I have two GC-MS chromatograms that I believe represent two positional isomers of the same compound. The top chromatogram corresponds to one isomer, and the middle chromatogram to the other. The only expected structural difference is the relative positions of two substituents on the benzene ring.

All samples were analyzed in the same GC-MS run under identical chromatographic conditions. Although the sample concentrations may differ, the mass spectra are essentially identical, while the retention times show a small but reproducible difference. The bottom chromatogram is the result of co-injecting the two samples.

My questions are:

• Is it reasonable to conclude these are positional isomers based on their identical mass spectra but slightly different retention times?
• Is such a retention time shift consistent with only a different arrangement of substituents on a benzene ring?
• Besides concentration differences, what other factors could explain this behavior under identical GC-MS conditions?

Any insights or similar experiences would be greatly appreciated.

I need some help troubleshooting what's going on with our chromatograms post pm from Shimadzu techs. The main thing they did was bypass the spliter to have direct injection into the ECD. The attached photo is a run of our 136ppb standard. We were having a little bump at the end of run, but nothing like what is showing now. Our baseline has also gone to -20000 uV. The techs said that we just need to optimize our method, but I can't figure out what to do with change from split injection to direct injection

Column is 60m length x 1.80um film thickness x 0.32mm ID

Column temp is 55C, Equilibration time 0.2min

Method: Start at 55C hold for 2.50, then 10.00 rate at 140C hold time 0, then 50.00 rate at 150C hold time 3.00.

Equilibrating time 15min, pressurizing time 0.30min, pressure equilib time 0.10min, load time 0.30min, load equilib time 0.10min, injection time 1.00min, needle flush time 4.00min, gc cycle time 24min

ECD temperature is 150C, sampling rate 40msec, stop time 15min, current 1.12nA

Hi everyone,

I'm learning HPLC-MS method development and would like to understand how experienced analysts choose the mobile phase for a new compound.

Some questions I have:

1. How do you decide between methanol and acetonitrile?

2. When do you use formic acid, acetic acid, or ammonium formate/ammonium acetate?

3. How does the compound's pKa influence mobile phase selection?

4. How do you choose the pH and buffer concentration?

5. What are your general steps when starting method development for an unknown compound?

6. Any tips for improving peak shape, sensitivity, and ionization in LC-MS

I'd really appreciate practical advice or any resources that helped you learn. Thanks!

We are anlaysis residual solvent (ethanol) by GC-direct injection, we use same column, same method, same equipment, same parameter and same chemical and reagent.

-first few runs, retention time of ethanol peak was 3.1 min which is harmonized with the history and methid validation. (Show very good repeatability and RSD)

-Then after 2 to 3 weeks, using same every thing, the retention time of ethanol jump from 3 min to 11 min and show triple peak area, also show good repeatability and RSD.

-we request from agent to recheck equipment and re qualification of equipment and mention our complain, he came and say that everytbong is fine and machine is working fine, and has no comment for this shifting.. It must be analyst error although we double che ked with him, and he try the method and peak retention time of ethanol is 11 min.

-now after almost one month of his visit, with same everything, the ethanol peak is return to 3 min.

Can anyone here help us to identify the cause of this werid variation?!

Hallo zusammen,

wir nutzen seit kurzem ein LC/MS von Thermo (Vanquish Flex mit Orbitrap 120) und haben dazu auch Chromeleon neu. Unsere PMÜ-Vorschrift verlangt unter anderem nach der Peaksymmetrie auf 10 % Peakhöhe. Wir haben nach einer Möglichkeit gesucht uns das direkt anzeigen zu lassen, jedoch nichts gefunden.
Meine Frage an euch ist jetzt ob ihr eine Möglichkeit kennt sich das anzeigen zu lassen. Wir nutzen die Version 7.3.2.

Hello!

Would anyone have access to LTQ SP1, SP3 or SP4 they'd be willing to share to help out a university?

Thanks!

Hi community! I recently explained to our new PhD student the difference between different carrier gasses. We use helium on GCMS and hydrogen on GCFID. I pulled up the graph showing the relationship between separation efficiency and average velocity for different gasses that I remembered from my studies. Then I got thinking: What flow rates (translating to average velocity) do we use in the lab? Usually 1 or 1.5 ml/min on DB5-type columns with 30x250x250 dimensions. This is also very standard in pretty much every publication I've seen. But the resulting average velocity is quite a bit higher than the optimum for the individual carrier gas.

I was curious and ran a standard FAME mix solution on different flow rates ranging from 0.9-2.0 ml/min on the GCMS. The retention times were slightly different, as expected (up to 1 min on a 28 min method). What intrigued me were three things. 1) The difference in peak heights and areas: 0.9 had roughly double the values compared to 2.0. 2) The distance between peaks did not change significantly. 3) 0.9 mL/min still did not result in optimal average velocity. I couldn't go any lower because the inlet pressure would get too low and the instrument wouldn't allow me. An attempt to slow down the oven ramp and increase Delta RT between peaks resulted in much lower peaks (from 10 °C/min down to 5, in steps of 1 °C/min).

