-
- admin
- Site Admin
-
- Posts: 647
- Joined: Wed Aug 11, 2004 12:19 am
By lily Tan on Monday, August 16, 2004 - 09:56 pm:
I am a beginner in HPLC. Trying to establish the method set for scopolamine. I am running using a brand new HPLC system with photodiode detector, isocratic pump, mobile phase is 65% Methanol, 35% MilliQ water;column C18. Tried running a serial dilution and blank. Notice a constant deep at around 2.5 min. for all concentrations -2ug/ml, 20ug/ml, 50ug/ml, 100ug/ml,200ug/ml(it will interfere my analysis). At first, we thought it was due to the system but the svc enginneer tried running a blank using his own column. No deep was seen in his results. We then change a brand new column. Still get the same problem. All the solvents I am using are HPLC grade. My HPLC is Waters , autosampler.
What I have to do to eliminate?
-------------------------------------------------------------------------------------------------------
By JM on Tuesday, August 17, 2004 - 01:45 am:
what is your sample diution media?? and the detector wevelength??
-------------------------------------------------------------------------------------------------------
By Ann on Tuesday, August 17, 2004 - 02:03 am:
By 'deep', do you mean a negative peak? Could this simply be the solvent front i.e. the change in absorbance caused by the unretained compounds in your sample matrix eluting from the column? If so, you would see this when injecting blank solutions and quite possibly, this may look different on another column.
What is the retention time of your analyte? If your analyte is eluting at the same time as this negative peak, it is probably easiest to slightly alter the composition of your mobile phase (reduce the amount of methanol slightly so that your analyte of interest elutes later, hopefully clear of the negative peak). You may also get better results (better retention of your analyte) if you controlled the pH of the mobile phase (try a buffer instead of water).
Here are a couple of references for scopolamine LC methods using C18 columns - abstracts containing the chromatographic conditions are available on PubMed http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed.
Ceyhan T et al. LC determination of atropine sulfate and scopolamine hydrobromide in pharmaceuticals.J Pharm Biomed Anal. 2001 Jun;25(3-4):399-406.
Pennington LJ, Schmidt WF Belladonna alkaloids and phenobarbital combination pharmaceuticals analysis I: High-performance liquid chromatographic determinations of hyoscyamine-atropine and scopolamine.J Pharm Sci. 1982 Aug;71(8):951-3
Good luck!
Ann
)
-------------------------------------------------------------------------------------------------------
By JA on Tuesday, August 17, 2004 - 12:11 pm:
From Ann's first reference, "Atropine sulfate and scopolamine hydrobromide were separated using a Bondapack C18 column by isocratic elution with flow rate 1.0 ml/min. The mobile phase composition was methanol, water, formic acid (65:35:1; v/v/v) and pH adjusted 8.3 with triethylamine."
What is the purpose of using both this weak acid and weak base in the MP? Is triethylamine-formate a buffer in it's own right?
I've seen some chiral normal-phase methods using for example both TFA and TEA and wondered the same.
-------------------------------------------------------------------------------------------------------
By lily Tan on Tuesday, August 17, 2004 - 06:24 pm:
Hi JM,
My sample dilution is Milli-Q water. The detector wavelength is 230 nm.
-------------------------------------------------------------------------------------------------------
By lily Tan on Tuesday, August 17, 2004 - 06:58 pm:
Hi Ann,
Thanks for all your suggestions. Not very sure if the peak that I am referring to is the same as what you mentioned cos' I don't have any idea how a negative peak looks like. Do you know which website will give more illustrations (preferably with chromatogram enclosed).
My RT for my analyte is around 2.7 or 2.8 min. My analyte is eluting almost the same time as this negative peak.
Thanks for the reference. I will tried out the method using the mobile phase composition recommended i.e.methanol:water:formic acid (165:35:1: v/v/v) adjusted to pH 8.3 by using triethylamine then filtered through 0.45 m nylon membrane filter and degassed in sonicator for 10 min the methanol, water, formic acid.
I don't have any triethylamine in the lab. Is NaOH good enough?
I have got some good basic knowledge from LCresources "getting started with HPLC". Any ideas from anyone if what other materials I can read up so that it can help me to build up a better foundation in this area.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Tuesday, August 17, 2004 - 07:27 pm:
I don't know... I would not use method with a pH of 8.3 on a classical silica column even if it has been published.
