Chromatography Forum

Problem: split peak on early eluting compounds; 1st injection is OK, second inj reveals peak broadening, then splits. Earliest eluting peak splits first, then the net earliest peak splits on all successive inj's
System: Shiamdzu 20
Normal phase, heptane/IPA Mobile phases
typical normal phase column

1. I eliminated method/column issue by switching (IPA flush first) to Reverse phase (water/ACN/new C18 column/injected agilent isocratic std) split peaks still remain on early eluters

2. I changed out 100ul sample loop, HPV rotor seal and needle, plunger seal on LPV (metering device, essentially) split peaks still remains.

Typically split peaks are chemistry/solubility related, I cant seem to figure this one out, Any thoughts? thanks

Do you take a new column each time you’re running this analysis or have you figured out how to clean the column so that you start up with normal peaks next time you run this method?
I think you’re dealing with some junk deposition on the column with each injection you make.
Maybe your mobile phase is too hydrophobic and some of the sample matrix stays on the column rather than eluting at some point.

Best Regards

Just to be sure: It's just the early eluting peaks in the chromatogram that split? Later eluting peaks are fine?
What's the sample solvent? Is it maybe too strong compared with the mobile phase?
Column dimensions and injection volume?

The last 2 peaks are not consistnet retention time, they shift all over the place but are gaussian.

column is C18 eclipse 5um 4.6x 150mm
RP method is run just so I can evaluate changes quicker, run time is 6 minutes. inj volume is 10ul. Sample diluent is methanol. MPA= water MPB =ACN. flow = 2ml/min
Garadient is 100% water for 4 min, then 100%B for the last 2 minutes.
Standard has 4 peaks, I run it all the time to rule out hardware issues, this is the first time I have gotten split peaks using this std.
I agree that "junk" is present somewhere, just need to find out where.

The C18 column is pretty old, it could be possible that I have solved the problem with the hardware changes, but have a poor C18 column. The problem is that the normal phase run is 70 minutes long, so its a bit of a gamble to switch now.

column is C18 eclipse 5um 4.6x 150mm
RP method is run just so I can evaluate changes quicker, run time is 6 minutes. inj volume is 10ul. Sample diluent is methanol. MPA= water MPB =ACN. flow = 2ml/min
Garadient is 100% water for 4 min, then 100%B for the last 2 minutes.
Standard has 4 peaks, I run it all the time to rule out hardware issues, this is the first time I have gotten split peaks using this std.

That's quite a strange method, to begin with.
- 100% water on a standard C18 might be not such a good idea. You might see phase collapse.
- Does your analytes elute in the 100% water step? Or later with 100% ACN? What are the capacity factors?
- Your solvent (100% MeOH) is a MUCH stronger eluent than your initial mobile phase (100% water). That's the standard way to produce split peaks on early eluters .

You said you're using a standard test mixture from Agilent? Then I'd try the test procedure associated with this standard mixture, I guess it should be some sort of water/acn isocratic mobile phase. Your strange gradient method might be part of the problem...