Hi Jagan,

I quite like ammonium acetate as it is, unlike any phosphate buffer, volatile (i.e. you can even use it on your LC-MS) and has a similar enough pH range (it is good between pH 3.8 and pH 5.8). Also, you can use it at wavelengths of 200 nm.

Good luck!

You can also use formic acid too.

thank you... I will look into it, since i need pH of 6.5.

In that case you'd probably want to try ammonium bicarbonate as a buffer. That starts at pH 6.6 and goes this 8.6. Though citrate would fit your bill, too, it's nit recommended below 220 nm.

Amemded on 09 March 2017

I have used sodium or potassium  phosphate buffer (at pH 2.1).   Ammonium ions in phosphate buffer seems not good for silica gells.   I have tested NH2-columns (aminopropyl-bonded silica gel) in vain.   CN-column is also not good to silica gel, and acetonitrile as an eluent is not also good.   Therefore, organic modifier should be alcohol.   Nitrogen atoms near silica-gel surface may be not good for silica gel structure.   Therefore, I have always used ODS (octadecyl silane) column.     Exceptionally I have used an avidin-bound affinity-silica-gel column (2.0 or 3.3 cm column length), however frequent home-made column-preparation is easily possible to perform since column length is short in this affinity-column case (please see file; Netherlands biotin and Wide range of Biotin).

I am sorry that Waters has recommended an NH2-HPLC method; i.e., K. Kobayashi, A.D. Powell, M. Toyoda, & Y. Saito. (1989) High-performance liquid chromatographic method for the simultaneous analysis of α-solanine and α-chaconine in potato plants cultured in vitro. J Chromatogr A, 462, 357-364.

Water's μBondapak NH2 column and a mobile phase of ethanol—acetonitrile—potassium dihydrogenphosphate (3:2:1) (pH ?) was used.   Since NH2 column accumulates or absorbs hydrophobic molecules, sample pre-treatment by ODS gels seems important by their method.

The life span of the column may be shorter than ODS, and please purchase the column frequently.   NH2-columns of Nomura Chemical, Seto, Aichi, Japan or Macherey-Nagel, Düren, Germany may be cheaper.

Further, I have found a research paper using ODS-RP-HPLC method.  

Drs. Robert J. Houben and Kommer Brunt (Netherlands Institute for Carbohydrate Research TNO, Rouaanstraat 27, 9723 CC Groningen, Netherlands) have reported an RP-HPLC method. “Determination of glycoalkaloids in potato tubers by reversed-phase high-performance liquid chromatography. J Chromatogr A (1994) 661, Pages 169-174".   ODS-columns of Nomura Chemical, Seto, Aichi, Japan and/or Macherey-Nagel, Düren, Germany is cheaper and well silanol-capped. 

@Sabine Ma'm i'll definitely look into the alternatives that you have mentioned

@Hayakawa sir I have to enquire for the column availability if feasible will definitely go for it

estimation of formic acid from sample ,i used phosphate buffer 2.6 ph 

220 nm

Is there any reason for pH 6.5? If you use HPLC, it is better to work with a C18-column at ca. pH 2, using 0.1% formic acid.

alkaloids are usualy basic analytes so low pH buffer/ mobile phase can cause basic analyte to become protonated. In this situation the basic analyte will be ionized, resulting in reduced retention on RP-HPLC that is why neutral or basic pH of mobile phase is necessary

15 March 2017

I agree with the opinion of Dr. Imre Molnár (Molnár-Institute for Applied Chromatography, Schneeglöckchenstraße 47, 10407 Berlin, Germany).   Please use the 0.1M sodium phosphate buffer (pH 2.0), then glycoalkaloids may be retained due to the increased hydrophobic interaction between glycoalkaloid and ODS (octadecyl- silane residues) gels.   I have not experienced the use of formic acid, which causes an irritation.   Please see the file in order to understand the separation of the representative alkaline protein of lysozyme.

Further, proteins and/or glycoproteins (and possibly glycoalkaloids) are changing their hydrophobicity or changing chemical conformation during purification.   Thus, these hydropobicity-changing molecules are only possible to be eluted from ODS-column via the gradient elution method.     

1)use as ammonium acetate any phosphate buffer, volatile use it at Lower pH range (it is good between pH 3.8 and pH 5.8).and (higher side pH range 8.2-10.2).

2)Ammonium carbonate (pH range lower side 5.9-6.9) and higher side (8.8-9.8).

3) ammonium formate( pH range 2.8-4.8)

4)TEA:Formic acid

I think you can also use phoshphate buffer saline( At varoiase) pH.In Maximum case the Acetonitrile , Methnaol & water are the basic solvent for method development.

thank you Ashwin vyas for the answer