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By Mary on Wednesday, June 2, 2004 - 08:25 am:
I also have noisy baseline problem with Waters florencence detector at low Ex wavelength. I thought it may be due to dirty flow cell, but cleaning flow cell didn't help. Could somebody give some suggestions? Thanks
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By Chris Pohl on Wednesday, June 2, 2004 - 12:01 pm:
It might be outgassing in your cell. How much backpressure do you have on your cell?
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By Uwe Neue on Wednesday, June 2, 2004 - 03:47 pm:
What is the difference between the excitation and emission wavelengths?
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By Mary on Thursday, June 3, 2004 - 09:21 am:
Thanks, Chris and Uwe. My noisy baseline problem solved by cleaning flow cell again with HNO3, acetone and IPA and also by decreaing sampling rate.
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By mary on Monday, June 14, 2004 - 04:15 pm:
hi Uwe,
while looking for answers, i would like to answer yours. If you are exciting the samples at a certain wavelength you are bombarding it with photons at certain excitation wavelength say 475nm, creating excitations of the electrons. As the excited electrons return to steady/normal state they can be detected by emission wavelength. You can find the exact emission wavelength by doing a full scan. Remember an excitation wavelength is specific to the molecule.
------------------------------------------------------------------------------------------------------------
By Carlos Teixeira on Tuesday, June 15, 2004 - 01:57 pm:
Dear Friends,
The reagents purity is more important for the good profile of the baseline. But, I would use similar conditions of inorganic componets in the Acetonitrile, too. (Octane sulphonic acid, sodium saltat).
How is the profile of the baseline in a blank-analysis?
Good elutions,
------------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, April 2, 2003 - 05:36 am:
I am developing a method involving detection of degradation products and low wavelengths, 210nm and 215nm..
The conditions were as follows:
Mobile Phase A: Octane sulphonic acid, sodium salt(2.0g/L)+0.5 ml H3PO3 and adusted to pH 3.0
Mobile Phase B : Acetonitrile
Temperature: 40 deg C
Sample and stds prepared in Mobile A:Mobile B (82:18)
Columns used: Symmetry C18 and Symmetry shield C8
Gradient Conditions with Flow rate 1.0ml/min:
Time(min)%A
0 82
12 75
15 60
25 60
I am experiencing significantly large wavy baseline at low wavelength. The degradation compounds can hardly be differentiated from the wavy baseline at 210nm and 215nm. The baselines were fine at higher wavelengths eg 245nm but the absorbances of the degradation compounds were low at these higher wavelengths.
Two HPLCs using separate columns gave the same wavy baselines at low wavelength. The wavy baseline was monitored running at Mobile A:Mobile B (82:18) and flow rate of 1ml/min for more than 10 hours with not much baseline improvement. The same wavy baseline at low wavelength was observed when the sample was injected and run at the above gradient showing that this was not due to the sample.
Can someone advise on the possible source of the problem?
------------------------------------------------------------------------------------------------------------
By Wanda Kruse on Wednesday, April 2, 2003 - 02:50 pm:
When analyses are at these wavelengths, purity of reagents are key. Acetonitrile sources vary in their uv cutoff, and supplier literature is not always reliable. You need to run your own scan. Secondly, Acetonitrile preservatives start to degrade within a couple days after opening the bottle. These degradants give a nasty baseline. Make sure your Phosphoric is HPLC grade, and the salts are of premium LC grade.
I have also had problems with MP impurities being caught by the guard column, and elute as the organic increases. For one of our analyses, we have had to put a cleanup column between MP A pump and the mixer. good luck!
------------------------------------------------------------------------------------------------------------
By Benjamin on Wednesday, April 2, 2003 - 02:52 pm:
Dear Anonymous;
Your baseline problems can be due to several factors. The purity of salts and reagents is critical. Usually H3PO4 HPLC grade and Octanesulfonic acid puriss from Fluka are very good. I also suspect that you could improve results keeping the buffer and ion par reagents constant during your gradient. Right now your gradient changes phosphate, octanesulfonic acid, and ACN.
Another factor to keep in mind is to give enough time to the column to reequilibrate at the end of each gradient.
Good Luck;
Benjamin
------------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, April 2, 2003 - 06:20 pm:
Thanks Wanda and Benjamin.
The Symmetry C18 and Symmetry Shield RP8 columns were 4.6x250mm.
We tried the C8 Symmetry 3.9x150mm today and did not experience wavy baseline problem. The baseline was good.
Would anyone know why this is happening?
