HPLC peak separation

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zacharyr
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HPLC peak separation

Hello all,

I am having trouble with peak separation. Attached is an example chromatogram.

My column is an Agilent 4.6 x 50 mm, XDB-C18 which contains silica particles of 1.8micrometer diameter. 
My mobile phase is as follows on the table: %A: 0.01M KH2PO4 at pH 3.00 and %B: Acetonitrile

Time
%A
%B

0.00
96.50
3.50

5.50
96.50
3.50

6.00
75.00
25.00

13.00
65.00
35.00

15.00
25.00
75.00

17.00
25.00
75.00

19.00
25.00
75.00

21.00
96.50
3.50

22.00
96.50
3.50

Flow rate is at 1.00mL/min, column temperature at 30 degrees Celcius, and injection volume of 10 microliters.

As stated above, an example chromatogram is attached.
How can I solve this issue??

Thanks, 
zacharyr

Dr. Analytical
Dr. Analytical's picture
Zacharyr:

Zacharyr:
Thanks for providing so much information.  It helps us to better understand your system.

Just a little more information would help:
What instrument are you using?
What wavelength is used for detection?
Which peaks are you interested in (which time ranges in your chromatogram)?

Now, on to your chromatography.
First, some settings that might need changing:
1. The injection volume is too large for such a short column.  I would normally only use 2 uL for a column like this.  Try several injections at smaller volumes, and see if that improves the chromatography.
2. The flow rate should be increased to 2.0 or 2.5 mL/min. to take advantage of the small particles.  This is why I asked about the equipment.  Can you tolerate an increase in pressure?

These two steps may improve the separation somewhat, depending on what peaks you want to separate.

Next, I would use a much shorter gradient.  This one is for a much longer column.  You have a high speed UHPLC column, and gradients are typically only about five minutes or less!  As a starting point, divide each of the times by 3 and try again.  Your last time would then be 7.3.

Other suggestions:
1. Replace the buffer with 0.1 % H3PO4.  It is about the same pH (a little lower), and much less trouble to make and less corrosive to the system.
2. Start with at least 5% B in your gradient.  Most C18 columns do not work well with less than 5 - 10% organic in the mobile phase.

Try these and send us results.  Good luck.
 

zacharyr
zacharyr's picture
 Dr. Analytical,

 Dr. Analytical,

Thank you for the quick reply, it is much appreciated.

Answers to a few of your questions:
I am using an Agilent 1260 HPLC, and the wavelength I am using for my detection is at 315nm.
I am interested in a particular peak, the one at 8.736. It is a bit hard to see in the attached picture above, so I have attached an edited one to make it easier to find. 

I am honestly not sure if I can tolerate the increase. I was told that we could handle up to 600bar, however, the machine came with the factory setting at 400bar as a max. My pump is an Agilent 1260 binary SL. 

If more information would be helpful, I am more than happy to provide you with whatever information you might need. I am new to HPLC, as well as my HPLC system, so any help is very much appreciated.

I will try your suggestions A.S.A.P. and will be back here with my findings. 

Thanks!

P.S. - If I am unable to increase flow rate, how much will peak shape and separation be affected? This is the only column we have where I work. We do not have anything longer or bigger.

zacharyr
zacharyr's picture
 Sorry for the double posted!

 Sorry for the double posted! Realized the above attachment did not work.
Here is my chromatogram with the peak that I am interested in.

Thanks,
zacharyr

Dr. Analytical
Dr. Analytical's picture
The picture still didn't come

The picture still didn't come through, but we can move along without it for now.

Since the peak you want comes out in the middle of the gradient, you do not need to start at 3.5%.  Replace the 3.5% values with, say 15%, and try that.  We can probably eliminate the initial hold altogether, but that will come later, once we fix the other problems.

The 1260  Binary SL will handle 600 bar.  They sometimes don't put that in the software defaults.  If you have all 1260 components, you are fine.  If you have a 1200 autosampler, then the limit is 400.  Otherwise, change the setting to 600 and give it a try.  Adjust the flow based on the maximum pressure observed during the gradient.

You can alway increase the column temperature - up to 50 - 60 C if necessary, to lower the pressure.  Do this in several injections, since peaks can sometimes change order when you change temperature.  Then start increasing the flow.

You could really benefit from some software, hardware, and technique training.  You are facing a difficult separation challenge and you can not be expected to solve this problem without some additional skills.

zacharyr
zacharyr's picture
 Dr. Analytical:

 Dr. Analytical:

Thank you for your help thus far, I am already seeing a nice improvement in the peak separation. 

I have made the changes you suggested, the attached chromatogram was done with the shortened gradient with an injection of 2uL at a flow rate of 2.0mL/min. The column temperature was at 50C, and I replaced the buffer with the 1% H3PO4. I'm actually very thankful/glad that you mentioned the H3PO4. It is much faster, easier, and actually cheaper to make. I replaced the 3.5% values with 15%.
It is interesting that even at 2.0mL/min I was not even close to exceeding 400, so I could actually probably increase it to 2.5, if that would help separation even more.

