% 3.369%
39 17.929 2977 2979 2985 VV 4 14080481 419380617 11.83% 3.002%
40 17.977 2985 2987 2991 VV 2 15541511 291653627 8.23% 2.088%

41 18.026 2991 2995 3000 VV 5 16873623 545313110 15.39% 3.904%
42 18.070 3000 3003 3014 VV 2 18488651 979243032 27.63% 7.011%
43 18.186 3014 3022 3029 VV 22643573 1157026247 32.65% 8.283%
44 18.265 3029 3035 3040 VV 4 26417033 711244133 20.07% 5.092%
45 18.331 3040 3046 3052 PV 4 5524812 131172181 3.70% 0.939%

46 18.379 3052 3054 3059 VV 2 5159063 61870125 1.75% 0.443%
47 18.416 3059 3061 3062 VV 2 970177 9271236 0.26% 0.066%
48 18.486 3062 3072 3076 VV 2 17191266 327417885 9.24% 2.344%
49 18.516 3076 3077 3081 VV 1293526 13147247 0.37% 0.094%
50 18.548 3081 3083 3087 VV 399627 5767862 0.16% 0.041%

51 18.602 3087 3092 3100 VV 3 362845 8175521 0.23% 0.059%
52 18.669 3100 3103 3107 VV 2 170337 2079766 0.06% 0.015%
53 18.729 3107 3113 3118 PV 3 1788250 31017162 0.88% 0.222%
54 18.782 3118 3122 3125 VV 828631 10405554 0.29% 0.074%
55 18.827 3125 3129 3133 VV 3 669822 10599182 0.30% 0.076%

56 18.868 3133 3136 3138 VV 432673 5618526 0.16% 0.040%
57 18.896 3138 3141 3143 VV 428368 5191545 0.15% 0.037%
58 18.935 3143 3147 3151 VV 1833196 25420976 0.72% 0.182%
59 19.001 3151 3158 3166 VV 5 2193735 67509135 1.90% 0.483%
60 19.117 3166 3178 3179 VV 5787624 99120819 2.80% 0.710%

61 19.205 3179 3192 3196 VV 3 15032056 544670622 15.37% 3.899%
62 19.248 3196 3199 3208 VV 3 9876790 195453939 5.51% 1.399%
63 19.661 3258 3268 3287 PV 224676 5498373 0.16% 0.039%
64 19.876 3293 3304 3316 VV 9793983 197440176 5.57% 1.414%
65 20.080 3332 3338 3352 VV 5 156887 5007344 0.14% 0.036%

66 20.393 3383 3391 3404 VV 3 323809 8470857 0.24% 0.061%
67 20.575 3409 3421 3434 PV 1661252 38217704 1.08% 0.274%
68 20.679 3434 3438 3453 VV 201432 4503268 0.13% 0.032%
69 21.492 3561 3574 3590 BV 4 115020 2867382 0.08% 0.021%
70 21.687 3599 3607 3623 BV 247420 4546364 0.13% 0.033%

71 22.202 3661 3693 3716 BV 4 148754 5818781 0.16% 0.042%
72 23.583 3916 3923 3929 BV 136578 2312090 0.07% 0.017%
73 23.903 3971 3977 3994 VV 2 149945 3116194 0.09% 0.022%
74 24.416 4056 4062 4086 VB 2 281649 5838493 0.16% 0.042%
75 25.218 4183 4196 4203 PV 295048 5139499 0.15% 0.037%

76 25.623 4251 4264 4266 PV 257203 4235189 0.12% 0.030%
77 25.665 4266 4271 4283 VV 2 439106 10637249 0.30% 0.076%
78 25.988 4314 4325 4340 BV 278189 5330856 0.15% 0.038%
79 26.730 4437 4449 4458 BV 236729 4251851 0.12% 0.030%
80 27.447 4553 4569 4577 PV 206644 6332657 0.18% 0.045%

81 27.666 4598 4605 4608 VV 2 686053 16139667 0.46% 0.116%
82 27.721 4608 4614 4622 VV 5979514 112563120 3.18% 0.806%
83 27.803 4622 4628 4641 VV 2 719547 21785987 0.61% 0.156%
84 27.919 4641 4647 4660 VV 2 459590 11409104 0.32% 0.082%
85 28.152 4677 4686 4698 VV 2 538292 13063268 0.37% 0.094%

86 28.350 4710 4719 4734 VV 868743 19361432 0.55% 0.139%
87 28.814 4791 4797 4804 PV 105571 1906369 0.05% 0.014%
88 29.869 4963 4973 5000 VV 2 670924 18258164 0.52% 0.131%
89 31.266 5199 5206 5226 VV 9 146008 4641472 0.13% 0.033%
90 32.811 5437 5464 5473 PV 9 88672 2482284 0.07% 0.018%



Sum of corrected areas: 13968023359

SPME.M Tue Oct 25 09:46:49 2016 CHEMSTATION

[Peter Apps]

Welcome to the forum

This looks strange:

FRONT INLET (SPLIT/SPLITLESS) BACK INLET (UNKNOWN)
Mode: Split
Initial temp: 250 'C (On)
Pressure: 6.88 psi (On)
Split ratio: 0.2:1
Split flow: 0.2 mL/min
Total flow: 3.9 mL/min
Gas saver: On
Saver flow: 20.0 mL/min
Saver time: 5.00 min

A split ratio of 0.2:1 requires gas flows that are lower than the EPC can control. It would be better to do a proper splitless injection starting with a splitless time of 2 min and adjusting according to peak shape.

I would not be surprised to find multiple peaks on a chromatogram from a microbial culture, but you should ask them to run a blank, and it must show only a few small peaks.