------------------------------------------------------------------------------------------------------------
By HW Mueller on Thursday, April 3, 2003 - 12:46 am:
What do you mean by "wavy baseline"? Noise or a succession of broad and/or narrow peaks coming out? If itｴs the latter it would mean your C-18 columns are just dirty. If your problem is noise.... maybe some strange coincidence?? I pass.
------------------------------------------------------------------------------------------------------------
By Wanda on Friday, April 4, 2003 - 07:54 am:
Sometimes you just gotta remake mobile phase even if you think you made it correctly. When I see a baseline I don't like, I remake the mobile phase, making sure the bottles are really rinsed well. sometimes there is residue in MP bottles which will impact your baseline. Secondly, I equilibrate for about 45 minutes about 50/50 to ensure the column is "wetted", then drop back to starting concentration. As far as the difference in the columns? There should be no difference in baselines.
------------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, July 9, 2003 - 03:31 pm:
The old HP1050 series A VWD detectors were prone to generating wavy baselines at low wavelengths when room temperature varied by +/- several degrees over time due to a poor HVAC system. The detectors were beneath a ceiling vent which blew out cool and then warm air periodically. Its caused by refractive index effects. Not sure if any of these factors could be affecting your system. The series C detectors and HP1100 detectors are much better in this respect.
------------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, July 10, 2003 - 12:51 am:
As Benjamin wrote, in case of ion pair gradient method it is very important to keep a constant oin pair/buffer concentration during the gradient. You should prepare mobile phase for B channel from ion pair reagent containing buffer and acetonitril The concentration of ion pair reagent have to be the same in A and B channel. The necessary equlibrium time of the system is much longer, than that without ion pair reagent. at least 15 min. equlibrium time (post time) is necessary between injections.
Anyway, the work with ion pair - gradient elution system is very difficult even at higher detection wavelength independently to the old or new HPLC.
Good luck.
Julia
------------------------------------------------------------------------------------------------------------
By Stuart Williams on Monday, July 21, 2003 - 06:53 am:
Dear Anonymous,
I guess that you are using an Agilent Quaternary pump system. If you are mixing from different pots - as you are - then you can see a wavy baseline even with isocratic mixing. This is why;
Mixing mechanisms;
Agilent Quat Pump; On each intake pump stroke it opens each eluant valve in turn for a proportional time - post pump mixing is poor, increased stroke volume can help, but this will not solve the problem, hence wavy baseline. I have added an extra static mixer and still seen only marginal improvement.
Agilent Binary Pump; A "continuous" flow from two independent pumps is mixed - hence flatter baseline. Good baseline.
Waters 2690; Eluants are "blended" in a low pressure valve before being pumped. Really good baseline.
------------------------------------------------------------------------------------------------------------
By ves on Friday, August 1, 2003 - 06:02 am:
I have a waters pda which does this with some peaks that absorb near the cut-off of 190 nm. The profile of the spectrum looks like a damping curve with a periodic "bounce" as the wavelength increases. An accordian-like bounce. I am using acetonitrile, but this appears to be electronic. The peak-to-peak are much too systematic to be spectroscopic.
------------------------------------------------------------------------------------------------------------
By Anonymous on Monday, August 4, 2003 - 10:00 am:
Hello!
I知 looking for a low volume high pressure mixer for an Agilent 1100 binary pump (for fast gradient separations with a microbore column). Does anybody know if Agilent or another manufacturer provides that mixer?
Thank you
------------------------------------------------------------------------------------------------------------
By Anonymous on Monday, August 4, 2003 - 10:02 am:
I知 looking for a low volume high pressure mixer for an Agilent 1100 binary pump (for fast gradient separations with a microbore column). Does anybody know if Agilent or another manufacturer provides that mixer?
------------------------------------------------------------------------------------------------------------
By Pequito on Sunday, November 23, 2003 - 07:59 pm:
We have experienced 'wavy baselines' using Waters PDA detectors - especially at low wavelengths. We have attributed this to changes in humidity. We now have a humidity controlled environment for our HPLC's however cylclic changes in humidity of only 2% result in corresponding fluctuations in the baseline (we compared baseline fluctuations with changes in %RH collected using a data logger). Therefore an inherent problem with this type of detector.
------------------------------------------------------------------------------------------------------------
By Anonymous on Monday, April 19, 2004 - 11:14 am:
I experience a similar effect in the Waters UV detector and it turned out to be the pre-amp board.
