Co-Elution of two peaks with almost same Retention Time

[mobunvar]

Two compounds are almost eluting together with Retention time of 3.21 min and 3.27 min. Flow rate is 0.1ml/min. I also tried 0.05 ml/min. But two drugs still not separated. pKa of the two compounds are 2.2 and 5.0. pH of the mobile phase is 2.3 and i also tried different pHs (2.5, 2.8, 3.2, 4.5) but no change. I am using Chromlith C18 column [100mm X 3mm]

Please any one can guide me how to separate those two compounds.

[Vlad Orlovsky]

Your compounds are not retained. At flow rate of 0.1 ml/min on 3x100 mm, column void is about 3.2-3.8 minutes. This is not chromatography. You have no mechanism of retention and no mater what flow rate you try you are not going to separate them. Flow rate has nothing to do with selectivity of your separation. Can you reveal structures? You might either need ion-pairing reagent, HILIC, or mixed-mode column. Please contact me by email if you would like to discuss this.

[mobunvar]

Peaks are eluted only after solvent front... I am working on RRLC and Column is chromlith. FLow rate is less because tubing of the system is very small.

Mr. Vlad, can u provide me ur email id..my email id is munvar.shaik@gmail.com

[HPLCaddict]

Peaks are eluted only after solvent front... I am working on RRLC and Column is chromlith. FLow rate is less because tubing of the system is very small.

???
The usual sense of using chromolith columns is to take advantage of their verly low backpressure and thus speeding up analysis by using very high flow rates. Using a monolithic column at 0.1mL/min is like, well, you wouldn't drive a Ferrari at 5 mph, would you?
As Vlad already pointed out, you're not doing chromatography as your peaks are not retained. First, adjust the chemistry (i.e. mobile and/or stationary phase), then you might take care of the flow rate...

[Vlad Orlovsky]

Look at list of this applications. Your goal is to find molecule similar to yours. If pKa of your compounds are 2 and 5 they are probably acidic and you might want to explore a different mechanism of retention:
http://www.sielc.com/Applications_By_Compound.html

you can contact me through our website.

[mobunvar]

that means I can't use 0.1 ml/min with Chromlith columns...I tried higher flow rate but as I increase the higher flow rate, area counts is decreasing.

I tried different combinations of Mobile phases with altering pH. I can see split peak but peaks are not separated completely. Along with these two compounds, i have two more compounds which needs to be analysed completely.
So previously I developed the method for these compounds using C18 (150 mm x 4.6 mm x 5microm) Purospher columns. All the four compounds were separated clearly. But when I used chromlith column, the sensitivity is increased predominantly so i want to shift my method to chromlith column and i am getting this, co-elution of first two compounds.

I didn't understand what shud i do

[Vlad Orlovsky]

Like I said, you have no retention and this is not a robust method. Any excipient , contaminant, solvent mismatch will affect your "non retention", because you have no controllable interaction and retention mechanism. You need to develop a method with k' of at least 1 and resolution of 1.8 (we usually shoot for much better resolution with mixed-mode columns). I would suggest that you try low organic (0-5%) in combination with low pH (pH 2 created with phosphoric, sulfuric, perchloric, methanesulfonic or trifluoroacetic acid). You goal is to use low pH to increase hydrophobicity of your compounds (assuming that they are organic acids of some sort with pKas you stated above)

[tom jupille]

I have several comments (some of which echo what Vlad has already said):

1. It is normal for area counts with UV detection to decrease when you increase the flow rate; the residence time in the flow cell is decreased.

2. The monolithic columns run at lower back pressure because a larger fraction of the column volume is accessible to solvent flow. That means that a smaller fraction of the column volume consists of stationary phase (they have a lower phase ratio than conventional columns), so you generally have to use a weaker mobile phase to get retention. You did not specify the mobile phase (aside from the pH). Is there any organic solvent in there? If so, how much?

3. By running at a very low flow rate, not only are you not making use of the advantages of the monoliths (high flow with reasonable pressure) you may very well be substantially below the optimum flow rate (so that plate counts are dramatically lowered).

4. Was the sensitivity with the Purospher good enough to meet your needs? If so, my advice would be to leave well enough alone.

[mobunvar]

My Mobile Phase conditiones are 0.05mM Heptanesulfonic sodium salt with 0.05% TEA pH adjusted to 2.3 with 85% Orthophosphoric acid and MeOH in the ration of 67:33. If I use this combination, first two peaks are eluting together and last two compounds are resolved better with good resolution.

With 0.1ml/min flow rate, peaks elute only after 6 mins and total running time is 10 mins.

I tried to decrease the MeoH percentage in mobile phase, as I go low, first two peaks are eluting as split peaks but my last peaks are getting worse.

With purospher, the sensitivity of the compounds is O.K but when I go for extraction, i need to use 1ml of Plasma which will be a drawback in the method. So I am trying for more sensitivity so that, I can reduce my plasma volume in extraction.

Right now, i am trying for gradient. After running the scout run (5%-50% in 20 mins, this was chosen with previous gradient run developed with Pursphere), first two peaks are completely separated with good resolution. But last two peaks become split peaks. So working on that to make them separate. But total run time has increased to 20 mins. This may be another draw back as im using RRLC. So still i am looking for suggestions so that it can help me in Isocratic run.

[Vlad Orlovsky]

You are using acidic ion-pairing reagent for retention of acidic compounds (unless two of your other compounds are hydrophilic bases). I am just trying to see what is rational for this weird combination in mobile phase. Looks like a soup to me, with no sense for any of the components. You problems is not RRLC but lack of understanding what is going on on the column. I would suggest to do gradient from 2% ACN to 50% ACN in 10 minutes with 0.1% of phosphoric acid. If you other two compounds are hydrophilic bases your best bet is mixed-mode or HILIC.

[unmgvar]

try to work at 0.8 ml/min for the chromolith
you must make sure that your data acquisition rate in the detector is fast
use 10 or 20 Hz speed