-------------------------------------------------------------------------------------------------------
By JM on Wednesday, August 18, 2004 - 12:46 am:
Hi Ann, The negative peak is of water as the sample media is different from the mobile phase. You can verify by injecting your dilution solvent ( Milli Q). To minimise these solvent peaks , use the Mobile phase as your final diluting solvent.
JM
-------------------------------------------------------------------------------------------------------
By JM on Wednesday, August 18, 2004 - 12:48 am:
oooops.... sorry!! it was for Lily,
JM
-------------------------------------------------------------------------------------------------------
By lily Tan on Wednesday, August 18, 2004 - 01:08 am:
Managed to get triethylamine. Tried out using the mobile phase composition recommended in the reference i.e.methanol:water:formic acid (165:35:1: v/v/v) adjusted to pH 8.3 by using triethylamine then filtered through 0.45 m nylon membrane filter. Flowrate: 1 ml. Running at isocratic condition. Currently using Symmetryshield RP18 4.6 x 250 column.
Still gotting the negative dip at RT 2.5, in fact much negative than earlier.
Really at a loss of what to do. Any suggestions from anyone?
-------------------------------------------------------------------------------------------------------
By HW Mueller on Wednesday, August 18, 2004 - 06:09 am:
Reduce your MeOH or change the pH to move the analytes further back and just enjoy the negative peak. I am sure Ann gave you a ref where this was worked out.
-------------------------------------------------------------------------------------------------------
By Ann on Wednesday, August 18, 2004 - 08:07 am:
Hi Lily
I completely agree with HW Mueller, if the negative peak is not interfering with the peak produced by your analyte of interest, then it is not a problem and you can ignore it. What is the retention time of your peak using the published method?
All the best, Ann
-------------------------------------------------------------------------------------------------------
By lily Tan on Wednesday, August 18, 2004 - 06:11 pm:
I happen to read one of the question posted by April on Wednesday, August 18, 2004 - 'unsure with some terms..." today. The peak that I got is exactly same as what was showed in the first example given by Tom under the definition of "solvent front".
Sorry to confuse all of you cos' all this while I was thinking I was having negative peak.
Ann and HW Mueller, in that case, do I still reduce MeOH or change the pH to correct this problem?
According to my colleague, the RT of my peak using the published method should be around 2.8 min. The solvent front always appears around 2.5 min. to 2.7 min.
Also tried out the suggestion given by JM using the published method but didn't work.
Nevertheless, really appreciate all of you for taking time to help me to troubleshoot.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Wednesday, August 18, 2004 - 11:39 pm:
Yes, move your analyte peaks away from the front, you can have all kinds of even changing interferences there.
-------------------------------------------------------------------------------------------------------
By lily Tan on Wednesday, August 18, 2004 - 11:58 pm:
Thanks, HW Mueller. Currently, I am using this mobile composition - methanol:water:formic acid (165:35:1: v/v/v). 100% isocratic. flowrate 1ml. Am I correct to say that when I reduce MeOH, I will increase my water vol accordingly so that I can maintain a final volume of 201ml.
How do you decide to make it more acidic or alkaline? Is there some principle or guide to consult?
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, August 19, 2004 - 03:25 pm:
lily Tan,
It is absolutely not recommended to run the SymmetryShield column at pH 8.3. You are in the process of killing the column, if you have not already done so. The Care&Use manual recommends to stay in the pH range from 2 to 8. Of course, it does not go dead immediately at pH 8.3, nor will it last forever at pH 8.0, but pH 8.3 will reduce the column lifetime.
A negative peak at the solvent front is nothing to worry about. It happens all the time. It may very well be related to how you dissolve your sample, but if there is no interference with your analysis, ther is nothing that you need to do.
If you want to stay with the method, adjust the pH to 7.0 instead of pH 8.3.
-------------------------------------------------------------------------------------------------------
By lily Tan on Thursday, August 19, 2004 - 10:17 pm:
Thanks, Uwe Neue. I have loaned a column from Waters that can run pH2 to 10. Will try it out today.
By the way, where I can get this "care and use manual". Is it available online?
-------------------------------------------------------------------------------------------------------
By HW Mueller on Friday, August 20, 2004 - 12:22 am:
Lily, adding those volumes of solvents you mention will most likely not give 201mL, still it´s ok to do it as you suggested, just be consistent (Most people probably do this as % vol before addition: 65mL MeOH + 34mL H2O + 1mL formic would be 65%/34%/1% v/v/v, respectively). .
On varying pH, this has been discussed extensively. In short, if you know your pKa/pKb you may start with a pH ~2 units away on the unionized side, if the retention is to high you change the pH to give more ionization...
