Dear board readers,
I´m searching for a hplc method for the determination of porphyrins, especially hemoglobin. I would like to analyse by rp-hplc.
Thanks a lot.

Hi Frank
What is your reason for determining haemoglobin? Are you interested in separating different haemoglobins from each other?
I carry out development work on the separation of about 10 different Hb's. How can I help?
Cheers
Che

Hi Che
I am interested in separating different haemoglobins from each other. Your guess was right in so far. The crucial part is the haem moiety.
By the way, I am also searching for a rp-method to determine Hb A1c; the marker for monitoring patients who suffer from diabetes mellitus.
I will appreciate any suggestions...
Thanx
Frank

I have not looked into this in about 15 years, so my comments may be slightly out of date. Hb A1c used to be determined with ion-exchange chromatography. There even were some clinical analyzers around to do this.

Yep, Uwe is correct again. As far as I am aware, Hbs are separated by ion exchange only. The minor differences in the protein sequences would not be able to be resolved by RP. I am project manager for the development of a new Hb analyser, which uses a weak ion exchanger and gradient elution. The functional spec stipulates separation of A1c from HbF, and other interfereces. I would think that separation by RP is impossible, by ion exchange it is difficult, but possible. If you are looking at the Aic level, as an indicator for diabetes, have you thought of 'affinity' chromatography, using a phenyl boronate as stationary phase. This is exeptional at separating glycated species from non glycated species. Present wisdom suggests A1c is about 40% of glycated species present in glycated blood. There are instruments on the market which use affinity, and others which use ion exchange. Can you tell me how you would attempt RP separation, I would be very interested if you could get any kind of separation.
Please reply
Che

First of all thanx to both of you (Uwe and Che).
I know that there are sophisticated analysers on the market, but I have to face a particular R&D problem. Analysing by Ion exchange chromatography will surely do, even it is difficult, as Che pointed out.
Maybe I will go further into literature and report my experiences. By now I must admit that both of you are fairly correct. But do we really have to give in?
We will see.
Frank

Hi Frank,
No, we don't have to give in, but unless we are working for ourselves, and/or have a bottomless pit of the folding stuff, sometimes not reinventing the wheel and maintaining our employment is preferable to a one in a million chance of finding a simple cure for that terrible disease! Although RP is ubiquitous, it is not a panacea!
I reiterate, if it is just glycated Hb you are looking at, affinity is superb, easy, cheap, quick, anything you like!
Hasta la victoria siempre!!
Che

Hi Che,
Alright then...of course it is not only HbA1c that causes the trouble...with that you `ve convinced me...ok, ok...
Actually, it ´s more the haem moiety with the centered Fe...
So I will try by ion exchange...and I apoligize in advance if it seemed a stupid question to you...
Anything `s possible but not essential...

Ciao bello et tante saluti
Frank

Hi again Frank,
no need to be sorry, I needed to be clear about the question (sometimes the question is more difficult to get right than the answer!).
Is it just the heme group you wish to detect, then reverse phase would be my choice, at the pH at which it is most stable, within the working pH of the column! starting at 50/50 acetonitrile/buffer.
However, I am puzzled, surely the heme group will be the same in each case? I don't know, not my area of expertise! Are you looking for oxygenated/deoxgenated/other adduct?/other species ie cat, dog etc. I will help, if I die trying!
Ciao
Che

Hi, I have analysed thousands of samples of haem groups from oxidatively modified myoglobin/ haemoglobin. The trick is to run the column at low pH. This will easily seperate the haem groups from the globin. I use a Zorbax 300SB C3 column (250mm x 4.6mm). (A Vydax C4 column does just as well).
Solvent A= Water with 0.1% Trifluoroacetic acid.
Solvent B=Acetonitrile with 0.1% TFA.
Start with ~35%B, leave this for a few minutes (~10) and slowly increase %B. The haems elute from 35-37%B and the globin elutes ~43%B (I get a good seperation of alpha and beta subunits). The only drawback is that you cannot analyse ligands (eg oxygen, CO) as this column runs at ~pH 2 which knocks off ligands and oxidises the iron to ferric.
Happy analysing!

Brandon