You can try a new method recently published . It does not use any counter ion and it makes the method very easy to handle.In the original paper the DHA is not described but we know it is eluted around 90 sec later than AA.Unlike methods described in different catalogs only for standards, it works very well in complex matrices.Another advantage is that this method is MS compatible.
Ann Biol Clin 2004,62:305-9.
By ravi talluri on Monday, May 10, 2004 - 09:11 pm:
I want to detect both the ascorbic acid and its oxidised product dehydroascorbic acid simultaneously by using hplc.can anyone suggest me regarding the mobile phase composition that i should use to detect them?
By HW Mueller on Tuesday, May 11, 2004 - 02:28 am:
DHA is an ill defined, unstable material. Nearly believable analyses of Asc and DHA involve the HPLC of Asc, than reduce a second identical sample and measure Asc again. The difference is a rough value for DHA. Derivatization of DHA is an option with which I have, as an example, not been satisfied. But I may see the matter as too "black" since human plasma/serum was usually involved. The work with Asc led me to believe (practically proven) that there is no DHA in plasma, and that claims to the contrary are really due to analytical artefacts.
(Asc determination in tomatoes via HPLC was a "breeze" in comparison, if you can´t find any of the million examples in the lit, send me an e-mail and I will dig into my lab journals...)
By ravi talluri on Tuesday, May 11, 2004 - 10:21 am:
Thank you mueller.
Actually i want to check whether the ascorbic acid is getting oxidised when put into solution (0.1mM DTT was added).
I am not clear regarding the mobile phase composition i have to use its because ascorbic acid is very hydrophilic so i speculate that it will have very less retention time.can i do any ion pairing agent to increase its reetntion time ?
By Patrik Appelblad on Tuesday, May 11, 2004 - 01:59 pm:
Ascorbic acid and DHA is good candidates for HILIC mode separation. Some time ago I produced an example of ascorbic acid separation on our ZIC-HILIC phase, and if you send me an email I can provide you with the data.
By HW Mueller on Wednesday, May 12, 2004 - 12:47 am:
OK, found my chroms on the tomatoes, it was one of these Aqu C-18 columns with phosphate buffer, 0.1 M at pH 2, no organics. With your, apparently, highly pure solution your only problem should be one of deciding among all the examples given.
The oxidation behavior of Asc has been studied extensively, in short: It oxidices very slowly at pH 4 and below, rapidly at pH7 and above (in H20). The oxidation needs some metal ions for catalysis but they are practically always present. Almost all common reducing agents will prevent oxidation. You can "observe" the whole processes beautifully in an ESR spectrometer (Asc radical).
By ravi talluri on Wednesday, May 12, 2004 - 10:25 pm:
once again thanks for your response and suggestions.
i too use C-18 columns.But ascorbic acid is a highly hydrophilic molecule so can i use my mobile phase with out any organics (i.e. only buffer)? Because in some papers i saw people using good amount of acetonitrile or some other organic solvent in their mobile phase.
By Uwe Neue on Thursday, May 13, 2004 - 04:17 pm:
you find many application examples at the following website:
By Zelechonok on Thursday, May 13, 2004 - 08:09 pm:
Ravi, most methods in literature require either ion-pairing reagent or give you very little retention with RP conditions. It is hard to study degradation products when you parent peak is not retentative. Much better result can be obtained with mixed-mode columns. Check the method developed for ascorbic acid. With simple mobile phase H2O/MeCN/AcOH significant retention was obtained(K=6).
By maris on Thursday, May 13, 2004 - 10:00 pm:
It's a long time you trying to market your production in those forum discussions. Surprisingly, when i tried to purchase your columns, it took more then three months to get a reply and finally one column of wrong geometry was supplied ( instead two I planned to purchase).
Are you serious?
Analytical R&D manager
By AllsepTech on Friday, May 14, 2004 - 04:41 am:
We want to apologize for any inconvenience with the ordering of our columns. As a person responsible for all customer service I want to inform you that we never received the order from Chemagis. In the beginning of December we provided you with the name of our distributor in your region. All columns (except free samples) are shipped according to the purchase order. We usually ship columns next day after receiving the order. Please contact us if you need assistance. (email@example.com)
By maris on Friday, May 14, 2004 - 09:55 am:
Probably it's a distributor's fault.The result is the same.
By ravi talluri on Friday, May 14, 2004 - 02:30 pm:
Thanks for your suggestion.
i will try with that mobile phase composition
By ravi talluri on Sunday, May 16, 2004 - 09:05 pm:
do you have any idea which ion pairing agent can i use for detecting the ascorbic acid.
By Zelechonok on Monday, May 17, 2004 - 10:25 am:
If you use Primesep B column you donâ€™t need any ion-pairing reagent. Mobile phase with MeCN/H2O/AcOH â€“ 10/90/0.25 it is all you need
By Uwe Neue on Monday, May 17, 2004 - 05:31 pm:
No ion-pairing reagent is needed for this compound. It is retained on a C18 column without difficulty. Look at this site:
By HW Mueller on Monday, May 17, 2004 - 11:31 pm:
how about using a C-18 (conventional, Aqua, polar....) and comparing it to that Allsep or Sielc, or whatever, which Zelechonok suggested, and tell us about the results, especially on tailing.
By Zelechonok on Tuesday, May 18, 2004 - 02:57 pm:
just compare two methods on Primesep B and Atlantis that Uwe mentioned.
Primesep B column:
mobile phase MeCN/H2O/AcOH – 10/90/0.25
k prime = 6
tailing factor 1.05.
mobile phase MeCN/H2O/TFA – 0/100/0.1
k prime about 1
tailing factor 1.12.
By HW Mueller on Wednesday, May 19, 2004 - 12:56 am:
What are the column´s sizes, age, stat. phase data, flow, temp., amt. injected; sample solvent, concentration, pH? What´s the pH of the mobile phases? How was the "tailing factor" determined? Everything was done with the same apparatus (connections!?), including integration and settings? (Incidentally, I am troubled by the use of TFA here, even if Waters suggested it)
to really compare the two columns you should, of course, at least use a buffer (the same type for both) then optimize the mobile phases for both columns.
By Zelechonok on Wednesday, May 19, 2004 - 04:09 am:
Dear HW Mueller
I don’t know all details of Waters application, I just assume they do a good job to optimize their method (maybe I am wrong…). I use for comparison data that available on their application note that Uwe provide us with.
We are using Agilent Chemstation software for tailing factor calculation. If you need more details on our method I can provide you with that. My e-mail is available in the title of this message. I have no time and intention to optimize Atlantis method.
By HW Mueller on Wednesday, May 19, 2004 - 07:19 am:
Well then, there is actually no basis for comparison of these data.
By Anonymous on Wednesday, May 19, 2004 - 10:17 am:
I think that if you assume that both manufacturers used the best, well optimized method you have enough data to make a comparison. Now it is up to scientist to decide what to do.
By MG on Thursday, May 20, 2004 - 01:43 pm:
Hypothetically, if I wanted to compare a HILIC Silica column versus an "AQ" type C18 for their abilities to retain uracil, the mobile phases would invariably be quite different. Yet I think it would be a useful comparison, to answer the question: Which column, when well optimized, does the better job with this compound?