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Friday, August 20, 2004 - 02:56 pm:
The Care and Use Manual comes in the column box with the column. Most people throw it away, when they take the column out of the box...
-------------------------------------------------------------------------------------------------------
By Lily Tan on Tuesday, August 24, 2004 - 02:29 am:
Just some updates: I am able to get my analyte peak after using another published method. The mobile phase that I am using now consists of : 20.9 ACN, 27.9 MeOH, 51.2 0.05M ammonium acetate.
Although I am still getting the solvent front at 2.5 min, the RT for my analyte is much later - 4 min. As a result, it did not interfere my component.
Thank you for your help from all of you. As a beginner, your invaluable suggestions are certainly very valuable and useful to me.
-------------------------------------------------------------------------------------------------------
By Ann on Tuesday, August 24, 2004 - 04:04 am:
Glad you got it sorted Lily, good luck with your method validation.
Ann
-------------------------------------------------------------------------------------------------------
By JA on Tuesday, August 24, 2004 - 01:48 pm:
I wondered if this was all down to a literature typo for the composition 165:35:1 MeOH/H2O/Formic acid? I know it's all sorted now but I would never have used such a bizarre ratio
For example, I highly doubt it'd work OK at 82.5/17.5/0.5 and not at 82/18/0.5.. it's a simple two-analyte mixture, which is why I posted previously 65:35:1.
On the column issue, Luna is pretty common and Phenomenex reckon it's stable from pH 1.5-10 (phosphate) for over 10,000 hours and demonstrate it for at least 2,000 hours (3 months continual flow). If you had this one maybe you could run your method and if it "died" before such time tell them you want a replacement
-------------------------------------------------------------------------------------------------------
By lily Tan on Tuesday, August 24, 2004 - 11:21 pm:
Thanks, Ann & JA.
lily
-------------------------------------------------------------------------------------------------------
By Ann on Wednesday, August 25, 2004 - 01:40 am:
You're welcome Lily)
BTW, I agree with JA that a Luna C18 might be worth trying, I've used them for several assays and found them to be very robust.
Ann
-------------------------------------------------------------------------------------------------------
By A.N. Onimis on Wednesday, August 25, 2004 - 07:44 pm:
JA & Ann, don't believe everything you read when you see a manufacturer state that the pH range of a silica based RP column is up to 10. Think about it.
I am a beginner in HPLC. Trying to establish the method set for scopolamine. I am running using a brand new HPLC system with photodiode detector, isocratic pump, mobile phase is 65% Methanol, 35% MilliQ water;column C18. Tried running a serial dilution and blank. Notice a constant deep at around 2.5 min. for all concentrations -2ug/ml, 20ug/ml, 50ug/ml, 100ug/ml,200ug/ml(it will interfere my analysis). At first, we thought it was due to the system but the svc enginneer tried running a blank using his own column. No deep was seen in his results. We then change a brand new column. Still get the same problem. All the solvents I am using are HPLC grade. My HPLC is Waters , autosampler.
What I have to do to eliminate?
-------------------------------------------------------------------------------------------------------
By JM on Tuesday, August 17, 2004 - 01:45 am:
what is your sample diution media?? and the detector wevelength??
-------------------------------------------------------------------------------------------------------
By Ann on Tuesday, August 17, 2004 - 02:03 am:
By 'deep', do you mean a negative peak? Could this simply be the solvent front i.e. the change in absorbance caused by the unretained compounds in your sample matrix eluting from the column? If so, you would see this when injecting blank solutions and quite possibly, this may look different on another column.
What is the retention time of your analyte? If your analyte is eluting at the same time as this negative peak, it is probably easiest to slightly alter the composition of your mobile phase (reduce the amount of methanol slightly so that your analyte of interest elutes later, hopefully clear of the negative peak). You may also get better results (better retention of your analyte) if you controlled the pH of the mobile phase (try a buffer instead of water).
Here are a couple of references for scopolamine LC methods using C18 columns - abstracts containing the chromatographic conditions are available on PubMed http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed.
Ceyhan T et al. LC determination of atropine sulfate and scopolamine hydrobromide in pharmaceuticals.J Pharm Biomed Anal. 2001 Jun;25(3-4):399-406.
Pennington LJ, Schmidt WF Belladonna alkaloids and phenobarbital combination pharmaceuticals analysis I: High-performance liquid chromatographic determinations of hyoscyamine-atropine and scopolamine.J Pharm Sci. 1982 Aug;71(8):951-3
Good luck!
