• State Verified Answer
  • +1 person also asked this people also asked this
  • Replies 31 replies
  • Answers 1 answer
  • Subscribers 99 subscribers
  • Views 8453 views
  • Users 0 members are here
Related

Agilent Knowledge Portal

Find maintenance, best practice, and troubleshooting articles.

Agilent Knowledge Portal

Phosphate buffer in HPLC

anto

Goodmorning,

since few months I am using phosphate buffer for my analysis (method from Agilent Application Solution "Analysis of water-soluble vitamins from multivitamin tablets"), I've never used it before. I have an Agilent 1260 HPLC binary pump and the column is Agilent poroshell EC-C18 2.7um, mobile phases are phosphate buffer pH 7 (channel A) and acetonitrile (channel B). I am looking for a washing protocol to prevent salt precipitation.

This is what I did untill now, but I don't know where I did a mistake: the column pressure is still too high (more than 400bar with 99% H2o and 1% ACN instead of 270 bar, I've already try to restore the column with different washes,..), I have to change it (but is quite new, I've bought it last august..)

-never stop analysis letting buffer in the system (es. overnight or during the weekend)

-after analysis wash the entire system with colum with Water (HPLC grade) 90% + ACN 10% for 20 min, then return to 100% ACN

Maybe 20 min is not enough?

 

Thank you!

 

 

25/03/19

Hi guys!

i'm still here hope you can help me. i really don't know what I'm doing wrong.

I have a new column (see previous part) and after more or less 40 runs it shows higher pressure: pressure rises from 300 bar (70%acn 0.5 ml/min) to 450 bar same conditions.

Here what I did (I've used your suggestions)

- prepare daily fresh phosphate buffer [for 0.5 ml of buffer, 0.6634g KH2PO4 (mw 136.08g/mol) + 1.328g K2HPO4 (mw 174.2 g/mol)], magnetic stirrer for 15 min, vacuum filtration 0.22um.

- sample filtration with 0.22um

- for cleaning the colum: I've created a method with these conditions (ACN & HPLC water)

     0 --> 60 min 15% ACN 0.1 ml/min

     0--> 7 hours slow gradient to 100% ACN with 30 min step (each step 5% ACN more)

     other 60 min at 100% acn 0.2 ml/min

at the end the pressure was ok (less than 300 bar) but when I check the initial condition (70% acn 0.5 ml/min) the pressure became higher

 

Where is the mistake??

What should I do now? wash with isopropanol 100%? change flow? the column is too new......

THAN YOU ALL!

 

Messaggio modificato da ANTONELLA GALEONE

Parents
  • 0

    I'm working for 2 years with this column, according to this Application Note, water soluble vitamins with much more troublesome matrix: animal feed premixes.

    I've modified it to 50 mM (even more concentrated, than Apllication Note, it is not very good for column in general) Sodium Phosphate pH=7, I have to because of my extraction process.

    It turned out impossible to prepare a buffer with sodium phosphate salts and to dissolve them fully. Maybe I mixed not long enough, but magnetic stirrer for 2 hours seems quiet enough, maybe my salts were not quality enough. Even after filtration it was clogging the column.

    I prepare it with NaOH (50%) solution and H3PO4 (85%) with no problem since then. I recommend for that ChemBuddy BufferMaker software if you have troubles with general chemistry calculations or just as lazy as I am.

     

    I would suggest you 30 ml each solution on a reversed column (0.1-0.4 ml per minute, you can't go 0.4 ml/min with viscous IPA and DMSO below 400 bar): start with acetonitrile-water (50:50), 1% H3PO4 washing, 3% H2O2 washing, DMSO washing, 0,5 g/l EDTA in 3% NH4OH (I have lots of inorganic salts in animal feed premixes), IPA-Hexane-IPA (you absolutely have to go with IPA, hexane is immiscible in the system with no IPA), 3% HCl washing, finish with acetonitrile-water (50:50) as I did it myself with 3 Agilent Poroshell columns EC-C18 2.7um 150mm 3mm with a guard 5mm 3mm 2.7um and it helped me with my matrix and didn't ruined all this 3 columns. But you should not do it regularly, if you have to, you should run a better sample preparation. You should try a solution or a set which is best dissolving your clogging material, there is no silver bullet, you should know your sample best.

    But the columns are pretty reliable as I see now.

  • 0 in reply to s.khatuntsev

    Thank you   for the reply,

    I’ll try to wash as you suggest!

    So dou think that a column guard could help when using buffer? The one that I use is made with K2HPO4 and KH2PO4.

    Nice to know ChemBuddy BufferMaker software!!

     

    Antonella Galeone

    Quality Control Lab Assistant

  • 0 in reply to anto

    I think you should double check your phosphate purity and salts weights or to prepare a buffer with KOH and H3PO4 of highest purity available for you.

    Guard column is just a small and replaceable portion of a column which clogs first. And unlike a column, a guard is much cheaper to replace if you clog it.

Reply Children
No Data
Was this helpful?
Privacy Statement
Terms of Use
Contact Us
Site Help