I also have noisy baseline problem with Waters florencence detector at low Ex wavelength. I thought it may be due to dirty flow cell, but cleaning flow cell didn't help. Could somebody give some suggestions? Thanks
------------------------------------------------------------------------------------------------------------
By Chris Pohl on Wednesday, June 2, 2004 - 12:01 pm:
It might be outgassing in your cell. How much backpressure do you have on your cell?
------------------------------------------------------------------------------------------------------------
By Uwe Neue on Wednesday, June 2, 2004 - 03:47 pm:
What is the difference between the excitation and emission wavelengths?
------------------------------------------------------------------------------------------------------------
By Mary on Thursday, June 3, 2004 - 09:21 am:
Thanks, Chris and Uwe. My noisy baseline problem solved by cleaning flow cell again with HNO3, acetone and IPA and also by decreaing sampling rate.
------------------------------------------------------------------------------------------------------------
By mary on Monday, June 14, 2004 - 04:15 pm:
hi Uwe,
while looking for answers, i would like to answer yours. If you are exciting the samples at a certain wavelength you are bombarding it with photons at certain excitation wavelength say 475nm, creating excitations of the electrons. As the excited electrons return to steady/normal state they can be detected by emission wavelength. You can find the exact emission wavelength by doing a full scan. Remember an excitation wavelength is specific to the molecule.
------------------------------------------------------------------------------------------------------------
By Carlos Teixeira on Tuesday, June 15, 2004 - 01:57 pm:
Dear Friends,
The reagents purity is more important for the good profile of the baseline. But, I would use similar conditions of inorganic componets in the Acetonitrile, too. (Octane sulphonic acid, sodium saltat).
How is the profile of the baseline in a blank-analysis?
Good elutions,
------------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, April 2, 2003 - 05:36 am:
I am developing a method involving detection of degradation products and low wavelengths, 210nm and 215nm..
The conditions were as follows:
Mobile Phase A: Octane sulphonic acid, sodium salt(2.0g/L)+0.5 ml H3PO3 and adusted to pH 3.0
Mobile Phase B : Acetonitrile
Temperature: 40 deg C
Sample and stds prepared in Mobile A:Mobile B (82:18)
Columns used: Symmetry C18 and Symmetry shield C8
Gradient Conditions with Flow rate 1.0ml/min:
Time(min)%A
0 82
12 75
15 60
25 60
I am experiencing significantly large wavy baseline at low wavelength. The degradation compounds can hardly be differentiated from the wavy baseline at 210nm and 215nm. The baselines were fine at higher wavelengths eg 245nm but the absorbances of the degradation compounds were low at these higher wavelengths.
Two HPLCs using separate columns gave the same wavy baselines at low wavelength. The wavy baseline was monitored running at Mobile A:Mobile B (82:18) and flow rate of 1ml/min for more than 10 hours with not much baseline improvement. The same wavy baseline at low wavelength was observed when the sample was injected and run at the above gradient showing that this was not due to the sample.
Can someone advise on the possible source of the problem?
------------------------------------------------------------------------------------------------------------
By Wanda Kruse on Wednesday, April 2, 2003 - 02:50 pm:
When analyses are at these wavelengths, purity of reagents are key. Acetonitrile sources vary in their uv cutoff, and supplier literature is not always reliable. You need to run your own scan. Secondly, Acetonitrile preservatives start to degrade within a couple days after opening the bottle. These degradants give a nasty baseline. Make sure your Phosphoric is HPLC grade, and the salts are of premium LC grade.
I have also had problems with MP impurities being caught by the guard column, and elute as the organic increases. For one of our analyses, we have had to put a cleanup column between MP A pump and the mixer. good luck!
------------------------------------------------------------------------------------------------------------
By Benjamin on Wednesday, April 2, 2003 - 02:52 pm:
Dear Anonymous;
Your baseline problems can be due to several factors. The purity of salts and reagents is critical. Usually H3PO4 HPLC grade and Octanesulfonic acid puriss from Fluka are very good. I also suspect that you could improve results keeping the buffer and ion par reagents constant during your gradient. Right now your gradient changes phosphate, octanesulfonic acid, and ACN.
Another factor to keep in mind is to give enough time to the column to reequilibrate at the end of each gradient.
Good Luck;
Benjamin
------------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, April 2, 2003 - 06:20 pm:
Thanks Wanda and Benjamin.
The Symmetry C18 and Symmetry Shield RP8 columns were 4.6x250mm.
We tried the C8 Symmetry 3.9x150mm today and did not experience wavy baseline problem. The baseline was good.