Due to the fact that the peak I'm interested in is an unknown molecule, I can only compare it to a standard that I believe to be very similar. However, the absorbance is different, so I am searching for the exact peak still as I write this. Come Sunday, possibly Monday, however, I believe I will have found it. I believe it is the peak at 1.053. However, I was interested in the absorbances that I saw for the early peaks (0.274-0.676). 
Is there any way I could further separate those early peaks? Would the peak even change places that much though? As you remember, I was interested in the middle peaks of my early chromatogram. I do not see why these peaks would suddenly come off at the very front.

Thank you again for your help. I feel as if I have learned alot more about HPLC in these few days that I have visited these forums. I am looking further into more learning materials for HPLC. I would definitely like to, and I really need to learn more.

Attached is the chromatogram. I am looking forward to your reply.

Kind regards,
zacharyr

Dr. Analytical
Dr. Analytical's picture
Zacharyr:

Zacharyr:
the new chromatogram certainly looks different, but that is a good thing.

When you switched to 15% acetonitrile for the starting condtions, you took all those peaks that were spread out at 3.5%, and pushed them all together at the beginning.  If you want to separate them out, then you wil need to reduce the % acetonitrile at the start.  Reduce to 10%, and try again.  Keep everything else constant, just replace the 15% with 10%.  You can also reduce this more, down to 5% if necessary.  This will spread out the peaks that you are seeing at the beginning.  I am suggesting that you do this in steps, so that you can see them move individually.  If you did it all at once, it might be difficult to see which peak is which.  If you need to separate those early peaks more, you may need to adjust the gradient.

Another good practice is to run a "blank" solution, so you can see if any of the peaks appear in the blank.  I am curious about the negative dip that appears just after 3 minutes.

Good luck.

zacharyr
zacharyr's picture
 Hello Dr. Analytical:

 Hello Dr. Analytical:
I have run a number of tests today testing out the gradient to get those peaks at the beginning. I gradually reduced the % acetonitrile to 5%, and did get somewhat better separation. The results I got from this separation are both interesting and confusing to me.

The following questions may be a bit confusing for other users, because I have to send the pictures to your (Dr. Analytical's) email. Maybe an administrator could help post the pictures I am sending to your email on this thread later? It just doesn't work for me. 

For clarity while reading my question: The picture by the name of "A" is my sample run with the 5% ACN instead of the 15% ACN. The picture named "A-SPIKE" is my sample spiked with a standard solution of PhIP -- a molecule I believe to be similar to the one that I am attempting to isolate. The 3rd picture is simply called "Peaks of Interest" and it is a chromatogram you have already seen. I have put a rectangle around peaks of interest.

Question
As you can see, in A (5% ACN instead of 15%), I got better separation. However, can I get still better separation? How?
I am a bit confused now because some "peaks" that appeared to be distinct, such as the peaks at .247 and .322 in "Peaks of Interest", actually separated alot. When I checked the absorption at those peaks, I found that those 2 peaks seem to be split into a mix of the peaks at .2, .3, and .6 in picture "A". How can I further separate these peaks in "A"? In fact, it seems as if most but not all of these two peaks just combined into the one at .6.

I am also interested in the big peak around 3 in picture "A". This seemed to be the peak at 1.053 in "peaks of interest".
When I injected the PhIP standard in picture "A-SPIKE", it essentially undid the good separation I got in "A". It actually clumped everything from .2-.6 in "A" into that big peak at .262 and .335 . What is even more confusing is that it also changed the form of the peaks around 3 minutes. As you can see, with the spike, the peak has now "rounded out" and isn't jagged like the peaks in A.
It's almost as if my standard mixed and came off in two places. I ran multiple spikes to make sure, and they all showed what can be seen in "A-SPIKE". 

I suppose the spikes helped me realize that I still am having issues with separation. I do not understand why the standard is clumping everything from .2-.6 together -- I'm guessing this shows that I still need better separation. I am hoping better separation will better clarify the data that I am getting.

Thanks -- this most recent post of mine I'm sure is hard to follow, I tried to keep things as clear as possible.

Looking forward to your response. Pics have been sent via email.

Regards, 
zacharyr

Dr. Analytical
Dr. Analytical's picture
zacharyr:

zacharyr:
I will try to attach the files to this message, so everyone else can see what we are talking about.

There may be several things going on here, and with complex samples, it is hard to interpret everything.

First, are you filtering ALL of your samples?  You should be using 0.22 um filter, or 0.1 if possible.  Are your samples clear?  If they are visibly cloudy, then you have been loading those materials onto the front of the column, and it may be close to dead.  I am concerned about this because many of the peaks do not have a good shape - they have a slight shoulder distortion on the back side.  This could be a second component, but when all the peaks have it, it often means that the column is being plugged up at the inlet.

Only hope is to reverse the column, put outlet to waste, and run a couple gradient cycles through it.  See if that helps.