Ann
)-------------------------------------------------------------------------------------------------------
By JA on Tuesday, August 17, 2004 - 12:11 pm:
From Ann's first reference, "Atropine sulfate and scopolamine hydrobromide were separated using a Bondapack C18 column by isocratic elution with flow rate 1.0 ml/min. The mobile phase composition was methanol, water, formic acid (65:35:1; v/v/v) and pH adjusted 8.3 with triethylamine."
What is the purpose of using both this weak acid and weak base in the MP? Is triethylamine-formate a buffer in it's own right?
I've seen some chiral normal-phase methods using for example both TFA and TEA and wondered the same.
-------------------------------------------------------------------------------------------------------
By lily Tan on Tuesday, August 17, 2004 - 06:24 pm:
Hi JM,
My sample dilution is Milli-Q water. The detector wavelength is 230 nm.
-------------------------------------------------------------------------------------------------------
By lily Tan on Tuesday, August 17, 2004 - 06:58 pm:
Hi Ann,
Thanks for all your suggestions. Not very sure if the peak that I am referring to is the same as what you mentioned cos' I don't have any idea how a negative peak looks like. Do you know which website will give more illustrations (preferably with chromatogram enclosed).
My RT for my analyte is around 2.7 or 2.8 min. My analyte is eluting almost the same time as this negative peak.
Thanks for the reference. I will tried out the method using the mobile phase composition recommended i.e.methanol:water:formic acid (165:35:1: v/v/v) adjusted to pH 8.3 by using triethylamine then filtered through 0.45 m nylon membrane filter and degassed in sonicator for 10 min the methanol, water, formic acid.
I don't have any triethylamine in the lab. Is NaOH good enough?
I have got some good basic knowledge from LCresources "getting started with HPLC". Any ideas from anyone if what other materials I can read up so that it can help me to build up a better foundation in this area.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Tuesday, August 17, 2004 - 07:27 pm:
I don't know... I would not use method with a pH of 8.3 on a classical silica column even if it has been published.
-------------------------------------------------------------------------------------------------------
By JM on Wednesday, August 18, 2004 - 12:46 am:
Hi Ann, The negative peak is of water as the sample media is different from the mobile phase. You can verify by injecting your dilution solvent ( Milli Q). To minimise these solvent peaks , use the Mobile phase as your final diluting solvent.
JM
-------------------------------------------------------------------------------------------------------
By JM on Wednesday, August 18, 2004 - 12:48 am:
oooops.... sorry!! it was for Lily,
JM
-------------------------------------------------------------------------------------------------------
By lily Tan on Wednesday, August 18, 2004 - 01:08 am:
Managed to get triethylamine. Tried out using the mobile phase composition recommended in the reference i.e.methanol:water:formic acid (165:35:1: v/v/v) adjusted to pH 8.3 by using triethylamine then filtered through 0.45 m nylon membrane filter. Flowrate: 1 ml. Running at isocratic condition. Currently using Symmetryshield RP18 4.6 x 250 column.
Still gotting the negative dip at RT 2.5, in fact much negative than earlier.
Really at a loss of what to do. Any suggestions from anyone?
-------------------------------------------------------------------------------------------------------
By HW Mueller on Wednesday, August 18, 2004 - 06:09 am:
Reduce your MeOH or change the pH to move the analytes further back and just enjoy the negative peak. I am sure Ann gave you a ref where this was worked out.
-------------------------------------------------------------------------------------------------------
By Ann on Wednesday, August 18, 2004 - 08:07 am:
Hi Lily
I completely agree with HW Mueller, if the negative peak is not interfering with the peak produced by your analyte of interest, then it is not a problem and you can ignore it. What is the retention time of your peak using the published method?
All the best, Ann
-------------------------------------------------------------------------------------------------------
By lily Tan on Wednesday, August 18, 2004 - 06:11 pm:
I happen to read one of the question posted by April on Wednesday, August 18, 2004 - 'unsure with some terms..." today. The peak that I got is exactly same as what was showed in the first example given by Tom under the definition of "solvent front".
Sorry to confuse all of you cos' all this while I was thinking I was having negative peak.
Ann and HW Mueller, in that case, do I still reduce MeOH or change the pH to correct this problem?
According to my colleague, the RT of my peak using the published method should be around 2.8 min. The solvent front always appears around 2.5 min. to 2.7 min.
Also tried out the suggestion given by JM using the published method but didn't work.
