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By carmen on Wednesday, June 9, 2004 - 05:36 am:
Hello friends,
I have a problem because I am analising benzoic acid on C-18, Phosfathe buffer, pH 6.6: Methanol, 90:10, but I always have 2 peaks at about 4 minutes. Which pH is the apropiate?
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By MG on Wednesday, June 9, 2004 - 06:34 am:
I would recommend analyzing benzoic acid at a more acidic pH. See also the recent post on the use of buffers with HPLC.
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By Anonymous on Wednesday, June 9, 2004 - 07:53 am:
Carmen: use your methanol-water but use phosphoric acid to keep the pH low to keep the benzoic acid protonated.
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By Anonymous on Wednesday, June 9, 2004 - 01:30 pm:
Inject less amount of your sample or increase your buffer capacity.
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By Uwe Neue on Wednesday, June 9, 2004 - 05:27 pm:
There should not be a problem, your conditions are fine. Your sample contains two compounds.
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By carmen on Thursday, June 10, 2004 - 12:17 am:
Finally, I have worked with pH= 6.2 and I have got only 1 peak. I have studied more about buffers and I have found is better to work with pH= PKa +/- 2. So, if pKa= 4.2, I could work with 6.2 or 2.2, but, 2.2 is two low for my column, C18, what do you think?
Thanks very much for your help.
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By Anonymous on Thursday, June 10, 2004 - 12:51 am:
Try looking at the RP-Amide type column (Agilent/Supelco) and using 0.1% TFA as buffer.
Peak shapes will be better and you can resolve sorbic acid and benzoic acid if you work with beverages etc.
HPLC Dave
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By MG on Thursday, June 10, 2004 - 06:23 am:
You'll get more retention at 2.2. Many C18's are rated down to pH 2.0.
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By RH on Thursday, June 10, 2004 - 10:06 am:
pH 2,2 should be no problem at all for a modern
C18 column. Below pH 2 you should start thinking
about a pH stable phase, also available on silica
base. Avoid very high temperature and high
phosphate buffer concentration at pH 2, this
method should be fine.
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 11, 2004 - 12:23 am:
If you want to analyse benzoic acid at low pH you can use phosphoric or sulfuric acid instead of buffer. And you don't have to go down to pH 2.2, around pH 2.5 is enough.
If with low pH two peaks are observed too, you can be sure you have two compounds (as Uwe noted earlier).
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By Anonymous on Friday, June 11, 2004 - 09:40 am:
I have had troubles analyzing benzoic acid before. My problem was because the sample solvent was 100% acetonitrile and the mobile phase was very high in water. This caused the peak to split, and be very broad. Diluting the sample with water stopped the peak splitting and efficiency problems. What are you using for you sample solvent/matrix?
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By Another Anonymous on Friday, June 11, 2004 - 06:34 pm:
To last Anonymous: another cure for you would've likely been keeping your aqueous injection volume very low.
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By carmen on Tuesday, June 15, 2004 - 10:05 am:
Hi anonymous, the solvent for sample is Methanol.
O
ther question: I have to acidity the mix Methanol-buffer or only the buffer to pH=6.2, and then mix with methanol to 10:90 methanol:buffer?
Last week I acid the mix and the results were OK., but today, I acid only the buffer with phosphoric to PH=6.2 and then I have mixed with methanol and the results were not very good, two peaks again.
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By cgg on Wednesday, June 16, 2004 - 01:39 am:
Hello Carmen,
PreferirÃa contartelo en Español. Si eres española, escrÃbeme: cegrunder@yahoo.es.
I´ve analised benzoic an benzaldehide at pH 3 in phosfate and acetonitrile.
Alwais you must acidific the buffer. It´s the only way to control pH conditions, because electrodes of pH can not be calibrated in other medium apart from water.The lectures of pH would increase error .
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By Anonymous on Wednesday, June 16, 2004 - 04:41 pm:
Benzoic acid and sorbic acid (actually benzoate and sorbate) may be easily separated on any number of RP columns because the salts are actually sufficiently hydrophobic to be retained with a just a small quantity of organic in the mobile phase. I do it frequently with an inertsil ods3 with an ammonium acetate buffer at approx pH 5.8 and ~10% MeCN. Running at a lower pH with a phosphate buffer (and higher organic content) works fine for either one as well, but not both unless you're running a phenyl or amide type column.
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By Anonymous on Friday, June 18, 2004 - 07:48 pm:
Carmen: your problem is caused by the fact that you dissolve the sample in methanol. dissolve the sample in water or mobile phase, and your problem with having two peaks will go away. (they are both benzoic acid, one is carried through the column with the excess methanol)
-------------------------------------------------------------------------------------------------------
By juddc on Friday, June 18, 2004 - 09:19 pm:
Anon2 is correct...alternately, you may be able to get away with a lower injection volume depending on your system volume and column geometry.
