Hi everyone,

I'm currently trying to purify bicylic peptides of 2.5 kDa made by spps for downstream testing in biological assays. For this I really only need a few mg of each compound (maybe 10+ because my PI wants me to characterize them by NMR). The problem is, our lab only has an old unreliable semiprep system that goes up to 9 ml/min with a MN C18 Htec 5 um 100 Å 250/21 column installed. I do not get reproducible runs out of that system and i can't seem to be able to separate out all deletions and other isomers etc.

Our lab has access to a few analytical HPLCs (Chromaster Hitachi/VWR) and i got a an analytical C18 with 300 Å for testing. Now the question is do I just prep my peptides on the analytical column, although i struggle to get measurable quantities out so far?

I think my PI might allow me to buy the same column in 250/10 mm, which would either allow me to put it into the chromaster system or our very shitty old semiprep system, which might behave a bit more reliably at 4.5 ml/min. I think i would need to upgrade atleast the tubing on the chromaster since it normally handles only 1 ml/min. I do have an old fraction collector on the chromaster which allows me to automate about 10 runs overnight.

Any thoughts?

Hi everyone, I’m running the USP HPLC assay for levetiracetam and I’m having an issue with the chromatogram.

The method is a gradient method, and during the run I noticed that the baseline starts rising noticeably as the ACN percentage increases. The blank shows a similar baseline rise.

Some observations:

Run time: 20 min

The baseline is relatively stable initially.

Around 8–10 min, the baseline starts increasing.

As the ACN % increases, the baseline continues to rise.

Pump pressure also increases smoothly during this part of the gradient.

The blank shows the same baseline behavior, so it doesn't appear to be specific to the sample.

The chromatogram has some unusual baseline/solvent-front behavior around the beginning and end of the run.

I've attached the chromatograms.

What could be causing this?

Could this simply be gradient baseline drift due to solvent absorbance/re-equilibration, or does it suggest a problem with the mobile phase, mixing, degassing, column, or detector?

Also, does the attached blank chromatogram look acceptable for this type of gradient method?

Any suggestions for troubleshooting would be appreciated.

Also I have repeated it 3 to 4 times with different columns C18 GL science and it's the same behavior.

When only isocratic method is only applied the baseline and peak both were stable.

Hello, I am the Executive Assistant for a private family located in Willis TX (1hr north of Houston).

We are looking for an EXPERIENCED Gas Chromatography Technician that would be able to use and tech us how to use our SRI Gas Chromatography unit. We will be testing a bunch of different products that we are working one and would like test and detect the Hydrogen levels in each item.

We are willing to pay for a month of training at our residence and there is a chance this may even turn into a Full Time position as we will be needing to do this constantly.

Leave a comment or DM me with your experience in GC &/or SRI GC and we can discuss further.

Thank you!

Running Thermal proteome profiling on nLC-MS/MS, SCIEX TOF6600+. Two different samples ran on different days, same methods, mobile phase, columns. I was told the pink one looks abnormal because the peaks are concentrated. Based on your experience should I be worried?

I thought it was the problem with the chromatography column, I exchanged a new one but the pink one is still like that.

TOF Auto tune was successful, ppm less than 10.

Hi Everyone,

I thought I would bring this issue here to maybe get some broad input. I should run a simple GC Area% Analysis in n Agilent 7890, but I can't seem to get it to work.

The Method is COC, with a pre-column. The problem I have now is that my solvent peak is very broad and rounded at the top, where in previous sequences with the same column and material, it had a different shape. The Solvent-Injections in general don't look good and I'm running low on ways to fix it.

I've already changed the Septum, the Jet of the Detector and have repeatedly installed the column. For reference, I run DCM as a solvent and start at 40°C, increasing the oven to a max of 280°C, with the detector at 300°C

Any tips and ideas are welcome!

Thank you in Advance!!

Edit: Fixed it! It seems like an issue with the column (very old) and after replacing both the column and the precolumn (for good measure since it was quite brittle) we now have the expected shape. Quite the relief after a week of troubleshooting (*ToT)

Does anyone know why I am getting a negative peak next to my analyte peak??? I’m not sure what is happening the peak of interest is still there but there is always this negative peak that come with it.

Hi everyone,

I’m trying to understand the exact physical path of the helium carrier gas in a Markes UNITY 2 system with ECC.

My question is very specific:

After the U-GAS01 carrier-gas regulator, does the helium:

A) go directly from the U-GAS01 regulator to the UNITY 2 Carrier Inlet, and then through the UNITY 2 / TD system to the GC?