Nevertheless, really appreciate all of you for taking time to help me to troubleshoot.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Wednesday, August 18, 2004 - 11:39 pm:
Yes, move your analyte peaks away from the front, you can have all kinds of even changing interferences there.
-------------------------------------------------------------------------------------------------------
By lily Tan on Wednesday, August 18, 2004 - 11:58 pm:
Thanks, HW Mueller. Currently, I am using this mobile composition - methanol:water:formic acid (165:35:1: v/v/v). 100% isocratic. flowrate 1ml. Am I correct to say that when I reduce MeOH, I will increase my water vol accordingly so that I can maintain a final volume of 201ml.
How do you decide to make it more acidic or alkaline? Is there some principle or guide to consult?
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, August 19, 2004 - 03:25 pm:
lily Tan,
It is absolutely not recommended to run the SymmetryShield column at pH 8.3. You are in the process of killing the column, if you have not already done so. The Care&Use manual recommends to stay in the pH range from 2 to 8. Of course, it does not go dead immediately at pH 8.3, nor will it last forever at pH 8.0, but pH 8.3 will reduce the column lifetime.
A negative peak at the solvent front is nothing to worry about. It happens all the time. It may very well be related to how you dissolve your sample, but if there is no interference with your analysis, ther is nothing that you need to do.
If you want to stay with the method, adjust the pH to 7.0 instead of pH 8.3.
-------------------------------------------------------------------------------------------------------
By lily Tan on Thursday, August 19, 2004 - 10:17 pm:
Thanks, Uwe Neue. I have loaned a column from Waters that can run pH2 to 10. Will try it out today.
By the way, where I can get this "care and use manual". Is it available online?
-------------------------------------------------------------------------------------------------------
By HW Mueller on Friday, August 20, 2004 - 12:22 am:
Lily, adding those volumes of solvents you mention will most likely not give 201mL, still it´s ok to do it as you suggested, just be consistent (Most people probably do this as % vol before addition: 65mL MeOH + 34mL H2O + 1mL formic would be 65%/34%/1% v/v/v, respectively). .
On varying pH, this has been discussed extensively. In short, if you know your pKa/pKb you may start with a pH ~2 units away on the unionized side, if the retention is to high you change the pH to give more ionization...
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Friday, August 20, 2004 - 02:56 pm:
The Care and Use Manual comes in the column box with the column. Most people throw it away, when they take the column out of the box...
-------------------------------------------------------------------------------------------------------
By Lily Tan on Tuesday, August 24, 2004 - 02:29 am:
Just some updates: I am able to get my analyte peak after using another published method. The mobile phase that I am using now consists of : 20.9 ACN, 27.9 MeOH, 51.2 0.05M ammonium acetate.
Although I am still getting the solvent front at 2.5 min, the RT for my analyte is much later - 4 min. As a result, it did not interfere my component.
Thank you for your help from all of you. As a beginner, your invaluable suggestions are certainly very valuable and useful to me.
-------------------------------------------------------------------------------------------------------
By Ann on Tuesday, August 24, 2004 - 04:04 am:
Glad you got it sorted Lily, good luck with your method validation.
Ann
-------------------------------------------------------------------------------------------------------
By JA on Tuesday, August 24, 2004 - 01:48 pm:
I wondered if this was all down to a literature typo for the composition 165:35:1 MeOH/H2O/Formic acid? I know it's all sorted now but I would never have used such a bizarre ratio
For example, I highly doubt it'd work OK at 82.5/17.5/0.5 and not at 82/18/0.5.. it's a simple two-analyte mixture, which is why I posted previously 65:35:1.
On the column issue, Luna is pretty common and Phenomenex reckon it's stable from pH 1.5-10 (phosphate) for over 10,000 hours and demonstrate it for at least 2,000 hours (3 months continual flow). If you had this one maybe you could run your method and if it "died" before such time tell them you want a replacement
-------------------------------------------------------------------------------------------------------
By lily Tan on Tuesday, August 24, 2004 - 11:21 pm:
Thanks, Ann & JA.
lily-------------------------------------------------------------------------------------------------------
By Ann on Wednesday, August 25, 2004 - 01:40 am:
You're welcome Lily
)BTW, I agree with JA that a Luna C18 might be worth trying, I've used them for several assays and found them to be very robust.
Ann
-------------------------------------------------------------------------------------------------------
By A.N. Onimis on Wednesday, August 25, 2004 - 07:44 pm:
JA & Ann, don't believe everything you read when you see a manufacturer state that the pH range of a silica based RP column is up to 10. Think about it.
