• State Verified Answer
  • +1 person also asked this people also asked this
  • Replies 31 replies
  • Answers 1 answer
  • Subscribers 100 subscribers
  • Views 7382 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

    Hello, you can check if the column is the cause of the high pressure excluding it from the system connecting with an "union" the hplc tubing.

     

    I usually exclude the tubing from the system from the detector to the pump checking the pressure and find where is the blockage.

    A column blocked with buffer is pretty dead....you can try, as last option, to wash it reverting it, starting with a low flow 0.5mL/min 100% water to 100% organic (Acn or methanol). Perform various washing session checking the backpressure.

     

    I use also to wash the entire system once a year like this

    1) exclude the column(to avoid damaging it) and connect the tubing with the union

    2)flush the system with HNO3 1% for 30 min, 5ml/min

    3)flush the system with hot water (70-80%C) for 30 min, 5ml/min

    3)flush the system with water checking the output from the flow cell until pH is no more acid

    4)bring back original condition and enjoy your cleaned system

     

    hope it helps

     

    greetings

Reply
  • 0

    Hello, you can check if the column is the cause of the high pressure excluding it from the system connecting with an "union" the hplc tubing.

     

    I usually exclude the tubing from the system from the detector to the pump checking the pressure and find where is the blockage.

    A column blocked with buffer is pretty dead....you can try, as last option, to wash it reverting it, starting with a low flow 0.5mL/min 100% water to 100% organic (Acn or methanol). Perform various washing session checking the backpressure.

     

    I use also to wash the entire system once a year like this

    1) exclude the column(to avoid damaging it) and connect the tubing with the union

    2)flush the system with HNO3 1% for 30 min, 5ml/min

    3)flush the system with hot water (70-80%C) for 30 min, 5ml/min

    3)flush the system with water checking the output from the flow cell until pH is no more acid

    4)bring back original condition and enjoy your cleaned system

     

    hope it helps

     

    greetings

Children
Was this helpful?
Privacy Statement
Terms of Use
Contact Us
Site Help