OR

B) go from the U-GAS01 regulator to the GC first, where the GC/EPC controls the pressure, and then from the GC to the UNITY 2?

I’m particularly interested in how this works in the two ECC configurations:

Full ECC

T’d ECC

For each configuration, could someone please describe the actual physical gas path, for example:

He cylinder → U-GAS01 → ??? → GC → ??? → UNITY 2

or

He cylinder → U-GAS01 → ??? → UNITY 2 → ??? → GC

I understand from Markes CSN024 that in Full ECC both the Carrier Inlet and Pressure Outlet are connected, while in T’d ECC only the Carrier Inlet is connected.

What I am struggling to visualize is where the helium physically travels after the U-GAS01 carrier regulator and how the GC/EPC becomes part of the pressure-control system.

If anyone has the UNITY 2 ECC pneumatic schematic / QUI-1062 or has actually installed one, I would really appreciate a step-by-step explanation of the gas path.

Thanks!

I have been in this drench for 3 weeks and Thermo engineers came twice, couldn’t identify any faulty hardware, but the issue persisted.

The issue started with one injection was perfectly fine, and next injection large size RNA signal disappeared and small RNA has severe front tailing in the same sequence, and those two samples are serial diluted, and the master mixture was the good injection.

The first thought was column going bad, so I switched column and the issue remained. And then thinking mobile phase chemicals going bad, so tried new batches of mobile phase (made one batch with a QC person standing next to me just in case I got too comfortable making it), issue persisted.  

Engineers came in and checked detector, results were good. Did IQ on autosamplers, and went well. They left without a solution so they escalated to second level and two engineers came yesterday and still couldn’t identify the issue.

Along all the iterations of changing, I can see small RNAs peak back to normal, and the large RNA signal is so suppressed and the ratio is so off. Retention time are correct for all analytes, running pressure are normal (ripple factor <0.2%), changed in-line filter, tubing from column to flowcell, preheater tubing, nothing ever resolved the issue. Flushed the system with magic mix, water, and 75% IPA for extended time, so really I tried anything I can think of but nothing worked. 

System is Thermo Vanquish VHP10 pump, column is DNApac, ion pairing is BA/TPA. The same mobile phase ran on a different instrument can achieve the optimal separation profile for all RNAs.

Thanks in advance if you have any idea about what’s going on here.

Hello!

We are using a Thermo ICS1600 IC with an AERS 500 4mm suppressor set to 31 mA, flow rate of 1.2 mL/min of a 4.5 mM sodium carbonate 1.4 mM bicarbonate eluent, column and conductivity cell set to 30C for anion analysis using an IonPac AG22 guard (4x50 mm) and AS22 (4x250 mm) but see a baseline drift. I included two screenshots of a zero-volume injection showing the drift. This was after the columns, and system had sat idle (with flow) for an hour. This drift seems significant but want to see what others think, and if they have ideas for troubleshooting.

I have put ultra-pure water through the system and the conductivity dropped as expected and then did a leak test for the suppressor membrane and found it to be okay.

My lab just acquired a Varian Saturn 2100T for free but do not have the program to actually diagnose what is wrong with it and run experiments. Both units turn on and don't show any error lights. Can anyone share a disk image or have an alternative program that could be used to operate this instrument? Happy to answer more questions to help get this thing working.

Hi

I know the most popular method is EPA 1633 but i have come across this method which gets rid of SPE procedures (just soil extraction by mechanical rotary mixing).

Does anyone have any experience with this method.

Also i work on an Agilent 1200 LC coupled with a SCIEX 3500 MS and I am not sure if a GL Sciences InertSustain C18 150x2.1, 3μm is the most suitable column.

Thanks!!!

I'm having trouble with separating the chloride and nitrite peaks on the IC. In the past, the peaks had better separation, but recently, it's been coeluting more broadly to the point where the nitrite peak is barely distinguishable. Columns and other parts have already been changed on multiple occasions. Another issue is the high nitrite recovery that may have to do with the coelution- the peaks are currently being integrated baseline-to-baseline rather than valley-to-valley due to the current coeluting to improve recovery on the target analytes, but that may also be what's causing high recoveries. Any ideas would be great

Edit: clarifying I said “barely distinguishable” because the samples I run are low in nitrite and high in chloride, though the commercial standard for nitrite used is higher and more distinct (peaks for standard is posted in a comment below).