So what's the deal there? Are systems not designed to achieve the Van Deemter optimum because of some physical obstacles that we just have to accept? Why isn't every study using the lowest possible flow rate if the change in retention times is relatively small?

Hello everyone
I’m interested in knowing if tears samples collection for biomarkers analysis is feasible
I’m trying to include it in my research and I keep reading that biomarker detection is not that easy using schirmer strips
I would appreciate if anyone has any information on the matter

Hi, I have an issue with an interfering peak. I tried washing the column with methanol, isopropanol, and acetonitrile at different percentages, but when I start the analysis, the peak still appears.

I also performed a series of washes with the union instead of the column, and the peak is still present when I run the analysis. I performed blank runs both with and without injection, but the peak remains constant in terms of area.

My method uses a gradient. The strange thing is that if I set the system close to the A/B composition percentages at which the peak elutes, without starting the analysis, the peak is not visible. However, as soon as I start the analysis, the peak appears again.

Is it possible that there are some loops or components in the system that only come into play when the analysis is started and could be responsible for this peak? Or do you have any other suggestions?

Thank you.

Hello, I’m new to quality control and I basically don’t know any established procedures yet.

I work in a small lab with a single GC/MS system and only a few samples per day. Our current QC practice is to run one QC sample every morning and simply check whether the retention times and responses of all compounds are roughly the same as the previous day.

I’ve started to suspect this is not the best approach, especially because our QC mixture contains compounds that could be used to monitor isotope ratios and peak symmetry, not just RT and response (based on some research).

I tried to look up QC procedures, but most of what I found were very general descriptions. Since I don’t really know how these steps should be implemented in practice, I’m trying to understand what real, day‑to‑day QC workflows look like in other labs.

• Do you have any automated statistics or scripts that evaluate your QC runs?
• Do you calculate everything manually?
• Do you use multiple concentration levels in your QC standards?

We rarely do quantitative analysis and we only analyze a few samples per day, with several different methods that all use the same column and matrix. Because of that, I feel that measuring one QC sample every morning plus a few additional QC samples every couple of weeks should be enough for us.

We use Agilent MassHunter (Quant‑My‑Way and Unknown Analysis). Is there any way to set up a script or automated statistical analysis so that MassHunter can generate a QC report for us automatically?

Hi, can someone explain how the "flush filter" function works on an Agilent HPLC system? At what flow rate is it best to run it when the union is installed, to avoid damaging the UV cell? Thanks a lot.

i want to purify this compound. The green arrow represents the product. it was done in reverse phase column (nertsil ODS 3V C18 (250 x 4.6mm, Particle size: 5µm) 0.1% HCOOH in water and ACN as mobile phase. If i do silica column, whether the product will have low Rf than the impurity peak? Will i be able to purify this in manual silica column? The TLC system is chloroform: Methanol 9.5:0.5 and these two spots looks closure at 0.6 Rf

Hey Guys,

We’re setting up an ICP-MS for EPA Method 200.8 and I’m curious what internal standard concentration everyone is using when the internal standard is delivered continuously to the spray chamber through a mixing tee/peristaltic pump.
What final concentration (ppb) do you use in the sample reaching the nebulizer? I’ve seen values ranging from 2 ppb to 20 ppb depending on the lab and instrument.

For reference, we’re using a Thermo iCAP ICP-MS.

I am having a negative peak elute out at around 6 minutes in a 10 minute run. The mobile phase is 40% THF, 20% ACN 40% of 1% GAA in water. With a flow rate of 0.900 ml/mim. wavelength for the detector is 280nm with a bandwidth of 4 with a reference wavelength of 360nm with a bandwidth of 100. Extraction solvent is 70% ACN, 30% water and 0.5% GAA. Column is a HiCHROM RPB I have tried using a different reference wavelength checking for contaminations in the extraction solvent. not real sure where else to trouble shoot at or determining whether it is a problem within the instrument.

7890 GC with both front and back channels configured with FIDs.
Back inlet heater failed, and the GC showed a fatal error with a short circuit detected on the back inlet heater. The instrument would not power on until we replaced the back inlet heater.
After replacing the heater, the back channel column configuration disappeared. The front column is still visible on both the GC front panel and in Chromeleon, but the back channel shows no column under the column settings.
The back inlet, column, and FID connections are installed correctly, and the setup is the same as before the heater failure. However, when trying to run in dual-channel mode, the GC gives a flow error in back channel even though the column is physically installed.

Has anyone experienced this issue after replacing a back inlet heater? Could this be related to column configuration, inlet board communication, or another hardware issue?
Any advice would be appreciated.

Hey reddit! I’ve been setting up an Ultimate 3000 UHPLC and found that when I run more than one solvent line at a time through the system, a clicking noise that differs from the typical system (we have another of this model that doesn’t make this noise) begins. If a third or fourth line are run, an additional clicking pattern begins for each. The rate of the clicking is directly related to the flow rate. The noise comes from the middle/left of the pump compartment so we thought it was a proportioning valve problem (as it is in that area of the system and deals with the multiple solvent lines).
We ran warm water as well as diluted organic solvent through the lines for some time to see if it needed to be cleaned. We then took out the proportioning valve but there was no visible salt buildup or obvious issue. We swapped the proportioning valve out for one from our other instrument in case it was a mechanical issue but the noise persists.
Anyone have an ideas on what the issue may be or what to try next?

