Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Chromatography Forum
Register Search Today's Posts Mark Forums Read

Chromatography Forum Discuss Chromatography techniques in this forum. Includes a HPLC forum, and other "liquid phase" separation techniques.


RP-HPLC bubbling advice needed

RP-HPLC bubbling advice needed - Chromatography Forum

RP-HPLC bubbling advice needed - Discuss Chromatography techniques in this forum. Includes a HPLC forum, and other "liquid phase" separation techniques.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 12-17-2008, 10:37 AM
Pipette Filler
Points: 1,216, Level: 20 Points: 1,216, Level: 20 Points: 1,216, Level: 20
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Posts: 4
Thanks: 0
Thanked 0 Times in 0 Posts
Default RP-HPLC bubbling advice needed



Hi all.

I have a RP-HPLC snag. While a sample containing 30% glycerol is being run through a Synergi MaxRP-C12 column with a linear acetonitrile gradient (water phase being Tris-0.05%TFA, pH 8.5) and the gradient reaches 45-50%, pressure goes down and a bubble forms blurring a substantial part of the picture. Glycerol-free samples run with no trouble. Glycerol is essential for solubility of proteolysis fragments in the sample, sample pH is 8.0 and is needed for proteinase K proteolysis. It would be great if someone could help sort this out. Thanks in advance.
Reply With Quote
  #2  
Old 01-06-2009, 06:43 PM
Pipette Filler
Points: 89, Level: 1 Points: 89, Level: 1 Points: 89, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: RP-HPLC bubbling advice needed

Have you tried cleaning up your sample after lysis using a SPE cartridge or some ziptips ? Proteolysis fragments are generaly soluble in some combination of ACN/H2O and some acid (Formic or Acetic)
Reply With Quote
  #3  
Old 01-25-2009, 12:41 AM
MoverZ's Avatar
Pipette Filler
Points: 108, Level: 2 Points: 108, Level: 2 Points: 108, Level: 2
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Posts: 8
Thanks: 0
Thanked 3 Times in 3 Posts
Default Re: RP-HPLC bubbling advice needed

Are you running your solvents through a degasser?
Reply With Quote
  #4  
Old 02-02-2009, 05:24 PM
Pipette Filler
Points: 1,216, Level: 20 Points: 1,216, Level: 20 Points: 1,216, Level: 20
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Posts: 4
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: RP-HPLC bubbling advice needed

Once again, thank you for your replies

Problem is I need my protein material soluble DURING proteolysis as well not just before loading it on the column. Glycerol proved to be the only reagent that kept solution clear without compromising my proteaseís stability. Sucrose could be another alternative but I doubt it could prevent bubbles forming due to its high viscosity. DMSO (up to 10%) didnít work.

We donít have a degasser so my colleague and I were experimenting blowing helium through our buffers using available material, which didnít help. The only protocol that worked well included a very steep linear gradient, 10 min at 1 ml/min 0-100% ACN, apparently not allowing enough time for bubbles to form but the peaks came out too stacked of course. Now that really became a problem when I started running BME-reduced samples so I finally opted for a step gradient. Thereís been no bubbles with the step protocol so far (and Iím groping for wood ) so right now Iím trying different protocols to separate both BME and urea from my reduced fragments, which is a bit tricky.

P.S. I wonder why I canít view all of the replies to my post, only the last two. There were a lot more, were they deleted?
Reply With Quote
  #5  
Old 02-02-2009, 08:35 PM
Pipette Filler
Points: 24, Level: 1 Points: 24, Level: 1 Points: 24, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: RP-HPLC bubbling advice needed

did you clean it?
Reply With Quote
  #6  
Old 02-03-2009, 12:11 AM
swanny's Avatar
Pipette Filler
Points: 494, Level: 9 Points: 494, Level: 9 Points: 494, Level: 9
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Location: Sydney Australia
Posts: 19
Thanks: 0
Thanked 3 Times in 3 Posts
Default Re: RP-HPLC bubbling advice needed

First of all, I think you need to find a degasser, wherever you can get one. Do you not have access to a vacuum line? What about one of those old, highly environmentally-insensitive tap-based vacuums? Degassed solutions will make a world of difference to your purification, and will extend the life of your column.

Next, even though you may need the glycerol for the proteolysis steps, you probably won't need it during the separation. Load with glycerol, by all means, but do the gradient without any, having made sure you've removed as much as possible in a washout step.

Have you considered other columns? Buffers?
Reply With Quote
The Following User Says Thank You to swanny For This Useful Post:
admin (02-03-2009)
  #7  
Old 02-08-2009, 05:42 PM
MoverZ's Avatar
Pipette Filler
Points: 108, Level: 2 Points: 108, Level: 2 Points: 108, Level: 2
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Posts: 8
Thanks: 0
Thanked 3 Times in 3 Posts
Default Re: RP-HPLC bubbling advice needed

I would expect that sparging with helium should be adequate in lieu of a degasser. So let's back up a second here... Do you visually see bubbles in the flow cell of your UV detector if you remove it and observe during the run? If it's just the pressure drop and chromatography issues there could be other explanations like a stuck check valve in one of your pumps etc.

I have never used glycerol myself, since I work entirely with small molecules, so I'm curious if it tends to act somewhat like a strong solvent in terms of elution.
Reply With Quote
  #8  
Old 02-09-2009, 10:53 PM
swanny's Avatar
Pipette Filler
Points: 494, Level: 9 Points: 494, Level: 9 Points: 494, Level: 9
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Location: Sydney Australia
Posts: 19
Thanks: 0
Thanked 3 Times in 3 Posts
Default Re: RP-HPLC bubbling advice needed

Another problem you might be having with your glycerol system is poor mixing. 30% is a heck of a lot of a fairly viscous solution, and when you mix it with acetonitrile, there's bound to be a great deal of "schlieren" mixing. Can you tell us the details of the HPLC system? Specifically, what's the size of the mixing chamber, and what flow rate are you using? How long does the gradient take?
Reply With Quote
Reply

Tags
advice , bubbling , needed , rphplc


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
RP-HPLC bubbling advice needed prishly Chromatography Forum 0 12-17-2008 10:27 AM
Advice needed for Western blotting Peter Ellis Protocols and Methods Forum 1 06-17-2008 09:41 PM
Career advice needed. WishfulThinker Physics Forum 2 07-11-2004 04:43 PM
Career advice needed (would be greatly appreciated) John Fairmont Protocols and Methods Forum 2 09-02-2003 03:17 AM
Career advice needed (would be greatly appreciated) Jayakumar, R Protocols and Methods Forum 0 08-22-2003 01:49 PM


All times are GMT. The time now is 03:13 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.15664 seconds with 16 queries