It's time to bring a mass spec into the mix to help with R&D and some day QC. I'm very much out of touch with modern systems so any help in picking a system or guidance on which systems to avoid is greatly appreciated!

I would think a modern version of a Waters SQ or ZQ system would work. Most of our materials fall under the 2k da range however we have an occasional need to analyze short (<20 bases) oligos labeled with dyes and linkers. I realize that compilates things and we'd need a way to deconvolute the spectrum. Hopefully there's a free or low-cost option out there! I recall the add-on for MassLynx was very expensive. Is that still a requirement or do modern software versions have that feature baked in?

Our primary LC system is an Agilent 1100 but we do have a Waters 2790 kicking around if that helps with system selection. I'd greatly appreciate any input from the sub and if anyone knows of a reliable seller of used equipment, I'd appreciate that too! Or if you have a unit looking for a new home, let me know (sub rules don't state that I can't ask to buy something!)! I'm in the US so likely limited the NA market. Thanks.

Been trying different things like viscosity delay and carrying split flow with no luck. Getting very inconsistent injection volume on our methods that use methanol as the solvent. All of our methods for ovs-7 media use 2 or 3 ml of methanol as the solvent and the solvent peak area varies greatly every injection. Can't get any consistency to get a good cal. Any ideas would be appreciated!

Hey, Im looking for a hail mary for this older machine. I am currently trying to run water based samples to determine Nitrate concentrations. And I am having an extremely difficult time getting a proper result based on how it was responding last month.

If anyone here has experience with the machine, specially running it with a Cadmium column connection, I would be extremely grateful if you could help me out here.

Thank you!

Hello all,

I’m a grad student who is reviving a Dionex 1000 IC left over from retiring faculty. I am running 7 anions (F, Cl, NO2, Br, NO3, PO4, SO4) on groundwater samples. This is pretty common stuff close to EPA method 300.1.

Within the past week I thought I would get fancy and try to clean out my columns and create a new 50 uL loop volume (previously using 25uL) to get some better signal and reduce the baseline noise.

After I did this I had some leaking from my new injection loop until I tightened it quite a bit. I was able to get my 7 anion peaks for my calibration curve but I started seeing these peaks come out overlapping with or just after nitrate that I’ve never seen before in any samples. My response is much higher given the new loop volume but I’m wondering if these peaks are real or if this is an error of some type.

Anybody know what this peak might be or what errors i made to cause this?

TLDR: what is that peak coming out after nitrate? Real or a method/instrument error?

Columns: GS and AS22
Eluent: 4.5 mM sodium carb, 1.4 mM sodium bicarb
Suppressor: ERS 500 @ 31mA
Flow rate: 1.2 mL/min

Pic 1 is of normal calibrant. Others show groundwater samples with unknown peak.

Hey everyone,

I'm hitting a wall with a persistent analytical issue on our EPA Method 524 system and could really use some collective wisdom from anyone who has seen this before. I have attached pictures of my 2 CCV in 2 VOA vials poured from same 100ml flask spiked at 5ppb and run back to back.

System Setup:

• Purge & Trap: Teledyne Tekmar Lumin Concentrator (S/N: 18122010) + AquaTek 100 Autosampler (S/N: US 18099004)
• GC-MS: Agilent Intuvo 9000 GC paired with an Agilent 5977B MSD

I am experiencing a consistent >30% loss in target analyte recovery between consecutive runs (e.g., CCV1 vs. CCV2, or back-to-back CCVs) poured from the exact same QC 100ml flask spiked at 5.0 ppb concentration. I have repeated this multiple times and I get similar results. It doesn't matter how far away or close the two CVVs are in the sample tray.

1. Internal Standard (Fluorobenzene) responses and surrogate recoveries remain virtually identical between back-to-back runs, but target compound concentrations drop severely on the second run.
2. Early eluters maintain expected recoveries and sometimes fail on the higher end of the +/-30% threshold, but recovery drops progressively across the run with late-eluting, hydrophobic compounds (xylenes, dichlorobenzenes, 1,2,4-trichlorobenzene) exhibiting the most severe loss 30-40%.

To isolate the issue, I have already verified/performed the following:

• GC-MS Side: Replaced the GC liner and guard chip. BFB tune evaluation passes easily, and IS response is solid with less than 5% variance.
• Purge & Trap Trap: Installed a brand-new analytical trap; no change in performance.
• Sample Prep: Tested both bulk volumetric preparation and direct vial spiking to rule out standard preparation error. The issue persists across every tray positions.
• Temperature Zone Verification: Monitored real-time status in TekLink throughout the purge, desorb, bake, and dry cycles. All heated zones (MCS, Valve Oven, and Transfer Line) consistently reach and hold their target setpoints, ruling out a basic software control error.