The differences between A and A-spike are disturbing.  It would appear that your spiking solution is not a single component, but contains a number of things.  It also is spiked at too high a level, so we can't really compare the two.  A more dilute spike solution would help.  However, it appears that your spike compound is probably coming out at the beginning (the two peaks), and/or at 3 minutes.  So, it isn't really clear which peak you are interested in.  Sorry, but these results are not helping your search.  Try a lower spike level (2X - 3X) after you check your column.

One other possibility is that you are leaving some things on the column.  Change the gradient so that all the 75% values are 95%.  Change the 6.3 minute time to 7.3 and add a minute to the following values as well.  If you see a lot of stuff eluting between 5 and 8 minutes, during a blank injection, then your column is contaminated.  Repeat the blank injections until the baselines are similar.

Then try your sample again.

If the peaks you want are indeed the ones coming out early, then this column is not going to work for you.  At 5% acn, you are at the low limit of elution strength.  I different column would be needed.  Exactly which one would depend on the structure of compounds you think are present.

Since you have a diode array, you could collect spectra (or look at them if you already have them), and try to identify which peaks are similar to your target compound.  With such a complex mixture you will ultimately need some other tools to help you, but we can still learn more from what you have.

zacharyr
zacharyr's picture
 Dr. Analytical:

 Dr. Analytical:

All of my samples are being filtered with a 0.2um filter. Actually very recently another student overloaded a column and after reversing it and cleaning it it was decided that that particular column was dead.
These runs that I am doing are being run on a newer column, which was reversed and cleaned very recently. After seeing the behavior of the other column while it was near-death and after it pretty much died, I highly doubt that the column is plugged (plus it was just recently reversed as it is).
I have a feeling this distortion comes from the sample just being so messy. I'm thinking I might have to clean it up a bit more before doing HPLC -- I'm currently looking into some extra S.P.E. steps I can take to do this.

I am planning on making a new standard solution tomorrow, so I will try a more dilute standard as well as a smaller spike. 

I ran a few blanks today and did not see anything that would suggest column contamination, but I could always run a few more to make sure.

I personally believe the early peaks are the ones of interest, so I believe I will need to look into other columns, although I'm not so sure funding will allow that at this point in time. Any suggestions on possible columns though? Structure should be as similar as possible to the molecule 2-amino-1-methyl-6-phenylimidazo(4,5b)pyridine.

I am going to begin collecting spectra as soon as possible.

Thanks,
zachary

zacharyr
zacharyr's picture
I actually just came across a

I actually just came across a few columns that might be of more use to me..? Would any of the following columns would be of more use for this particular separation that I am trying?

Both are much longer with bigger particle sizes:

- HAISIL 100 C18 (5um); 250 x 4.6mm 
- Econosphere C18 (5um); 250 x 4.6mm

Dr. Analytical
Dr. Analytical's picture
Neither of these is likely to

Neither of these is likely to be of any use.  You need to move away from a C18 column toward something with more polar functionality.  There are some columns that retain amines, but they are complicated to work with, and expensive.

If you have no other choices, these could always be tried.

zacharyr
zacharyr's picture
 Alright -- I'll be trying

 Alright -- I'll be trying those columns anyway, but that is too bad. I doubt I'll be able to get my hands on any of those amine-retaining columns any time soon, so I'll have to make the best of what I have.

We'll see what I can do. Thanks for the help!

zacharyr
zacharyr's picture
 Found another HAISIL 250 x 4

 Found another HAISIL 250 x 4.6mm column today with just silica packing. Do you have any rough ideas as to how that would affect separation? I'm thinking it could possibly be of a bit more use than the C18?

Best,
Zach

Dr. Analytical
Dr. Analytical's picture
Now you have something

Now you have something completely different to work with!  (Don't know if it will work, but it will be different!)

Here is an article that may get you started with some different phases: "A Global Approach to HPLC Column Selection Using Reversed-Phase and HILIC Modes: What To Try When C18 Doesn't Work," LGGC North America, Vol. 28 (3), 234-244. It is available online at:
digital.findanalytichem.com/nxtbooks/advanstar/lcgc-na0310/index.php

You should start with the recommendations for silica.  These columns are slow to equilibrate (allow one hour before using the first time) and gradients do not always work well, so start with isocratic and hopefully you can get something to work.  These columns are also not as stable, so if you use a near neutural pH buffer, keep the pH at about 6.5.   Ammonium formte works well for this.

Final question: what was this column used for in the past?  If it was used as a normal phase system (with something like hexane), you should wash it in IPA first, then acetonitrile, then the final mixture.

Good luck.  Will look forward to seeing the results.

zacharyr
zacharyr's picture
 Thanks for the article!

 Thanks for the article! There are a few things I am going to try before using the silica column -- mainly things that should clean the sample up a bit more. If the results are still sketchy, however, the silica column may be the way to go.

When I do get around to using the silica column, I'll be sure to post up the results.

This site has been an enormous help as I have started my chromatography. As always, many thanks!

-Zachary R.