Paracetamol - Peak area and buffer

[ultima_86]

That was strange, I have not seen that case until yesterday. I did the assay analysis of Paracetamol and I happened to use the wrong buffer.
My official Mobile phase is the mixture of Buffer:Methanol with ratio 86:14. The buffer is prepared by adding 20ml CH3COOH to 840ml H2O then adjust pH 7.0 with KOH. I used the C18 column, the temperature is 40 Celsius. Normally, the RT is 7 min with the peak area about 500000.
That day, I happened to used the line for Water instead of the one for the Buffer. But the result didn't change anything. Everything from RT, Peak area, tailing, T.Plate to the reproducibility was good. Surprised, I tried some more buffer to check the result (The MP ratio between buffer and CH3OH was always 86:14)
Firstly, I added a bit CH3COOH (about 3ml) to my current buffer (pH 7.0) in order to lower the pH value. I got the pH 5.5 after doing that. Then I run the analysis again and got the same result. I also used NaH2PO4 0.05M at pH 3.0 and once again I got the same result.
But when I used the (NH4)2HPO4 0.05M adjusted pH 9.5 with NH4OH, everything is the same except the AREA was double. That is all for my experiment and here are my questions:
1. Why does the Paracetamol 's RT never ( or changed little) when I used many kind of buffer and even Water?
2. How can the buffer make my peak 's area double ( but the shape is still very good)?
I am looking forward receiving all your answers. Thanks a lot!

[carls]

My two cents:

Paracetamol is a very weak base (pKa ~10) so no change in RT is expected until the pH of the mobile phase is increased to ~9-9.5. The organic solvent in your mobile phase affects the pKa of the buffer (reduces it, maybe to pH 9.3) and the analyte (increases it, maybe to 10.4) so perhaps you were on the edge of a pH value that would cause a change in retention.

20mL acetic acid/L H2O (2 v/v%) is quite a high concentration (~300mM) and raises the UV cutoff of your mobile phase into the 250nm region which is also the UV max for paracetamol.

Using phosphate/ammonium for pH 9.5 reduced the background UV absorbance

[ultima_86]

Thanks for your replies,but I still don't understand what is "UV cutoff",I have looked for dictionary but couldn't find it.
And if I use phosphate/amonium pH 9.5 the UV absorbance is reduced,why is the area of paracetamol in Amonium pH 9.5 double?According to what you said,I think the area must be halve? Please help me understand,I am still confused!

[carls]

In analytical chemistry the UV cutoff is defined as the wavenlength where the solvent/solution (mobile phase) absorbance = 1AU. It is not recommended to work near the UV cutoff as most of the analytical signal (90% of UV signal) is absorbed by the mobile phase. In your case you could be working in a range where the absorbance is closer to 2AU (1% transmittance) which is generally beyond the linear range of any UV detector. If you remove the background absorbance by replacing 300mM acetic acid with 50mM phopsphate (UV cutoff <200nm) you could see an increase in signal.

Another possibile explaination for the change in UV intensity is the molar extinction coefficient of paracetamol increases at higher pH. You could easily check this with a benchtop UV spectrometer.

http://www.scribd.com/doc/77132690/73/U ... -modifiers

[ultima_86]

In analytical chemistry the UV cutoff is defined as the wavelength where the solvent/solution (mobile phase) absorbance = 1AU. It is not recommended to work near the UV cutoff as most of the analytical signal (90% of UV signal) is absorbed by the mobile phase. In your case you could be working in a range where the absorbance is closer to 2AU (1% transmittance) which is generally beyond the linear range of any UV detector. If you remove the background absorbance by replacing 300mM acetic acid with 50mM photsphate (UV cutoff <200nm) you could see an increase in signal.

Thanks for your link and explanation. Please help me fix if I don't understand exactly. If I use Phosphate which have UV cutoff < 200 nm as my buffer and choose the Wavelength 200nm, I won't be able to see any peak since almost the analytical signal is absorbed by the mobile phase. Which means, if I use the isocratic, will I see the straight line and no peak?
If I want to use the mentioned Phosphate buffer, which wavelength should I choose? How about 20nm far from the UV cutoff (180nm or 220mn) or I must need a farther distance?
You said that "If I remove the background absorbance by replacing 300mM acetic acid with 50mM photsphate (UV cutoff <200nm), I could see an increase in signal" which means the peak area will be bigger? Thank you very much!

[carls]

Use 50mM pH 7 phosphate buffer to mix with the ACN (86:14 buffer:ACN, right?)

Use 248nm for detection since this is the UV max for paracetamol.

[ultima_86]

Thank Carls, I finally understand. By the way, can you help me answer this question. Sometimes, the procedure ask me to use the Water and Organic solvent only, but I don't know the reason. Can you teach me which case ( or which compound) could I use water instead of Buffer. Or it is simply the practical experiments as I used to use Water:Methanol = 86:14 instead Buffer:Methanol (same ratio) in do the assay of Paracetamol and happened to get the same result.
Up to know, I have thought that in case the pKa of my substance is more than 9 I will be able to use water to get the similar result (RT, Area, Tailing, T.Plate and peak shape) when I use Buffer. But sometimes, the peak shape is not nice, it is broaden, asymmetric or even splitting. I'm so grateful to you for your replies!

[carls]

Paracetamol should be well behaved and not require a buffer. As you mention its pKa is ~10 so it will not be ionized in water. However, buffering the pH of the mobile phase is always a good idea to ensure all compounds in your sample (not just the target analyte) behave reproducibly.

I cannot explain why the peak shape of paracetamol would look better with a buffer than without. I suppose having a buffer present could affect the charge environment near the sorbent surface which could change H-bonding characteristics (this is pure speculation). Perhaps there is something else that is different in the other methods you use such as a stronger injection solvent that gives the poor peak shape.

[tom jupille]

having a buffer present could affect the charge environment near the sorbent surface which could change H-bonding characteristics (this is pure speculation).

Not speculation at all; you hit the nail on the head. Buffering is not just about the analyte! Silanols are somewhat acidic and you need sufficient buffering to keep their ionization under control. This is especially important with older "Type A" columns.

[carls]

I agree controlling silanol ionization is one consideration for choice of buffer pH. The speculative part of my statement above was that buffer ions modify the H-bonding behavior of the sorbent by accumulating near the surface and thus screening access to H-bonding sites. The buffer cation obviously competes with any basic analytes (the analyte discussed in this post is neutral/weakly acidic) for ion exchange sites but I simply have not checked the literature to see if screening of H-bonding (aside from occupying the dissociated silanols which themseleves have H-bonding character) by a buffer has been discussed/explored.