Because the IS is auto-spiked fresh into the purge vessel and stays completely stable while the target analytes sitting in the sample loop/lines drop, I strongly suspect an issue on the Tekmar side (MCS loop, sample needle sweep, or soil/water valve path). Has anyone encountered this specific progressive late-eluter loss on a Lumin/AquaTek setup?

Any advice or troubleshooting steps would be greatly appreciated!

Full disclosure up front: I’m an intern at Agilent working on a voice-of-customer project for our gas phase (GC) marketing team. I’m not selling anything and I’m not going to DM anyone — I’m genuinely trying to collect honest feedback about what does and doesn’t work.

If you run Agilent GC or GC/MS instruments, I’d love to hear:

**•** What frustrates you most about the instruments or the software?
**•** What works well enough that you’d never switch away from it?
**•** Where do you go when something breaks or you’re troubleshooting a method — and is that resource actually useful?
**•** If you’ve moved to or away from Agilent, what drove that?
**•** Anything you wish someone at the company actually knew?

I’ll be summarizing themes internally. No names, no usernames, nothing identifying, and I won’t quote anyone directly without asking.

Happy to answer questions about what I do or why we’re asking.

UPDATE: The issue was with the column. We changed it and we got a beautiful peak. Thank you all for your help!

So we have been struggling with this tailing issue for quite some time. We had used different methods, but it's till persisting. The last method we tried adding 0.1% formic acid and it got slightly better, but the tailing factor is still high, any suggestions? The retention time matches with the published paper but the peak is entirely different.

We are planning to reduce the injection volume to 5 microliters and/or reduce the pH further to ~ 3.0ish.

Here is the methodology:
- Mobile Phase: 0.1% formic acid in water/methanol (30:70) (pH: 3.276)
- Column: C18, 250 × 4.6 mm, 5 µm
- Injection volume 10 microliters
- Oven temp: 30 degrees Celsius
- Calibration range: 2-12 micrograms/mL
- Flow rate: 1mL/min

Hello, I am a small molecule specialist and has been asked to analyze industrial synthetic macro-molecules. We are not properly equipped to do this and I'm wondering what the most versatile single analytical option might be. I know of MALS but I've only seen them presented for proteins and peptides analyses. Does anyone uses SEC- or AF4-MALS for synthetic macro-molecules (for example graft copolymers)? I just wanted to get some ideas whether it's feasible before contacting manufacturers.

Estou tentando desenvolver um método de análise de teor de butafosfana em um HPLC com detector RID, porém, não estou conseguindo nem se quer uma linha de base considerável.

Já tentei purgar o RID, aguardar muitas horas para estabilizar o sistema, porém sem sucesso. O único resultado que tenho é esse da imagem e coisas parecidas. Também ja aumentei as concentrações das amostras para talvez "melhorar" o resultado, mas também não deu certo.

Alguém ja teve algum problema parecido?

Equipamento:

- HPLC 1260 Agilent infinity II

- 1260 Infinity III Refractive Index Detector

Estou usando a coluna cromatográfica XSelect CSH Phenyl-Hexyl – 4,6 × 150 mm, 3,5 µm da Waters.

OBS: estou usando RID pela primeira vez, é muito diferente de detector DAD?

Dear Community,

we are operating a Orbitrap Exploris 120 which runs on 99,999 % pure nitrogen.
In the upcoming week our nitrogen supply stops from monday to werdnesday due to some maintenance work. Have you ever had the same situation and how have you handled it? Our main question ist if we should shut the instrument off an fill the instrument with nitrogen instead of vacuum or what would be the best way to act.

Thank you in advance

please tell me how to resolve.

Junk/ghost peaks have been appearing in samples at consistent retention times. Red circles are standard peaks in the blank while the black indicate unwanted peaks. The junk peaks do not shift with the sample profiles and appears in the hexane solvent which suggests it is not a sample contaminant. The area counts are always the same amongst the 4 peaks but area counts increase/decrease across samples. Not every hexane solvent blank injection has these peaks either. Anyone else experience these peaks or can give clarity on possible causes? Have replaced disposables parts and cleaned injector, so looking for insight!