Chromatography Forum

dear all,

iam using tetrabutyl ammonium hydroxide solution for ananlysis of MAgnesium ascorbyl phosphate and sodium ascorbyl phosphate.
the problem is if it is a new c18 column iam getting good peak shape and resolution, after 4- 5 analysis the column goes bad condition and it give split peak.

please suggest for cleaning procedure for the same.

First off all please follow the column care instructions of the manufacturer and do the Initial QC test again to see if the column still gives good Resolution an number of theoretical plates.
We Need more Details like column Dimension, flow rate, back pressure etc. to be able to give you a recommendation.

you are using the tetra butyl ammonium hydroxide solution it is highly basic near to 11.5 to 12.5 now please check the pH range of your column. If it is 2 to 7 or 7.5 it may be damage the column stationary phase.
If you want to continue the same mobile phase switch the column known as waters X-terra, X-bridge or Zorbax Extand or Inertsil sustain.
Give us your complete method detail which may help us to predict the actual cause of the column damage.

Regards

Praveen

dear,

iam adjusting pH to 7.0 with Orthophosphotic acid or Triethyl amine.
pH range of column is 1.5 to 10.5 and is there any best method to wash out Ion pair buffer and get back original phase.

sorry to provide column details,

Purospher star RP-18 e 250 x 4.6mm,5um.

method details are

Column: Purospher star RP-18e 250 x 4.6mm,5um
Buffer: A: 3.7g of Tetrabutyl ammonium hydroxide solution(25% methaol) in 1000ml of water (55%)
B: methanol(45%)
Flow: 0.8ml/min
UV:260nm
Conc:0.5mg/ml

The concentration of Bu4OH (~15 mM) seems high. Instead of using it as a buffer, I would prefer using an appropriate concentration for ion pairing (1 mM) and add buffer ~ 25-50 mM.

Incidentally phosphate is hard on silica-based columns, particularly above pH 6. In your particular application, the choice for mobile phase pH is important. For instance, the phosphoryl group has one pKa around 2, and another near 7. If your mobile phase pH is around 7, retention will be very sensitive to changes in mobile phase pH.

At the end of the day, run a gradient from 5-80% MeOH. Hold at 80% MeOH until UV absorbance approaches 0.

Allow plenty of time for (re)equilibration after storage.

wash the column with 400 ml distilled water then wash it with 400 ml acetonitrile HPLC grade .

ph/Amr Tarek
Instrumental Unit Head at Otsuka pharmaceuticals