Chromatography Forum

Hi ALL

Please HELP and sorry for such a long post

I have a problem with some chromatography using a Phenomenex Luna 5u c18(2) – 250*4.6mm column.

I am detecting for the following components:

Paracetamol - (10.1mins) - UV
Caffeine – (12.6) – UV
Phenylephrine HCL – (2.7) - FLD
Guaifenesin – (15.1) - FLD
Guaiacol - (16.0) – FLD
---------------------------------

METHOD is as follows:

The mobile phase: gradient system as follows:

Time A% B%
0mins 95 5
2mins 95 5
10mins 55 45
17mins 50 50
17.01mins 95 5

A - Water: formic acid 0.05%
B - Methanol:formic acid 0.05%

Run time: 20mins; Flow rate: 1.0ml/min; Injection: 3ul, Temp 35C
Detection: UV:275; FLD: ex 275nm em 300nm

Samples and standard made up in 0.1M hydrochloric acid (the samples have to be made up in this)
------------------------------------------------------------------------------

As you cam see from the following chromatograms.

http://www.davidwalkerhplc.zoomshare.com/1.shtml/HPLC

(1) In the first chromatogram the components are properly separated, with excellent Gaussian peaks

(2) The second chromatogram is the noise for the above standard: with slight retaining of the standards, but in general OK.

(3) The third Chromatogram is an appalling standard after only a few weeks column use.

(4) The fourth chromatogram is the noise that corresponds to the above standard

From these chromatograms it can be seen that over time the components are sticking to the column as the number of injections increase. As it can be seen from the noise of the last chromatogram.

So my question is: WHY?

Is it because the column is been destroyed by the low pH due to the samples/standards been made up in 0.1M HCL. If this is the case any suggestions of what column I could use to stop this?

OR - Is it the components are been ionized by the low pH of the 0.1M HCl.
If the components are been ionized, what buffer would you suggest I make up the samples/standards in?

Any suggestion would be fantastic help

Kind Regards

Dave

I notice also a significant loss of retention. The Luna c18(2) column is quite resistant to acid; I don't think your sample in 0.1 N HCl is the problem. (Although if it were my method, I'd prefer a different acid.) However the column is very hydrophobic and susceptible to dewetting under highly aqueous conditions. If that is the problem, re-wetting will cure it. Flush the column with degassed acetonitrile for half an hour. Unfortunately, if dewetting is the problem, it will return. Consider using a polar-embedded or polar-endcapped column instead. If you can't change columns, better degassing can help. Also, at the end of the day, rinse and store the column in acetonitrile. (For some reason, methanol just doesn't do as good a job at re-wetting.)

Why don't you try a buffer instead of just formic? I like 10 mmolar ammonium formate adjusted to pH3.5 with formic. Mike use phosphate buffer for your application. I use formate buffer because doing LCMS.

I've been using a polar embedded column which works very well for a lot of applications. A few are shown at

http://users.chartertn.net/slittle/

section on matrix effects.

I find a precolumn works very well with the column. When things go bad, just throw it away and usually fixes. Still use the precolumn filter before the precolumn. I've had a few problems with precolumns, but the manufacturer has addressed them.

I notice that you only have 3 minutes of re-equilibration @ 1 mL/min. Your 4.6 x 250 mm column has a volume of roughly 3.5 mL. Obviously, this is not nearly enough time to requilibrate the column (even accounting for injector delay as it sets up for next injection).

Your gradient only goes to 50% MeOH at the end. Perhaps there is material which is not being washed off the column and is building up over time. Running the gradient up higher to ensure everything is being eluted might help.

Just a couple thoughts.

Firstly thank you all for your comments.

I have tried back flushing the column with acetonitrile/water combinations and then over night flushing with acetronitrile/water forwards at 0.5ml/min. SO the column should be 'regenerated'.

I tried the same standards (kept in the freeze for a week) and everything was fine, except the caffeine had similar tailing at 12.6mins. (chromatogram 3) The noise had a similar lump where the gradient of methanol kicks in at 10mins, everything else was fine.

I know over time, the same effect is going to happen too the other components.

It would appear that the componets are been held on the column.

My sample prep is as follows:

Weight the standards into volumetric flask, add 10ml MeOH; shake make up with 0.1M HCl. Then transfer 5ml to a 50ml flask and make up with HCL.

The reason for acid is the samples are capsule and the acid is required to dissolve the capsule, releasing the actives. The samples have a similar sample prep as above.

The same components have been analyzed successfully with this same method, but made up in MeOH:water, as the acid was not needed, as these samples were not capsules.

Could I at this point add a buffer at the final dilution, instead of the acid? Any suggestions what I could use? This may count act the possible ionization. JAMES LITTLE could I add the phosphate buffer at this point? Or is it better added with the phase?

OR am I way off the mark?

Please Help….I’m a bit lost

Thanks

Dave

P.S

I will extend the run time, with an extra MeOH step. Thanks Hugh

Dave,
I have a Luna C18(2) of the exact same dimensions, and have used it for analyses of guaifenesin and caffeine under similar conditions (water/formic acid/ ACN mobile phase). I don't think the column is your problem. I think the last poster is probably right about you needing more equilibration time- I find on my column @1mL/min it takes about 7 minutes to re-equilibrate. Also, notice how the noise increases as the concentration of methanol in the mobile phase increases. Maybe there's a way to clean your methanol up? That might help.

If you try those things, and still have that shoulder coming out in front of the peaks, maybe it is the column then. I was seeing front-side shouldering for pretty much every peak I was getting a couple weeks ago- I had either clogged the entrance frit or created a void by reverse flushing. If this is what happened to your column, you may be able to fix it by opening it up, giving the frit an ulrasonic bath and smearing some C18 slurry in the top with a spatula. Although, I guess it voids your warranty, it's kind of a desparate move.

Hope that helps, and good luck!

In general, it is good to run with a buffer for rugged methods. Several LC sites on the internet have calculators for determining buffers and the limits of pH appropriate for the selected acid.

I've never used HCl for any ot our analyses. Usually Formic, acetic, phosphoric, and less often trifluoroacetic acid. Always thought HCl not particularly good if stainless steel in the system.

Also the use of buffer system would buffer your acidic sample upon injection.

What about using one of these other acids such as phosphoric to dissolve your capsule.

Again, I don't particularly like phosphoric for LCMS since such a significant ionization problem. Also, trifluoroacetic not too good for LCMS. Usually use formic and acetic.

Good luck.

James,

Why don't you look up the buffer capacity of 0.1% formic acid on one of your internet calculator sites- this is the usual strength employed. Perhaps 0.05% is a bit weak. Maybe the result will suprise you.