Amoni acetate and Phosphate buffer

[ultima_86]

Hi everyone,my troubles occur when I was trying to analize Clarithromycin (Identify only).The procedure require the Phosphate buffer with pH 4.0 (Conc. 0.04M)and the RT around 17 minute.The ratio of buffer and Methanol is 58:42 and I use C18 column,flow rate 1.0 ml/min, wavelength at 210 nm.
The problen is the RT not stable (from 16 to 19 min with each injection).I tried double the Con.c of buffer but it was no use. I also thought of the buffer range of Phosphate so I use Amoni Acetate instead and adjust to pH 4.0 with Acetic acid but I got no peak at minute 17. I only got a negative peak at minute 5.
So I would like to ask you the way to have a stable RT in my analyses and
Can you help me explain why Amoni Acetate buffer which have buffer range suitable with the required pH but can't give a good result whereas the Phosphate buffer can? (though the RT still have problem)

[carls]

clarithromycin is a basic analyte with a pKa of ~8.5. Using a buffer at pH 4 or 2.5 wont make a difference in retention since the analyte is fully protonated at both pHs. Clarithromycin does not have a good UV chromophore which is why you dont see it with acetate buffer. Try pH 2.5 Kphosphate 25mM. How old is the column? Any special modifiers used previously (TFA, TEA, etc.)? Dirty samples run?

[ultima_86]

Thanks for your replies!
The column is quite new and I added no modifier.My sample is clear,I think!
But can I ask you some more questions?
1. How do you know the properties of Clarithromycin such as pKa and the sensity with UV detector? Do you refer in books, website or it is simply your pratical experience? If they are from books or web,please give me the address.
2. Is there difference between Kphosphate and Naphosphate because according to my knowledge the buffer range of them almost the same. And sometimes I use Naphosphate instead of Kphosphate but the results are the same (area, RT, ect..). If there are something else beside which I mention above,please let me know! You replies will be appriciated!

[carls]

For physical properties I use a variety of sources but chemspider is a good strating point. Occaisionally I find drugbank.ca to have useful information:

http://www.chemspider.com/

http://www.drugbank.ca/drugs/

Na and K phosphate definately have the same pKa/buffering range. In some cases K can give better performance than Na due to K having a higher affinity for the ionized silanols thus blocking access to them.

For UV absorbance I rely on experience but generally look for aromatic groups or extended conjugation. Also, carbonyl groups gives some UV absorbance.

[ultima_86]

For physical properties I use a variety of sources but chemspider is a good strating point. Occaisionally I find drugbank.ca to have useful information:

http://www.chemspider.com/

http://www.drugbank.ca/drugs/

Na and K phosphate definately have the same pKa/buffering range. In some cases K can give better performance than Na due to K having a higher affinity for the ionized silanols thus blocking access to them.

For UV absorbance I rely on experience but generally look for aromatic groups or extended conjugation. Also, carbonyl groups gives some UV absorbance.

Thank you very much! Now I can develop my knowledge more with the two given links from you. Wish you all the best will come to you. I am so grateful to you with your valuable information!

[Klaus I.]

Please have in mind, that Clarithromycin degradetas very fast in solution. The degradation product ist almost invisible in UV (compared to the also very low-absorbant Clarithromycin).
Depending on the conditions, it is possible that Clarithromycin will be completely degraded during one day, also if you use a cooled AS.