Hello friends,
I have a problem because I am analising benzoic acid on C-18, Phosfathe buffer, pH 6.6: Methanol, 90:10, but I always have 2 peaks at about 4 minutes. Which pH is the apropiate?
-------------------------------------------------------------------------------------------------------
By MG on Wednesday, June 9, 2004 - 06:34 am:
I would recommend analyzing benzoic acid at a more acidic pH. See also the recent post on the use of buffers with HPLC.
-------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, June 9, 2004 - 07:53 am:
Carmen: use your methanol-water but use phosphoric acid to keep the pH low to keep the benzoic acid protonated.
-------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, June 9, 2004 - 01:30 pm:
Inject less amount of your sample or increase your buffer capacity.
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Wednesday, June 9, 2004 - 05:27 pm:
There should not be a problem, your conditions are fine. Your sample contains two compounds.
-------------------------------------------------------------------------------------------------------
By carmen on Thursday, June 10, 2004 - 12:17 am:
Finally, I have worked with pH= 6.2 and I have got only 1 peak. I have studied more about buffers and I have found is better to work with pH= PKa +/- 2. So, if pKa= 4.2, I could work with 6.2 or 2.2, but, 2.2 is two low for my column, C18, what do you think?
Thanks very much for your help.
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, June 10, 2004 - 12:51 am:
Try looking at the RP-Amide type column (Agilent/Supelco) and using 0.1% TFA as buffer.
Peak shapes will be better and you can resolve sorbic acid and benzoic acid if you work with beverages etc.
HPLC Dave
-------------------------------------------------------------------------------------------------------
By MG on Thursday, June 10, 2004 - 06:23 am:
You'll get more retention at 2.2. Many C18's are rated down to pH 2.0.
-------------------------------------------------------------------------------------------------------
By RH on Thursday, June 10, 2004 - 10:06 am:
pH 2,2 should be no problem at all for a modern
C18 column. Below pH 2 you should start thinking
about a pH stable phase, also available on silica
base. Avoid very high temperature and high
phosphate buffer concentration at pH 2, this
method should be fine.
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 11, 2004 - 12:23 am:
If you want to analyse benzoic acid at low pH you can use phosphoric or sulfuric acid instead of buffer. And you don't have to go down to pH 2.2, around pH 2.5 is enough.
If with low pH two peaks are observed too, you can be sure you have two compounds (as Uwe noted earlier).
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 11, 2004 - 09:40 am:
I have had troubles analyzing benzoic acid before. My problem was because the sample solvent was 100% acetonitrile and the mobile phase was very high in water. This caused the peak to split, and be very broad. Diluting the sample with water stopped the peak splitting and efficiency problems. What are you using for you sample solvent/matrix?
-------------------------------------------------------------------------------------------------------
By Another Anonymous on Friday, June 11, 2004 - 06:34 pm:
To last Anonymous: another cure for you would've likely been keeping your aqueous injection volume very low.
-------------------------------------------------------------------------------------------------------
By carmen on Tuesday, June 15, 2004 - 10:05 am:
Hi anonymous, the solvent for sample is Methanol.
O
ther question: I have to acidity the mix Methanol-buffer or only the buffer to pH=6.2, and then mix with methanol to 10:90 methanol:buffer?
Last week I acid the mix and the results were OK., but today, I acid only the buffer with phosphoric to PH=6.2 and then I have mixed with methanol and the results were not very good, two peaks again.
-------------------------------------------------------------------------------------------------------
By cgg on Wednesday, June 16, 2004 - 01:39 am:
Hello Carmen,
PreferirÃa contartelo en Español. Si eres española, escrÃbeme: cegrunder@yahoo.es.
I´ve analised benzoic an benzaldehide at pH 3 in phosfate and acetonitrile.
Alwais you must acidific the buffer. It´s the only way to control pH conditions, because electrodes of pH can not be calibrated in other medium apart from water.The lectures of pH would increase error .
-------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, June 16, 2004 - 04:41 pm:
Benzoic acid and sorbic acid (actually benzoate and sorbate) may be easily separated on any number of RP columns because the salts are actually sufficiently hydrophobic to be retained with a just a small quantity of organic in the mobile phase. I do it frequently with an inertsil ods3 with an ammonium acetate buffer at approx pH 5.8 and ~10% MeCN. Running at a lower pH with a phosphate buffer (and higher organic content) works fine for either one as well, but not both unless you're running a phenyl or amide type column.
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 18, 2004 - 07:48 pm:
Carmen: your problem is caused by the fact that you dissolve the sample in methanol. dissolve the sample in water or mobile phase, and your problem with having two peaks will go away. (they are both benzoic acid, one is carried through the column with the excess methanol)
-------------------------------------------------------------------------------------------------------
By juddc on Friday, June 18, 2004 - 09:19 pm:
Anon2 is correct...alternately, you may be able to get away with a lower injection volume depending on your system volume and column geometry.
