New EP method for paracetamol

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

16 posts Page 1 of 2
Hi all,
I will run the new EP method for paracetamol this week.
We have feedback from other sites that it generates very high backpressures ,without specifying them, and they have trouble running in conventional HPLC.
We dont own a UHPLC.
Has anyone tried this method?
I want to be ready for any problems i may be facing.

Thanks a lot
based on the column parameters (2.7 um, 2.1x100 mm) flow and mobile phase (0.3 ml/min, MeOH/H2O, 30°C) one could expect about 290 bar only for column (at the end of gradient, without precolumn). So overall maybe expect about 320 bar.
If that's beyond the capabilities of your system, maybe you need to reduce the flow and recalculate the gradient. Or increase the column diameter, and recalculate the flow and gradient.
But that may be not yet allowed by 2.2.46...
Sounds like an all-too-common issue with some EP, USP, other compendial methods.

Of course our QA Director felt the USP was without reproach and we needed to follow it as written, but in reality USP was not really appropriate for actives formulated into consumer products like ours at low levels. Those fights make me happy to be retired now.
Hello.
I haven't tried new ep' paracetamol yet but run lots other methods using 2.7 uM solid core columns [1] f.e. poroshell and nucleoshell. 0.3 ml/min and 100*2.1 column is OK for agilent 1260, pressure should be much less than 600 bar.

[1] https://www.hplc.eu/halo.htm
Best regards,
Dmitriy A. Perlow
I haven't seen the method yet so i dont know if a core shell column is used.
I only know that a precolumn is "required" and that in other sites they have problems with the backpressure generated.
You 1260 withstands 600 bar?
Is it a binary pump?
Hollow wrote:
based on the column parameters (2.7 um, 2.1x100 mm) flow and mobile phase (0.3 ml/min, MeOH/H2O, 30°C) one could expect about 290 bar only for column (at the end of gradient, without precolumn). So overall maybe expect about 320 bar.
If that's beyond the capabilities of your system, maybe you need to reduce the flow and recalculate the gradient. Or increase the column diameter, and recalculate the flow and gradient.
But that may be not yet allowed by 2.2.46...



Could you tell me the way of calculating the approximate pressure please?
common formula for backpressure across a packed column, particular from book "HPLC Columns" by Uwe Neue

p = (4000 * Lc * n * F) / (pi *dc^2 * dp^2)

all in SI units !
4000: measure for column permeability
Lc: column length [m]
n: viscosity [Pa.s]
F: flow [m3/s]
dc: column diameter [m]
dp: particle diameter [m]

viscosity from tables in book "Practical HPLC Method Development" by Snyder, Glajch, Kirkland
Hi jcwagner,

Hollow types quicker than I do, I had the same reference in mind for the pressure.

Both the quaternary and the binary pumps that shipped with the Agilent 1260 LC systems are rated to 600 bar.
MattM
you may want to check online tools like the one from Waters
http://www.waters.com/waters/promotionD ... =134694974

or the one by University of Geneva
https://epgl.unige.ch/labs/fanal/hplc-calculator
The EP monograph says:
Precolumn: size: l = 0.005 m, Ø = 2.1 mm; stationary phase: end-capped solid core octadecylsilyl silica gel for chromatography R (2.7 µm).
Column: size: l = 0.10 m, Ø = 2.1 mm; stationary phase: end-capped solid core octadecylsilyl silica gel for chromatography R (2.7 µm);

So yes, it's a core-shell column and EP wants you to use a precolumn.

The knowledgebase gives some more information:
Precolumn: Halo C18 is suitable; Column: Halo C18 is suitable; Dwell volume used for the development of the method = 1.07mL.

The dwell volume stated (1.07 mL) doesn't seem to point to a "genuine" UHPLC, they should have much lower dwell volumes. Could be a 600 bar HPLC, though.
Things I would try if the pressure is too high for your equipment:
- Omit the precolumn. EP 2.2.46 allows you to change column size +/-70% and omitting the precolumn means nothing else than shortening the column. Gives you ~5% less backpressure. An when analysing an API your samples sholuld be so pure that a precolumn is not really needed.
- Increase the temperature. EP 2.2.46 allows +5°C for gradient methods.

These two are rather straightforward without further fiddling with flow-rate and gradient timepoints. You just have to check if the selectivity of the method is somewhat compromised by the temperature change. And of course, you must pass the defined SST with the adjusted method.
Thanks a lot for the answers!

i am very glad that EP finally states the dwell volume of the instrument. I had very tough time sometimes explaining to my supervisors why our chromatogram is not the same as theirs.
I will definitely ommit precolumn for the first time , it isnt necessary, just buffer salt, paracetamol and 2 impurities. I don't have one anyway.

I am not aware of Halo columns, does someone use them?

By the way i don't think that EP allows such a change in lengths for gradients any more. Chech it out if you want.

Thanks again!!
I have used Halo columns a few times. They are produced by Advanced Materials Technologies. In the U.S., mac-mod distributes HALO columns. Or, follow this link if you are not in the U.S.:

https://www.advanced-materials-tech.com ... stributor/
MattM
jcwagner wrote:
By the way i don't think that EP allows such a change in lengths for gradients any more. Chech it out if you want.


Check EP 2.2.46, change in length is allowed also for gradients.
This chapter is in revision at the time, seems to be harmonized with USP 621, which will give us even more freedom 8) .
Yes you are right!
About the paracetamol analysis:
I run this morning, poroshell column, no pre column, changed temp from 30 to 35C(column) , upper temperature 390 bars in Agilent 1260 .
My biggest problem was the leaks from the connections , mainly before column.
Tomorrow i hope i won't see leak shutdown when i arrive at work.
Column equilibration takes long, i let 20 min.
Do you use peek connections and tubing with such pressures?

Thanks for the help
I can think of one brand of PEEK fitting that would work:

https://www.idex-hs.com/store/fluidics/ ... value=4379

However, there are many other steel and composite fittings alternatives from, for example, IDEX, Optimize Technologies, Agilent, Thermo, Supelco, and so forth. These will work with stainless steel capillaries well.

Standard PEEK fittings are not worth taking a chance on in the long run. Likewise, there is a grade of PEEK tubing that can work at ca. 480 bar,

https://www.idex-hs.com/store/fluidics/ ... value=4336

it may make better sense to stick with stainless steel, though.
MattM
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