Ghost peaks with same intensity in every injection

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

17 posts Page 1 of 2
Hi All,

I'm having some trouble with my HPLC runs:
I'm getting some ghost peaks (2 or 3) in almost every injection with the same intensity.

I get it with samples, blanks and no-injection runs.

Even though I'm using a gradient (H2O + 0,1% TFA : Acetonitrile; actually I'm running with different gradients, e.g. 10%AcN -> 95% AcN or 40% AcN -> 90% AcN), it is NOT due to solvent mixtures since it started appearing some days ago (and have plenty of previous runs with the same method without these peaks).

Im using a Phenomenex Luna C18 (5um)

I tried several things:

Preparing new mobile phase (problem persists)
Preparing mobile phase without TFA (problem persists)
Changing pre-column (problem persists)
Changing column (same column, problem persists)
Running with no column and pre-column (NO PEAKS)
Running with no column (NO PEAKS)

To my eyes, everything points out to be something precipitated in the column, but when I changed it for another one with the same specifications and got the same results I just couldn't believe it.

Any other suggestion?
What can it be?

Thanks in advance!
Mostly it is related to contamination of the system. Do you have proteins in your sample matrix?? Your column is fine, don't change it. Take the column out of the system and flush the system first with hot water, followed by distilled water and methanol, than ACN and Isopropanol. You can add a litle bit of sulfuric acid to the hot water but flush carefully with pure water. Good luck
Gerhard Kratz, Kratz_Gerhard@web.de
Thanks Gerhard,

I have only organic molecules, no proteins and very low salt content (no buffers either).

I'll try what you suggest and tell you later.

Thanks again!
It is likely something that has been retained on your column from the sample matrix. Eventually, the concentration will become so high that it will be dumped into your chromatograms (even blank or no injection chromatograms). If you are currently happy with your chromatogram except these ghost peaks you can add a 95% IPA column wash at the end of every injection.
HPLC chemist wrote:
It is likely something that has been retained on your column from the sample matrix. Eventually, the concentration will become so high that it will be dumped into your chromatograms (even blank or no injection chromatograms). If you are currently happy with your chromatogram except these ghost peaks you can add a 95% IPA column wash at the end of every injection.

I thought about this option (something retained), but shouldn't it dissappear with several blank runs? Peak intensity SHOULD be smaller with sequent runs as far as I know.

Anyway, including a column wash is a good practice.

Gerhard Kratz wrote:
Mostly it is related to contamination of the system. Do you have proteins in your sample matrix?? Your column is fine, don't change it. Take the column out of the system and flush the system first with hot water, followed by distilled water and methanol, than ACN and Isopropanol. You can add a litle bit of sulfuric acid to the hot water but flush carefully with pure water. Good luck

I'm washing the system, and checked that the flushing tubing of the pumps (that should be full of IPA) was in fact empty.

There was air in there, but could this be the source of contaminants?
also thinking of contamination, not column

do you have another source of water?
if not, maybe filter it through a C18 SPE-cartridge.

Also make sure to take off the solvent bottle filters and re-check. If problem is gone, you may re-add the bottle filters or better change with new ones.

also have a look at the corresponding Troubleshooting-Wizard entries
http://www.lcresources.com/resources/TSWiz/hs100.htm and
http://www.lcresources.com/resources/TSWiz/hs400.htm
That's a common enough problem that we actually did a video tutorial about it a few years ago:
viewtopic.php?f=31&t=19085
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
If you have the same issue even for no-volume injections that kind of narrows it down to the detector- is your flow cell contaminated or perhaps the lamp has reached the end of its useful lifetime? When was the system last given a PM service? The following wash may help:

1. Remove column and replace with union. Take sinkers off all lines and store in 50:50 Organic:Water.
2. Prime ALL lines in hot water for 5 minutes each. Then add a few vials of hot water to sample plate/tray. Make up a simple Sample Set which does about 30 full-loop injections of your vials, set run time to 0.5 minute per injection and have flow rate at about 0.3ml/min using your mobile phase line(s) eg100% Line A1, 50% A1, 50% B1.
3. Let the water flow through system for an additional hour afterwards.
4. Prepare a 30:70 Phosphoric Acid:Water solution, dip all lines EXCEPT seal wash (leave this line in your standard seal wash vessel) into this and again prime lines as per Step 2 and repeat Step 3 with the vials of acid, this time leaving the acid flowing through the system for a good 3-4 hours.
5. Put lines back into water, Step 2 and 3 again, now leave the water flow all night to completely remove all traces of acid.
6. Next day, put sinkers back on all lines, replace washes and mobile phase, prime all lines for at least 15 mins each, purge injection system and try a few trial injections of blank, standard etc.

Good luck!
If you have the same issue even for no-volume injections that kind of narrows it down to the detector
No, it doesn't.

Remember, this is a gradient separation. What can happen is that non-polar garbage from the "A" reservoir can accumulate on the head of the column during the pre-gradient equilibration. That garbage is then eluted during the gradient when the mobile phase gets sufficiently strong. Watch the video I referenced for a more complete explanation.

