02-14-04 21:48
No 488653
High Bees!
Swin got a 2nd hand analytical HPLC into her hands. The apparatus was set up (Pump -> Rhedyne 6way-valve -> spherisorb column -> UV/VIS detector) and connected to a PC via an serial Analog/Digital-Converter...
Distilled water is used as solvent for the first test runs, but will be replaced by MeOH or MeOH/H2O during the next weeks...
So i started the first trial with 1,3ml/min flow rate - a pressure of 2500psi build up, and some extremely diluted ascorbic acid solution was injected. - The result was a very nice peak at a retention time of about 5 minutes. (detection @ 270nm).
The second trial was made with a Ascorbit acid/acetyl salicylic acid mixture. - The ASA-Peak came 2 minutes after the vitamine c peak.
Swim tried also to inject solutions of
- 2C-B.HCl
- TCP.HCl
- PCP.HCl
- PCM.HCl
, but none of these substances caused a peak at 270nm.
Now my questions - are our favorite amine´s hydrochlorides not detectable with UV, or do i use the false wavelenght?
What column would be the best for our purposes?
Does anybee have some general tips regarding HPLC-Analysis? SWIm is primary an organic bee
, but the analytic stuff seems interesting too 
Have a nice weekend!
xicori
(Distinctive Doe)
02-14-04 22:10
No 488657
Anything with a benzene ring should bee easily detectable with a UV detector.
a pressure of 2500psi
My memory has faded a bit, but that sounds to high. You can wreck your column if the pressure is to high.
There is alot of info online about HPLC columns and separations.
I am(or strive to bee) 100% patriotic to all people on Earth.
(Moderator)
02-15-04 01:48
No 488689
Amines are difficult to separate on silica-based columns, sometimes they don't elute at all from the column. You have to use a buffer with a pH around 4 and excess competing ammonium salts in it and/or use special columns with a more dense derivatization (e.g. phenyl / C14 / endcapped or special amine-columns). 270 nm is fine for methoxys, for unsubstituted phenyls I would use a shorter wavelength of 260 nm.
(Hive Bee)
02-15-04 02:52
No 488709
Finding a good elution gradient requires a bit of experimentation indeed.
I have been quite pleased with the following elution gradient on a reversed phase column:
Time Solvent A Solvent B
(min.)
0 100 % 0 %
30 0 % 100 %
35 0 % 100 %
Solvent A: H20 + 0,1 % trifluoroacetic acid
Solvent B: 8 / 2 CH3CN / H20 + 0,1 % trifluoroacetic acid
Phenylalanine methyl ester, for example eluted at 11,4 minutes (flow rate 1 ml/min). The wave length was 254 nm.
When the HPLC is started up, it is generally a good idea to purge the column for a while. The pressure and the signal from the UV detector (which has to 'warm up') has to bee stabilized (since this is your private HPLC, you won't need to worry about the garbage that sticks on the column from the previous user
: the 5 minutes solvent B at the end of the analysis generally clears the column).
(Hive Bee)
02-18-04 21:13
No 489577
Even for short path, thin diameter analytical columns...2500psi is a lot higher than i would expect. Check out an online manual for your specific column to get operating pressures, and set the pressure cutoff at 10% less than the highest operating pressure.
As an aside - running a column with pure H2O, when it expects a mix of MeOH/H2O can cause very high pressures, even 1% HPLC grade MeOH can make a huge difference to the operating pressure. Remember, you should also let it run for a while to equillibrate proprely.
Fan of Shulgin
Peace, love and empathy
(Hive Bee)
02-22-04 19:49
No 490413
You injected amine hydrochlorides and used plain water as solvent. Result: Your (protonated) amines will be most likely eluted in the injection peak, as the interaction with the mobile phase is much stronger than with the solid phase, if you used RP-spherisorb material. If you used NP-material (as the high pressure you've obtained in your HPLC-system suggests), the amines may be strongly adsorbed to the solid phase and may not leave your column at all.
Fur further info look at the HPLC textbook by Kazakevitch and McNair, http://hplc.chem.shu.edu/NEW/HPLC_Book/i
Quidquid agis, prudenter agas et respice finem!
(Moderator)
02-22-04 20:29
No 490416
There is a noticeable retention of those protonated amines on RP. This retention might even so high that you need a more lipophilic solvent mixture for them to elute.
(Hive Bee)
03-09-04 20:30
No 493999
High!
Thanks for the help, and the good tips!
SWIM changed the solvent to MeOH - Now Coffeine gives a very nice peak too, but the amine hydrochlorides still dont eluate...
There is basically no "injection peak" at my system...
Swim thinks that he needs a different column that can be used over a wide range of applications (Amine-salts, but also polar organic stuff - ketones, essential oils, and so on)... but there is a real jungle of hplc-colums out there on the market, and SWIM finds no overview what column is suitable for what purposes....
Could you give swim a column tip? :)
Would any of these types fulfill the needs?:
Nucleosil 100 7-C18, 250mm
Typ ODS 2 / 5 µm
ODS 1 / 5 µm
Nucleosil 100 5-C8 150mm
Partisphere C-18, 125mm
Nucleosil 100-5 NH 2, 250mm
Nucleosil 100-10 SA, 250mm
best wishes,
xicori
(Moderator)
03-09-04 21:10
No 494009
If you are using the starting eluent mixture as the solvent there shouldn't be a visible injection peak. Injection peaks are either air bubbles or result from solvent absorption if the solvent is different from the eluent.
For amines you want as much of the surface of the silica covered by the lipophilic substituents. This is usually better with shorter chain lengths (phenyl, C8 - C14) and can be further increased by endcapping. And/or use an ammonium buffer.
For everything else a cheap RP18, 250 mm length, 100∠ pore size, and 5 µm particle size should do it... The shorter the column the faster the runs and the lower the resolution.
(Hive Bee)
04-30-04 22:03
No 504039
Try using an acidic eluent (e.g. H2O, MeOH or MeCN, H3PO4) and add an ion pair reagent (e.g. sodium hexyl-1-sulphonic acid, 0.005 M) to the solvent system.
The acidic eluent will protonate the amine and the ion pair reagent will form an neutral complex, therefore a good seperation on a RP column is possible. This method works fine for the seperation of alkaloids and other basic compounds.
The tendency is to push it as far as you can