It is poor practice to try to fix bad chromatography by playing with data acquisition and processing settings.

Is the GC-MS lab accredited ?

Peter

[tkubowicz]

Hello

Below it is your inlet settings:

FRONT INLET (SPLIT/SPLITLESS) BACK INLET (UNKNOWN)
Mode: Split
Initial temp: 250 'C (On)
Pressure: 6.88 psi (On)
Split ratio: 0.2:1
Split flow: 0.2 mL/min
Total flow: 3.9 mL/min
Gas saver: On
Saver flow: 20.0 mL/min
Saver time: 5.00 min

Few things:
1. Split ratio 0.2 is weird value - I don't really understand who and why set it up like that. Try to use spilt 2:1, 5:1, 10:1 but not 0.2:1
2.Total flow is 3.9ml/min and gas saver is on after 5 min to 20ml/min...so it is gas waster because flow is going from 3.9 to 20 ml/min

I'd recommend for problems with tailing peaks:
1.Check inlet - replace liner, septa, o-ring
2.Cut about 5-10cm column from inlet side
3.Install column and run your method

Good luck

Regards

Tomasz Kubowicz

[iuliachiciudean]

Thank you so much for your replay!!
I will try suggest them this.
Do you think that this can also be caused by a the fact that the column is not properly clean or something before runs?
I am sorry if my questions are a little unappropriated (stupid) but, as i said, I have no experience with GC method.
Thank you!

[tkubowicz]

Hi

There are no stupid questions...only answers are stupid
I'd recommend as in previous post:
1.Cut 5-10 cm of column from inlet side (from MSD as well if you want to do it properly)
2.Clean inlet, put new liner, septa etc.
3.Bake out your column - leave overnight with flow 1-10ml/min, oven 200-280deg depends on column specification
4.Run blank - no injection, just method without any injection

Ideally you should see only slight basline drift because of temperature and possible bleeding effect. But for 2-3 runs the profile should be the same.

Regards

Tomasz Kubowicz

[James_Ball]

As mentioned above, I would not recommend a split ratio of less than 5:1 on a 6890. Newer 7890s can handle a 2:1, but the older 6890 will be more stable at 5:1 or greater.

Tailing can also happen if the column is inserted too far into the injection port. Often I will verify the depth by taking one of the gold seals that go on the bottom of the inlet and placing it on the end of the column to test before actually installing it. The tip of the column should only be 1-2mm above the surface of the seal. Also the column must be cut level and smooth to prevent tailing of peaks.

[Rndirk]

Do you give them the samples as an agar plate or something? Do you know how do they handle it prior to SPME?

Chances are the lab has no clue how to handle this matrix. It's weird that you get a bunch of raw data as a result. Did you communicate with them prior to the analysis, what your sample is and what do you expect from them?

I agree that a blank measurement would be helpful. Preferably a sample that is as close as possible to yours, but without the analyte of interest.

[iuliachiciudean]

Hi Rndirk,

No, the sample weren’t agar plates. Were 100 ml of aqueous minimal medium (only microelements and some mineral salts in double distillated water) + 0,5 mL hexadecane + bacterial cells.

From what they told me they mixed my samples with 900mL H2O. I don’t really understand why this step is necessary, but it shouldn’t affect my measurements very much.

I told them exactly what my samples are and I explain them all the logic behind the experiment.

They should be used to this kind of analyses since their main interest is the detestation of different pollutants in the environment (soil or water samples). From what they told me they are working with alkanes as well.

Yes, is a mystery to me why I have soooo many picks? It supposed to be only one, very high, pick for hexadecane and it was supposed to look like a pick (sharp) not like a plateau kind of shape.

I will talk to them again tomorrow to see what they proposed we should do.

Anyhow, your answers are very helpful! At least I can suggest them something in order for this to work as it should be.

Thank you!
Best regards,
Iulia

[Peter Apps]

The problem begins with the nature of the sample you prepare. As you might have noticed 0.5 g of hexadecane will not dissolve in 100 ml of aqueous solution, or in 1 l after it has been diluted - it will float around on the top, so no telling whether they actually had any of it in the sub-sample that they analysed.

Peter

[Peter Apps]

Double post - sorry.
Peter

[iuliachiciudean]

Hi Peter,

Yes I know it floats. That is why we usually extract hexadecane with hexane.
But they told me that we don't need an extraction step if we are using SPME.
I haven't use it before so I have no clue how this SPME works. Since is called Solid-phase microextraction, maybe is not even working for aqueous samples.

Best regards!

[Peter Apps]

Problem 1. There is no way to get a representative subsample of water with hexadecane floating on top of it. The only way to get a valid result is to analyse the whole of the sample that you submit. So you would extract your 100 ml sample with hexane, and then dilute the hexane solution of hexadecane.

Problem 2. SPME is the wrong technique for heterogeneous samples with 0.4 % of an insoluble analyte. So go back to hexane extraction.

Peter

[Rndirk]

To clarify, SPME consists of a fiber that extracts anything that prefer to stick to the fiber compared to water. Typically: trace analysis in homogeneous, aqueous samples.

So as Peter said, this is the wrong technique for quantitative analysis of your sample. Your analyte is not present in trace amounts, it's not even dissolved. Besides there's a lot of other stuff that will stick to the fiber.

[iuliachiciudean]

Ok.
To conclude, this is the wrong method for my alkane extract.
The main idea is that I should go back to hexane extraction.

And also, maybe changing the lab for doing the GC-MS measurements will be good as well.

Thank you very much for all your help!

Everything is clearer now.

With respect,
Iulia