Chromatography Forum

I am analyzing Poly (L-lysine hydrobromide) (Mw= 500-2000) in water at 0.1%.
I am not sure why I see such a jagged HPLC chromatogram, and why I see peaks for more than one fraction of PLL in the mass spectrum of a peak. Additionally, I'm not sure why the HPLC peaks are not equally spaced (is this only due to the fact that the gradient is not steep enough?)

thank you for any help you may be able to offer

Posting the chromatogram and one spectrum may help all of us to.. help you.

Thanks for your reply. I had intended to post spectra in my first post, but I was not and am still unable to figure out how to do it.

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Thanks for your reply. I had intended to post spectra in my first post, but I was not and am still unable to figure out how to do it.

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See ''Sticky: Embedding Chromatograms and Reports'' in the forum index.

Thanks again for your help!!! I am new to MS analysis

Again, I'm hoping to respond to the following questions:

First I have to identify multiple-charged peaks of Poly L-lysine. This seems straight forward. My PI seemed to indicate that there would be not triply charged pk for 2-mer unit. I would think that there could be. And is is possible to have peaks with many more charges for Poly L-lysine?



Next, I need to identify the reason multiple fractions of Poly L-Lysine are visible in some of the mass spectra.

My PI indicated that multiple fractions of PLL are visible in some of the mass spectra. It seems to me that if there was carryover on the reverse phase C18 column, that I would see evidence of this carryover between peaks. but when i look at mass spectra between peaks, there is no evidence of various lower mw PLL fraction peaks. I would have thought that what he believes to be various fractions are just fragmentation of the separated mw fractions. (any ideas would be helpful)

And lastly he wants to know why the peaks in the chromatograms are not equally spaced
I don't know why my PI keeps asking about whether the peaks are equally spaced or not. He mentioned that when the gradient is steep enough the peaks will be equally spaced.

The first gradient did not separate the fractions of Poly L-lysien (PLL). The separation did not go well. ( I guess because the gradient was too steep??and the various mw fractions of PLL coeluted? Why then, are there 3 peaks. It does not seem that each peak contains higher sequentially higher MW fraction clusters???

I HPLC methods are described below:

Gradient method 1:
The first method run was a steep linear gradient from 10 to 60% MeOH; 80 to 30% dH20; 10% 0.1%PFPA M.P. composition over 6 mins.
The PPL fractions coeluted. The separation did not go well.

Gradient method 2:
A more gradual linear gradient method was run from 10 to 60% MeOH; 80 to 30% dH20; 10% 0.1%PFPA M.P. composition over 12 mins.

Gradient method 3:
A linear gradient method was run from 20 to 60% MeOH; 70 to 30% dH20; 10% 0.1%PFPA M.P. composition over 6 mins.


gradient 1


peak 1 (0.96 min)


peak 2 (3.97 min)


peak 3 (4.88 min)



gradient 2


gradient 3


peak1 (3.07 min)


peak 2(4.05 min)


peak 3 (4.77 min)


peak 4 (5.27 min)


peak 5 (5.62 min)


peak 6 (5.88 min)


peak 7 (6.10 min)


peak 8 (630 min)



Thank you very much for any suggestions you may be able to offer!!!!

Multiple charges are likely above mw 1000 but that’s compound
dependent ( I have to admit though that I don’t have experience
with PLL, I work with polymers mainly)

Regarding the spectra.
I think you are doing MS/MS (in source CID) with the current
instrument settings. Which LC-MS do you have?

If it’s a Waters decreasing the “cone voltageâ€

Thanks so much for your reply!!!!

PI mean Project Investigator (my advisor )

I have no idea why he is asking about equally spaced peaks. Maybe he just wants me to figure out why it happens when gradient parameters are adjusted.

I didn't add any sodium so i don't think i'lll see any Na cations?. The original sample was a PPL -HBr.

I'm using MS not MS/MS, but I am seeing a lot of fragmentation so i agree it looks like MS/MS. I will try adjusting the voltage.

Thanks again!!!!! It was very nice of you to help me! very best, Annie

• LC system: A Waters Alliance HT HPLC 2795 with a Waters 2996 Photodiode array detector was used for all analyses. MassLynx v.4.0 software and the MLQuantify application were used to process the data. A RP C18 Waters Symmetry column (3.5μm, 4.6 x 50mm), which was used for all experiments, was external to the instrument. A temperature controlled column compartment was not used. The flow rate for all experiments was 1.0 ml/min., and the injection volume was 10μl.

The analysis was performed online, by feeding the liquid eluting from the LC column directly to the electrospray mass detector.

• Mass detector: Waters Micromass ZQ single quadrupole mass spectrometer; electrospray ionization (ESI); nebulizing gas: N2; MS Tune software within MassLynx was used for instrument parameter control and data analysis. The following voltage parameters were set within the range: 1. Core (V) between 20 and 70V 2. Extractor (V) between 3 and 10 V. The Capillary voltage was set to 3.50 KV and the RF lens to 0.5 V, and remained constant. The source temperature was 100°C and the detector temperature was 300°C.
Mass collection was set from 100 – 2000 m/z. The Run duration was 1.1 minutes in continuum data collection format with a scan time of 10s.

The second and last separations are perfectly fine - they serve the purpose. Most oligomer separations look this way...

I didn't add any sodium so i don't think i'lll see any Na cations?. The original sample was a PPL -HBr.

You can pick up Na when using glassware in your sample preparation.
Adduct formation is also compound dependent. A easy way to verify this is to dope the mobile phase (or maybe even by direct infusion) with another cation (Li, K, etc) and see what happens...

great suggestion. thank you!