Would anyone know why this is happening?
------------------------------------------------------------------------------------------------------------
By HW Mueller on Thursday, April 3, 2003 - 12:46 am:
What do you mean by "wavy baseline"? Noise or a succession of broad and/or narrow peaks coming out? If itｴs the latter it would mean your C-18 columns are just dirty. If your problem is noise.... maybe some strange coincidence?? I pass.
------------------------------------------------------------------------------------------------------------
By Wanda on Friday, April 4, 2003 - 07:54 am:
Sometimes you just gotta remake mobile phase even if you think you made it correctly. When I see a baseline I don't like, I remake the mobile phase, making sure the bottles are really rinsed well. sometimes there is residue in MP bottles which will impact your baseline. Secondly, I equilibrate for about 45 minutes about 50/50 to ensure the column is "wetted", then drop back to starting concentration. As far as the difference in the columns? There should be no difference in baselines.
------------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, July 9, 2003 - 03:31 pm:
The old HP1050 series A VWD detectors were prone to generating wavy baselines at low wavelengths when room temperature varied by +/- several degrees over time due to a poor HVAC system. The detectors were beneath a ceiling vent which blew out cool and then warm air periodically. Its caused by refractive index effects. Not sure if any of these factors could be affecting your system. The series C detectors and HP1100 detectors are much better in this respect.
------------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, July 10, 2003 - 12:51 am:
As Benjamin wrote, in case of ion pair gradient method it is very important to keep a constant oin pair/buffer concentration during the gradient. You should prepare mobile phase for B channel from ion pair reagent containing buffer and acetonitril The concentration of ion pair reagent have to be the same in A and B channel. The necessary equlibrium time of the system is much longer, than that without ion pair reagent. at least 15 min. equlibrium time (post time) is necessary between injections.
Anyway, the work with ion pair - gradient elution system is very difficult even at higher detection wavelength independently to the old or new HPLC.
Good luck.
Julia
------------------------------------------------------------------------------------------------------------
By Stuart Williams on Monday, July 21, 2003 - 06:53 am:
Dear Anonymous,
I guess that you are using an Agilent Quaternary pump system. If you are mixing from different pots - as you are - then you can see a wavy baseline even with isocratic mixing. This is why;
Mixing mechanisms;
Agilent Quat Pump; On each intake pump stroke it opens each eluant valve in turn for a proportional time - post pump mixing is poor, increased stroke volume can help, but this will not solve the problem, hence wavy baseline. I have added an extra static mixer and still seen only marginal improvement.
Agilent Binary Pump; A "continuous" flow from two independent pumps is mixed - hence flatter baseline. Good baseline.
Waters 2690; Eluants are "blended" in a low pressure valve before being pumped. Really good baseline.
------------------------------------------------------------------------------------------------------------
By ves on Friday, August 1, 2003 - 06:02 am:
I have a waters pda which does this with some peaks that absorb near the cut-off of 190 nm. The profile of the spectrum looks like a damping curve with a periodic "bounce" as the wavelength increases. An accordian-like bounce. I am using acetonitrile, but this appears to be electronic. The peak-to-peak are much too systematic to be spectroscopic.
------------------------------------------------------------------------------------------------------------
By Anonymous on Monday, August 4, 2003 - 10:00 am:
Hello!
I知 looking for a low volume high pressure mixer for an Agilent 1100 binary pump (for fast gradient separations with a microbore column). Does anybody know if Agilent or another manufacturer provides that mixer?
Thank you
------------------------------------------------------------------------------------------------------------
By Anonymous on Monday, August 4, 2003 - 10:02 am:
I知 looking for a low volume high pressure mixer for an Agilent 1100 binary pump (for fast gradient separations with a microbore column). Does anybody know if Agilent or another manufacturer provides that mixer?
------------------------------------------------------------------------------------------------------------
By Pequito on Sunday, November 23, 2003 - 07:59 pm:
We have experienced 'wavy baselines' using Waters PDA detectors - especially at low wavelengths. We have attributed this to changes in humidity. We now have a humidity controlled environment for our HPLC's however cylclic changes in humidity of only 2% result in corresponding fluctuations in the baseline (we compared baseline fluctuations with changes in %RH collected using a data logger). Therefore an inherent problem with this type of detector.
------------------------------------------------------------------------------------------------------------
By Anonymous on Monday, April 19, 2004 - 11:14 am:
I experience a similar effect in the Waters UV detector and it turned out to be the pre-amp board.
