Hello,
Recently I used reverse phase HPLC with a C18 column to purify a spider toxin, using acetonitrile+0.1% TFA and water+0.1% TFA as mobile phase. The elution protocol worked very steady until recently. The retention time of the spider toxin kept delayed further and further. It used to be eluted at ~ 21% acetonitrile, now it is eluted at 95% acetonitrile.
I tried to regenerate the C18 column by wash with Methanal-Chrloroform-Methanol as instructed by the column manual. It did not solve the problem. Later I change the security guard cartridge of the column because it may get dirty and cause the delay. But it still did not solve the problem.
Do you guys have any solutions for my problem? Is the beads of the column getting bad? Any suggestion will be appreciated.
0
3 replies to this topic
#2
Adrian K
-
- Global Moderators
-
- 684 posts
Legendary Graduate Beggar
24
Excellent
Excellent
Posted 23 October 2012 - 05:45 PM
Check pump calibration and possible loose tubing.
Also, check your system pressure, it might be inconsistent.
Also, check your system pressure, it might be inconsistent.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#3
mdfenko
-
- Active Members
-
- 2,240 posts
an elder
77
Excellent
Excellent
Posted 24 October 2012 - 07:42 AM
is your mobile phase prepared fresh or used over several days?
how do you degas? are you sparging with helium?
the tfa may be evaporating. try fresh mobile phase (if you haven't already done so).
how do you degas? are you sparging with helium?
the tfa may be evaporating. try fresh mobile phase (if you haven't already done so).
talent does what it can
genius does what it must
i do what i get paid to do
genius does what it must
i do what i get paid to do
#4
ionchannelbk
-
- Members
-
- 2 posts
member
0
Neutral
Neutral
Posted 24 October 2012 - 08:14 AM
Thanks for reply, Adrian and mdfenko.
The mobile phase was always fresh. Actually I have to make it fresh. I ran Prep-HPLC, which consumes 4L mobile phase in 2 hours. I degas it by vacuum which looks working well for my many previous runs.
Now I think it is worth checking the pumps and the mixer.
The mobile phase was always fresh. Actually I have to make it fresh. I ran Prep-HPLC, which consumes 4L mobile phase in 2 hours. I degas it by vacuum which looks working well for my many previous runs.
Now I think it is worth checking the pumps and the mixer.
Also tagged with one or more of these keywords: HPLC, protein separation
 |
Protocols and Techniques Forums →
Protein and Proteomics →
Peptide HPLC Sample PrepStarted by Guest_Kb Homes_* , 22 Apr 2013  Peptide, purification, HPLC
|
|
|
|
Protocols and Techniques Forums →
Biochemistry →
Lost of ReproducibilityStarted by Guest_Zeenat Tinwala_* , 09 Sep 2012 PGA, LC/MS, HPLC
|
|
|
||
Protocols and Techniques Forums →
Protein and Proteomics →
AKTA/Unicorn compatibility with Windows 7?Started by Guest_vsoy_* , 12 Jun 2012 software, akta, fplc, hplc
|
|
|
||
Protocols and Techniques Forums →
Protein Expression and Purification →
Weak affinity chromatographyStarted by Guest_rjp_* , 20 Apr 2012 affinity, chromatography, IgM and 2 more...
|
|
|
||
Protocols and Techniques Forums →
Protein and Proteomics →
Weak Affinity ChromatographyStarted by Guest_rjp_* , 18 Apr 2012 chromatography, biochemistry and 2 more...
|
|
|
