separation of sulfonamides on C18

[Girvuch]

I must separate 10 sulfonamides on a C18 column of 250x4 after precolumn derivatization with fluorescamine and i have problems with a couple of pairs.

[Bryan Evans]

Below is a high throuput analysis of sulfonamides separated on
3 different ODS phases (Cadenza CD-C18, Unison UK-C18 and
Cadenza CL-C18):

http://www.silvertonesciences.com/files/TI258E.pdf

[Harald]

Found this application on Hypersil GOLD aQ. Peak shapes looked super.

Part No.: 25305-154630
Phase: 5μm Hypersil GOLD aQ
Geometry: 150 x 4.6 mm
Flow rate: 1 ml/min
Temp.: 30 °C
Inj vol.: 5 μl
Detection: UV 270
Mobile Phase: Water+0.1%formic acid;ACN+0.1%formic acid; 10 to 100% B in 15
1. Sulphaguanidine
2. Sulphanilamide
3. Sulphathiazole
4. Sulphamerazine
5. Sulphamonomethoxine
6. Sulphaquinoxaline[/img]

[Mary Carson]

I suspect those applications listed are without the fluorescamine derivatization...

[Bryan Evans]

I suspect those applications listed are without the fluorescamine derivatization...

Very true - Imtakt's data is for underivatized sulfonamides.

What are your experimental conditions? Which compounds are not
separated?

[Girvuch]

I must separate: SDZ, SMZ, SMR, STZ, SMP, SQX, SMZL, SIZ, SDM, SMXL after derivatization pre-column with fluorescamine in a common C18( column (or i have a C 8 ) of 250x 4 mm. I can not separate SDZ from STZ and SMP from SMZL. Thanks a lot for the answers.

[cyanogen78]

I think that I can help you...I have separated 13 of sulphonamide derivates with fluorescamine 2 years ago. First - the mobile phase has to be phosphate bufer ph 3.0, 20mM; gradient has to be programmed with acetonitrile and little adition of methanol. pH is necessary for stability of derivates - its important to obatin a lower LOD. But the column is more important - do you have C18 with smaller ID, for example 2.1, ot do you have column with 3.0 or 3.5um particles. It will be easy if you have suh columns. Otherwise you can use a standard 20x4.6mm ID also but it will take more analysis time. In my opinion Zorbax C18 will be the most stable and reliable in these acidic conditions and will have longest lifetime too...

[Girvuch]

Now, i have only a standart 250 x 4 mm C18 column. But i will try to get a diferent one, but i not sure i could.
So you say i must try with a gradient of AcN, little MeOH and buffer pH3...and that i will have long time analysis...more than 40 minutes? i do the derivatization reaction in the loop programing the autosampler...and it takes more than 30 minutes ....so i will have more than an hour for each sample... but my problem now is to separate the compounds...so i will try the gradient you say.
Thanks a lot for your answer.

[cyanogen78]

If you have this column only (may be from Merck) try to do this: start with 10% AcN and 90% of the phosphate buffer pH=3.0. Use linear gradient to 70-80% AcN in 20minutes. Flow=1ml/min. Excitation wavelenght = 405nm, emission wavelenght = 490nm. Let see how it will look... What pump do you have - binary or ternary/quaternary?
When we developed the method for sulphonamides I remember that a precolumn derivatization was with fluorescamine at 60degrees celsius for 30minutes. On what HPLC do you work?

[Girvuch]

I have no problems with the derivatization reaction at room temperature during 30 minutes (i have programmed the autosampler to do it). I work with 405/495 excitation/emision and i have rather good peaks at 3.5 ppb.
I have a cuaternary pump, and an HPLC Agilent 1100.
I have a Merck Lichrospher 100 RP-18 5 um column, i tried gradient with MeOH/AcH 2% from 10 to 90 % of MeOH in 40 minutes, but even in this conditions i could not separate STZ from SDZ and SMP from SMZL.
I will try the gradient you say with buffer pH 3.
I found a BDS C18 100x 4mm 3 um column, it can help?

[MaryCarson]

I knew this analysis sounded familiar. There are at least 3 published methods for sulfas by precolumn fluorescence derivatization. The latest one includes 12 sulfas, though it uses FMOC, not fluorescamine for derivatization:
J. Sep. Sci. 2007, 30, 2647 – 2655.

This earlier paper only does 8 sulfas, but does use fluorescamine:
Anal Bioanal Chem (2003) 376 : 534–541.

[cyanogen78]

...and this paper separates 11 SA's with fluorescamine precolumn derivatization:
Journal of Chromatography A Volume 871, Issues 1-2, 25 February 2000, Pages 37-42
OK, Girvuch, I think that Lichrospher column is short for this analysis but BDS can be sufficiently long because it has smaller particles. You will have higher system pressure with it BDS don't care its OK. I propose these experiments:
1. Do a gradient staring from 10 to 85% AcN in 20 minutes.How many peaks do you see?
2. If you have not success with the initial gradient program, try to vary a gradient time (increase) or AcN final percentage (decrease).
3. If you still have problems - try to use a constant small amount of methanol in the mobile phase. You have a quaternary 1100 Series pump so you can easy program this, its sufficiently precize and reliable. So put a bottle with methanol on the top and set up a ternary gradient that starts with 5% methanol and 5% AcN (90%buffer) and finish with 5% methanol and 80% (15%buffer) AcN.

After work day don't forget to wash the system with water/AcN 70:30.
It will be easy if we can see the chromatograms here check how can you post scanned images on the forum.

[Girvuch]

Sorry i did not answer before, but i had to use the HPLC for others determinations.
I used the BDS C18 100x 4mm 3 um column, it was an excelent advice....with it i could separate another sulfa....so i had 9 peaks of 10 compounds...then as i could not separate SMP from SMZL trying diferent gradients ...i put a C8 150mmx 4mm column in serie with the BDS C18...and i had the 10 peaks! SMP separate from SMZL but the resolution was not good enought
Now my questions is: do you think i could separate all the sulfas (the 10) with a BDS C18 3 um column of 150 mm instead of 100 mm??
Thanks a lot!