Everything Elutes Early in One Peak

[mant0]

Hello. Maybe a somewhat basic question. I am having issues with my chromatography on an LC-MS run. I have tried running two samples, one consisting of 3 standard peptides in solution and the other a BSA tryptic digest.

Running on a C18 RP column, Water>AcN (FA) solvent system, 5%B - 50% B gradient.

For both samples, I get predominantly one large peak at 3 mins that contains all my analyte ions of interest. For my standard peptide solutions, I also see the tree peptides separated at different RTs later on in my gradient but the intensities of these peaks is much lower. The majority of my sample seems to coelute early in my gradient. I am not above the column load capacity by my calculations. Thoughts?

[mattmullaney]

Hi--what are the column dimensions, injection volume and sample solvent? Also, total analysis run time?

[mant0]

nanoflow column 75um X 15 cm so CV of ~0.6 uL and injection volume of 5 uL and my gradient was 0-50% over 10 minutes. My gradient is on the short side but should be fine for my sample. The sample is in 0.1% FA.

[Jiri Urban]

I would play with gradient more (2% ACN at start, longer gradient time - does it change if you do your gradient in 30 or 60 min?, and so on). Are you sure your pump works well?

I am not sure if 5 ul injection volume on such a small column is the right thing. Have you tested lower injection volume?

[mattmullaney]

Hi again, mant0,

I apologize, had some HPLC-unrelated plumbing problems at home...

I concur with Jiri...while with those column dimensions and a well-functioning pump or pumps, the gradient program ought not to be a problem...but it is a nice idea to test it out by lengthening the program to see if you get better separation.

Five microliters on a column of those dimensions is A Lot of Volume. I'd definitely try cutting back to 1 uL...just to see what happens. Typically I've used no more than 1 uL on 2.1 mm x 100 mm columns, though I freely admit these were small molecules I was separating--and not peptides.

Please see what you think, and Best Of Luck!

[mant0]

Thanks Matt and Jiri for the replies. Ive tried it with a 1 uL injection and I get the same results as well as with a longer 30 min gradient.

Here is one of the results I got today.



So Im still seeing this huge peak at the start of my gradient where all my peptides coelute

But then later on the gradient but at a lower intensity I see the same peptides separated.



If I had any type of leak in my nanoflow system, could that cause this type of issue? I'm pretty confused.

[mattmullaney]

Wow, okay...

What you've shown is a 1 uL inj of a standard tripeptide mix...one "blob" peak at 3 minutes and 3 small ones much later on in the extended gradient...what's weird is that the gradient slope has changed quite a bit, though the "blob" hasn't changed in retention time (I'd have thought it would have moved to a little bit later in time)...how does the "blob" peak area for the 1 uL injection compare to that of the 5 uL injection(s)?

As to a leak, my answer is "maybe", but it would almost have to be between the injector and the column for this kind of weirdness, I'd think. I'd look at the peak areas of the blob's first...then maybe set isocratic conditions to about one minute before where the "blob" elutes, say at two minutes, and just sit and wait to see if you can get any separation at all...

...and I'll hope Tom comes to our rescue!

[tom jupille]

Unfortunately, I don't have any answers, but some observations:

1. You didn't specify the flow rate, but that 3 minute peak looks like a typical "solvent front" (elution at t0) -- which would imply a flow rate somewhere around 200 nL/min and in which case most of your sample is washing right straight through the column without interacting with the stationary phase.

2. Along that line of thought, if you had a bad column with a massive channel such that most of the sample bypassed the stationary phase entirely, you might get just that effect. Sooo -- have you tried replacing the column?

3. Even 1 microliter is a *huge* injection volume for that size column. That would be the equivalent of injecting 2-3 mL on a "conventional" 150 x 4.6 mm column. The fact that your diluent 0.1% formic acid is weaker than your mobile phase (5% ACN in 0.1% formic acid, right?) helps somewhat, but it's still a gigantic volume overload.

If this were my problem and changing the column didn't help, I'd try increasing the concentration by a factor of 10 and dropping the injection volume by that same factor (thus keeping the total mass load constant) and look at the ratios of the "blob" versus "retained" peaks. If it shifts in favor of the retained peaks, then you've confirmed that it's volume overload. If it doesn't shift, then I'm out of ideas.

[mattmullaney]

Mant0 and Tom Jupille,

I am thoroughly embarrassed...I allowed the scale of the instrument to "throw" me. After sitting down and doing my own set of calculations I'm in agreement with Tom. Basics will always be basics, though, which I somehow forgot for a bit--probably was carelessness on my part.

A column-volume for a 0.075 mm x 150 mm column, leaving out stationary phase "bulk," is ca. 0.66 uL (and I missed this above and apologize to mant0). If the eluent flow was 0.2 uL/min, 3 min of run-time would pass .6 uL of eluent...approximately one column-volume...another way to say this is what Tom said, no retention of analyte(s). It's as if there was no stationary phase in the column...throwing in the porosity of the stationary phase (unless the S.P. is non-porous silica like Kovasil) the "blob" k' is still only slightly greater than one...no real "chromatography" is being done.

Either there is a massive leak between the injector and column inlet, or there is a gigantic channel in the column's stationary phase.

During the course of the evening I came across a cool book:

More Practical Problem Solving in HPLC Author is Stavros Kromidas, ISBN: 978-3-527-31113-2, Paperback, 309 pages, January 2005, Wiley VCH

Chapter 2.2 is pretty much excellent reading...maximum injection volume suggested for a column of approximately the same dimensions as the one you are using is 30 nL (some downscaling for an analytical scale guy like me!) and the column mass load suggested is 6 ng (also quite small to me). Suggested flow rate for eluent was 350 nL/min. It's good to live by a good Library

I apologize Mant0, I think, but this was kind of a fun problem for me to help with...very educational. Hope this Helps...my thanks for listening to my opinions, and Best of Luck again!

Learning Doesn't Ever Really Stop...and Tom, my thanks and appreciation for coming to our rescue!

[Jiri Urban]

There is probably nothing to add now