A broad, "mid-gradient hump", however is a detector response to refractive index mismatch.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Ah fair enough I didn't consider the gradient part. I saw the video and I have even had this problem myself with a related substances gradient method when our buffered mobile phase made from water where the filters weren't changed regularly was accumulating a lot of crud when the gradient became more organic and hence led to a lot of crud peaks.

Its a tough problem to eliminate. I could only minimise it in the end with the use of a ghost peak trap between the pump and the mixer which acted as a kind of filter for these low level contaminants, it never fully got rid of the issue though.

Good luck OP.
Well, the problem was in fact contamination.

I don't know which was the source, but it seems that something was clogging the system, since after the cleaning procedure, the pressure dropped by 30% in every run (since gradients were used, I got the same pattern, but with much less pressure), and I now have NEAT BLANKS =D.

This is what I did:

1) Removed the column and replaced with union
2) Flushed both pumps and system with water (200 ml)
3) Flushed with 10% Nitric acid in water (100 ml)
4) Flushed with water (100 ml)
5) Flushed with IPA:Water 50:50 (50 ml)
6) Flushed with IPA (50 ml)
7) Flushed with Methanol:Water 50:50 (150 ml)
8) Flushed with Methanol:Acetonitrile:Water 40:40:20 (100 ml)
9) Flushed with Acetonitrile:Water 70:30 (150 ml)

Everything at high flowrates (5 ml/min total; 2.5 ml/min per pump)

After this, I connected the column and got very fluctutating pressure lectures, so I:

1) Disconnected the column and replaced with union
2) Flushed thoroughly with water (both pumps) until pressure stabilized
3) Soaked both pumps and purged with Acetonitrile:Water 50:50 until pressure stabilized
3) Soaked and purged each pump with it's respective solvent (Pump A: Water + 0,1% TFA, Pump B: Acetonitrile) until pressure stabilized
4) Connected the column and stabilize with 50% of each pump.

PROBLEM SOLVED! =D

Now I have a neat system. I replaced both bottles of mobile phase and prepared everything from scratch.

Of course I had to run some blanks before getting a neat one, but now the system is running great!

Thank you all for your time and suggestions, I couldn't see the video yet, but I sure will.

Kind regards to everyone!
Ghost peaks in gradient HPLC are mostly caused by traces of impurities in your mobile phase. Particularly the aqueous mobile phase should be checked here.
Often ubiquitous plasticizers such as dibutyl phthalate are causing such effects.

A simple solution is the application of a online cleaning column for the mobile phase like the Ghost Trap DS (Shimadzu), Ghost Buster Colum (Welch) or the Ghost-Guard-LC (ANALYTE). These are commercial available columns for the online cleaning of the mobile phase.

Try such an cleaning column !! This could help you running ideal gradients!
I think that it is the contamination in the system.
You can link directly the pump and the detector and run without injection. If peak not be detected, mean that the contamination comes from somewhere in the system.
Try to directly link each module to detector, you will find out the problem.
With me, maybe it comes from one of some rotor seals. Could you give me the system information you run?
Hope it will be useful!
polakenzen wrote:
Well, the problem was in fact contamination.

I don't know which was the source, but it seems that something was clogging the system, since after the cleaning procedure, the pressure dropped by 30% in every run (since gradients were used, I got the same pattern, but with much less pressure), and I now have NEAT BLANKS =D.

This is what I did:

1) Removed the column and replaced with union
2) Flushed both pumps and system with water (200 ml)
3) Flushed with 10% Nitric acid in water (100 ml)
4) Flushed with water (100 ml)
5) Flushed with IPA:Water 50:50 (50 ml)
6) Flushed with IPA (50 ml)
7) Flushed with Methanol:Water 50:50 (150 ml)
8) Flushed with Methanol:Acetonitrile:Water 40:40:20 (100 ml)
9) Flushed with Acetonitrile:Water 70:30 (150 ml)

Everything at high flowrates (5 ml/min total; 2.5 ml/min per pump)

After this, I connected the column and got very fluctutating pressure lectures, so I:

1) Disconnected the column and replaced with union
2) Flushed thoroughly with water (both pumps) until pressure stabilized
3) Soaked both pumps and purged with Acetonitrile:Water 50:50 until pressure stabilized
3) Soaked and purged each pump with it's respective solvent (Pump A: Water + 0,1% TFA, Pump B: Acetonitrile) until pressure stabilized
4) Connected the column and stabilize with 50% of each pump.

PROBLEM SOLVED! =D

Now I have a neat system. I replaced both bottles of mobile phase and prepared everything from scratch.

Of course I had to run some blanks before getting a neat one, but now the system is running great!

Thank you all for your time and suggestions, I couldn't see the video yet, but I sure will.

Kind regards to everyone!


Hi there Polakenzen good day ~

I faced a similar issue recently (without column the blanks injections are fine but with column, the ghost peaks appeared. Thinking that its the column issue, replace with a new one and it appeared again)

Anyway, for the cleaning procedure that you have done above, does the problem disappear all together until now (no more ghost peaks) or did you clean the system again as per your suggestion above during this period of time?

Look forward to hear kindly from you !

Thanks a lot ~

Sincerely,
Ken
Look at http://analyte.de. The "Ghost-Gaurd-LC" could help you!
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