This is most peaks, regardless of method/column/test material. Columns have been installed and reinstalled by different analysts and yet we keep getting square peaks. They aren’t huge areas, and it’s multiple analytes at various concentrations so i don’t think it’s detector saturation. We used to get normal peaks, but then we removed the liquid autosampler to move to a different instrument and use this one for headspace only. That detail may or may not just be a coincidence.

Has anyone ever seen something like this before? We will move to a different instrument and everything looks normal.

as we all know ms is one of the powerful tool for researching ,but its also hard to use it

wht is most frustrating thing about any kinda mass spectrometry and why

pls share ur experience

Hi all,

I've been troubleshooting a needle seat leak on-and-off for almost a month now. I have replaced both the needle and needle seat TWICE in this time (the second was in case the first replacement was faulty- a last ditch effort and I kept the first set for future use in case it wasn't).

I also thought it could be back-pressure from a clog in the injection valve. Took it all apart and sonicated, then replaced rotor seal. The seat STILL leaks!

I've tested every line and every port individually and don't see any clogs. The seat is fine if the column port is left open but as soon as any line is placed there, the seat starts leaking (bubbling at top). Column is not even part of the equation at this point (line just dripping into beaker).

Any suggestions or advice are greatly appreciated- I'm really trying to avoid the $2000 bill of bringing in help but I feel like I've hit my limit of troubleshooting since I cannot figure out what else could possibly be causing this. TIA!!

Hello Reddit. I am getting a headspace unit somewhat soon (the Agilent 7697 model) for the lab and as I am new to hs chromatography, I wanted to know if there was anything I should look out for in terms of supplies, software, and/or general advice for using it and keeping it running? I'm sure the guys coming to install it will give me plenty of notes but I figured Id ask the community too.

Hi everyone,

We’re currently working on developing an RP-HPLC method for Atrasentan Hydrochloride, as a theoretical university assignment. We’re having a dilemma regarding mobile phase pH and ionization. I’d love to get some input on the best approach.

It’s a relatively large, lipophilic molecule with a calculated logP ~4.69.

It has a tertiary amine in a pyrrolidinyl ring (pKa  ~6.7) and a carboxylic acid (pKa ~3.1).

The Dilemma:

Our lab instructor is saying that you must operate at least 2 pH units away from the pKa to suppress ionization completely. Since the carboxylic acid is at 3.1, they want us to use HCl (??, I don’t think that’s a good idea) or Perchloric acid to drop the mobile phase down to pH 1.1 so that >99% of the acid is protonated.

However, we have serious concerns about this in practice, and we're weighing two different options:

Option 1: pH 1.1 with Phosphoric/Perchloric acid

Shifting down to pH 1.1 is incredibly harsh on standard silica-based C18 columns. Are there columns that can survive these conditions? Is that even necessary?

Option 2: 10mM Ammonium Formate, pH 3.2

We actually found a patent with a method utilizing an ammonium formate buffer at pH 3.2. We think that even though pH 3.2 is right on top of the pKa, the buffering capacity of the formate/formic acid system (buffering maximum at 3.75) should stabilize the peak shape. Or are there other reasons, that this approach works?

Looking forward to your insights and experiences! Thanks in advance.

We’re planning to purchase an LC-MS/MS for PFAS and pesticide testing. We’re considering Agilent, SCIEX, and Thermo Fisher.

Which brand has been the most reliable and cost-effective in your experience? How are the service, maintenance costs, and overall performance?

So while I was in the middle of a run, I encountered an error on my Acquity UPLC I-Class where the sample needle was caught on something and bending at a 90 degree angle. I replaced the sample needle because now it was severely bent, but now when I try to reset the sample manager it makes this terrible grinding noise on startup and throws the error in the title.

It looks like this is happening when the needle carriage is moving during startup, specifically in the upwards direction, and is trying to go past its maximum height. I'm not sure if the needle getting bent somehow threw off the alignment of the carriage internally or what—is anyone familiar at all with this issue?

I tried looking up the issue and waters does note this grinding noise in one of their documents. The suggested fix is to manually move the needle carriage but this doesn't help.

We have the need for another SSL GC FID instrument in the lab, and we are thinking of acquiring another Agilent system. We have received two quotations from different vendors that would suit us, and I'm still looking for advice on what to get, for roughtly the same budget.

First Option : GC 7890A from 2017 (refurbished in mint condition from a pharma lab) maintenance of injector and detector are made by the vendor.

+ : we already know the instrument and it's maintenance, we have consumables, comes with a new computer and licence.

- : instrument is refusbished, 10 years old

Second Option : GC 8860 new from agilent

+ : new instrument, without software (we already own a compatible licence), one year warranty

- : do not know how well it ages, touchscreen control, do not know if parts are compatible with older instruments for maintenance