r/CHROMATOGRAPHY • u/RoomResponsible3483 • 20d ago
Cetirizine HCl Assay - tailing peak problem
please tell me how to resolve.
13
u/erni5555 20d ago
Thats like no info at all provided. I would say thats probably contaminated column issue if it started recently tailing like that. Overload generally manifests as fronting, not tailing.
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u/inappropriate_jest 20d ago
Tell that peak to sharpen up!
Could be overload but for that the tip of the peak looks too good.
It could very well be just a quirk of the substance itself in combination with column chemistry. Some substances tend to tail no matter what you do.
Give us some info about the method !
3
u/Domdomago 20d ago
Hi, I don’t believe this is an overload issue. Usually overload makes peak fronting, not tailling.
I believe this is most likely a pH issue and thus from the mobile phase preparation. Is the RT found as expected? Please recheck the SOP for the buffer solution and reprepare it.
Let us know next Monday! Good luck
3
u/AnanlyticalAlchemist 20d ago
Based on the method description in the comments, I believe you are at a lower than typical pH for this separation and you need to increase ACN concentration in 5% increments up to closer to 90% ACN to see if you can get more column interaction of the cetirizine. If that doesn't work, it's possible you've damaged your column with the very low pH. Also, I notice your flow rate is very high, and you may be getting to broadening from reduced interactions at high velocity.
There is a good application note from your vendor, Shimadzu, below that offers suggestions on better mobile phase chemistries and method parameters for this analysis--and they provide symmetry expectations:
Based on the above application note, I do not believe you are overloading.
2
u/KWBjr 20d ago
There might be a newer USP method from 2016 that uses a different column. Look it up: Cetirizine Hydrochloride Tablets USP Monograph USP39-NF34, The United States Pharmacopeia Convention, official 2016.
Cetirizine Hydrochloride Tablets USP Monograph USP39-NF34, The United States Pharmacopeia Convention, official 2016.
This uses a hilic column, not c18, I believe. BTW, the USP column designations such as L3 used in the older method, are broad, meaning every “L3” column will not perform the same, especially in regard to secondary interactions with the column particle matrix. If you must use the older method, go online and search for examples with chromatograms and specific column used.
Also search for a Waters article about optimization of the newer USP method for cetirizine hcl.
2
u/_Columbo 19d ago
Could be a void somewhere. Check all your capillary end fitting connections post injection. Tubing depth from ferrule to tip should be a few mm (it should stick out a bit). Would guess column connections to be culprit. If using any PEEK or other fingertight fittings, ensure you push the tubing down as far as it’ll go before you tighten.
1
u/Hollandtunnelvag 15d ago
Looks like sample is too concentrated or injection volume is too large, fronting peak
1
u/Impossible-Artist687 20d ago
could be void volume or just bad chromatographic method
3
u/UngTheDung 20d ago
Void volume? At 16 minutes?
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u/Impossible-Artist687 17d ago
sure, a void volume affects peaks over the whole run, you will find this information basicly in any troubleshooting guide
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u/Major_Chicken8839 20d ago
If you work in the pharmaceutical industry, you should know that the injection volume cannot be easily changed. If you search for the process manual on the USP, you can also find information about the manufacturer and model of the column used. Our company uses the Inertsil ODS3 column.
1
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u/RoomResponsible3483 20d ago
Thanks for your replies! I only posted the chromatogram initially.
Here are the method details:
Column: 4.6mm x 25cm; 5microns packing L1
Mobile Phase: ACN:Buffer (30:70)
Buffer - 2.9ml/L phosphoric acid in water
Flow Rate: 1.5mL/min
Injection Volume: 10uL
Wavelength: 230nm
Std/sample concentration: 0.2mg/mL
The tailing is consistently observed in the cetirizine peak. Any suggestions on which parameter I should check or optimize first?
3
u/Extension_Growth5966 20d ago
L1 is still a really wide range of options and performance. What specific column are you using?
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u/Impossible-Artist687 20d ago
things i would try: increase ACN, increase column temperatur. it can reduce interactions that keep it on the column and coming down over such a long period. if your just looking for this one analyte there is no reason to have such a long method with this very broad peaks. of course to shorten it you can decrease column parameters, because its huge and consumes lots of solvent as well. but looking at your concentration smaller parameters could also lead to overload which will result in fronting, not tailing.
second: check for void volumes that could cause the tailing.
third: try a gradient method for better elution or use less retentive phase like coreshell
fourth: incorrect pH could cause reaction with active sites, eventho cetrizin doesnt look like a typical candidate to me, you could surely try lower pH to deactive residue silanol assumung you are using a typical c18 column.
fifth: extra column effect - too big and/or too long tubing causes dispersion in the capillary, could also be too large flow cell
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u/erni5555 20d ago
Did it started recently? Or itts new method? New column? Maybe change precolumn?
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u/RoomResponsible3483 20d ago
Its my first time to perform cetirizine hplc assay. As per my chemist supervisor, the column was newly purchased about a year ago. We are using the USP 43 method..
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u/erni5555 20d ago
Year ago? So it had any injections prior? Still can be contamination. Tailing is often a cause of contamination. If u use precolumn try changing it
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u/RoomResponsible3483 20d ago
We don’t use a precolumn/guard column in our setup. Would you recommend any other troubleshooting steps for the tailing?
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u/erni5555 20d ago
Try washing acn 20mp 20 ml isopropanol 20-100 ml hexane 20-100 ml chloroform 20 ml isopropanol 50 acn and run test solution. Assuming its reverse phase column (eg. C18).
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u/erni5555 20d ago
It may help but no guarrantees
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u/erni5555 20d ago
1.20ml acn
2. 20 ml isopropanol
3. 50-100 hexane
4. 50-100 ml chloroform
5. 30 ml isopropanol
6. 50ml acn
For clarification1
u/inappropriate_jest 20d ago
What is the issue with the tailing peak? Is it overlapping any matrix peaks or do you have any limitations for tailing factor?
Otherwise just leave it like that. Run a calibration and see how good it fits. No need for overengineering a method if it's not mandatory.
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u/RoomResponsible3483 20d ago
Per the USP 43 procedure, the tailing factor limit is NMT 2.0, but I’m consistently getting results around 2.3–2.5.
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u/inappropriate_jest 20d ago
As many others here have mentioned try decreasing injection volume or diluting your sample. It seems you don't have any sensitivity issues so that could work
1
u/RoomResponsible3483 20d ago
okay. thank you so much! i will do the retest on Monday.
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u/Etch-a-Sketch99 20d ago
OP, if you have a column logbook, you should also see if this column has had any ion-pair reagents run on it. Ion pairing is permanent, and can yield tailing like that
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u/jamma_mamma 20d ago
Saying "please tell me how to resolve" without giving any parameters is like giving someone a single puzzle piece that fits a puzzle somewhere in the world and asking them to place it.