-
- Posts: 7
- Joined: Sun Jul 27, 2014 2:18 am
I have no images present but let me explain my current issue.
We have an HPLC method. It runs the usual Acetonitrile/methanol/acidified water (for our acid compound). At 0.002mg/ml standard injections, the heights are roughly at 0.02 AU, give or take, and obviously, they're thinner and taller at the beginning. The peaks were roughly 0.15-0.3 minutes wide at baseline. 10uL injection
This is using a 15cm C18 column (Ace).
So I needed to develop a UPLC method for it, and decided to use a 50mm Excel Super 2 C18. I made some 0.003mg/ml standards, but my peaks aren't as tall, and still quite wide, in fact almost identical widths to the HPLC chromatogram. Most of my peaks elute at 0.4 - 2 minutes, and another at 3.2 minutes (incredibly non polar so cannot get it closer without ruining resolution of other peaks). 1.5-2uL injections, ACN:Acidified water, no methanol.
They're not as tall either, around 0.01 AU ish tall, half of the 0.002mg/ml standards on HPLC. Methanol addition makes no difference.
I then tried an Ultracore 2.5 and the peaks got taller, roughly 0.025 AU, and much thinner, but still not the usual textbook UPLC really thin 1-2 second wide peaks thin/tall. 1.5uL injections
My question is thus;
Are UPLC peaks always taller than HPLC peaks, and thinner? Band broadening wise, the column should be fine (but will be getting a replacement one in case) and our system has been professionally installed, no dead volumes in the fastenings or anything, and all correct UPLC 0.12mm diameter tubing. Method runs fast at 0.9ml/min with 450bar pressure and only 45°C so not much time for axial diffusion to occur and broaden the band.
My logic is that our machine is a UHPLC, and can run HPLC methods. The detector detects the HPLC peaks identically to the HPLC machines, despite being a UHPLC. This to me means the detector is in no way "more powerful" than an HPLC detector? If it did the response would increase. I can run the same standard on an HPLC detector and a UHPLC detector and get the same response factor, peak height, and area (by same I mean within reason very similar to within 0.3% RSD easily)
So I have 0.002mg/ml with a 10uL injection for HPLC, that totals 0.00002mg going through the detector of each compound, which yields response A.
I also have 0.003mg/ml standards with a 2uL injection (sometimes 1.5) for UPLC which totals 0.000006mg total going through, which yields response B, and my heights are only a little off the HPLC's.
So relatively speaking, my UPLC peaks ARE taller, if I were to inject 2uL on the HPLC method the peaks would be tiny. But I was always under the impression UPLC peaks are always taller? Or is this salesmanship stating so and when practical comes into play, it isn't always textbook?
We have ruled out system malfunction band broadening, it is happening in the columns.
Usually the symptom is tailing, but could it be that the Excel Super 2 has a particular retentive capability on our compounds and thusly holds on and causes band broadening? We have a Excel 2 C18 (Not super) which from my knowledge just means it has less pH range and the peaks looked identical, so chances of having 2 columns with duff packing from 2 silica batches is...rare.
The Ultracore unfortunately does not give the required separation, but the peaks look much nicer, which, kind of answering my own question, suggests it's simply the Excel 2's chemistry doesn't play ball nicely with our compounds, while the ultracore 2.5 did.
The Ultracore 2.5 still only matched HPLC height of the peaks though, and the widths were slightly better but not to levels UPLC literature boasts about.
As another aside, 50mm columns have less theoretical plates, ergo the van deemter equaton the HETP will be lower, so more selectivity/resolution power, but surely less height, given the name of height equivalent of theoretical plates.
Have I misunderstood anything?
I sincerely apologise for not having pictures, I may be able to upload them later on. You'll have to take my descriptions for now, it's 3:35 am and don't have them to hand.
Overall I am happy with my method, I have fully resolved peaks and the method has been truncated from the usual HPLC half hour to 3.2 minutes. I just really expected these amazing super tall super thin 1-2 second wide peaks. My peaks at 0.6-1 minutes on the Excel are 0.1-0.15 mins wide, and the Ultracore at 0.6-1 minutes are around 0.05-0.1 minutes wide, so better than HPLC, but not by much.
Apologies for long post.
EDIT: I should point out this ISN'T a method transfer but a new method, it just so happens that 70% of the compounds of interest are in our original HPLC method so pretty much my first development phases were with similar columns but UPLC versions, and with most of the standards being the same compound.
I understand a method transfer would be a very easy set of equations simply taking into account column volume, I.D. and L/dp, and flowrate.
Also, I understand UPLC can run slower flowrates, and obviously, the longer the compound passes through the column, the more sensetivity, however, conversely, the slower the flowrate, the more time for B term (gradient/axial diffusion) to take place, so the peaks widen considerably, and the method ends up being 10 minutes long and the peaks become 0.5-0.6 minutes wide at the end, worse than HPLC. I use the water to slow down and separate the peaks, as our compounds are slightly hydrophobic - moderately hydrophobic. My method does have 20% more water than the HPLC method for better resolutions and separation. Could it be the water and hydrophobicity making the band broaden?
Also I need to use a 50mm column, or 75mm at worse. We're going fully UPLC and 10cm+ is HPLC. I have had advice of simply get a longer column but that really isn't an answer.
We have an HPLC method. It runs the usual Acetonitrile/methanol/acidified water (for our acid compound). At 0.002mg/ml standard injections, the heights are roughly at 0.02 AU, give or take, and obviously, they're thinner and taller at the beginning. The peaks were roughly 0.15-0.3 minutes wide at baseline. 10uL injection
This is using a 15cm C18 column (Ace).
So I needed to develop a UPLC method for it, and decided to use a 50mm Excel Super 2 C18. I made some 0.003mg/ml standards, but my peaks aren't as tall, and still quite wide, in fact almost identical widths to the HPLC chromatogram. Most of my peaks elute at 0.4 - 2 minutes, and another at 3.2 minutes (incredibly non polar so cannot get it closer without ruining resolution of other peaks). 1.5-2uL injections, ACN:Acidified water, no methanol.
They're not as tall either, around 0.01 AU ish tall, half of the 0.002mg/ml standards on HPLC. Methanol addition makes no difference.
I then tried an Ultracore 2.5 and the peaks got taller, roughly 0.025 AU, and much thinner, but still not the usual textbook UPLC really thin 1-2 second wide peaks thin/tall. 1.5uL injections
My question is thus;
Are UPLC peaks always taller than HPLC peaks, and thinner? Band broadening wise, the column should be fine (but will be getting a replacement one in case) and our system has been professionally installed, no dead volumes in the fastenings or anything, and all correct UPLC 0.12mm diameter tubing. Method runs fast at 0.9ml/min with 450bar pressure and only 45°C so not much time for axial diffusion to occur and broaden the band.
My logic is that our machine is a UHPLC, and can run HPLC methods. The detector detects the HPLC peaks identically to the HPLC machines, despite being a UHPLC. This to me means the detector is in no way "more powerful" than an HPLC detector? If it did the response would increase. I can run the same standard on an HPLC detector and a UHPLC detector and get the same response factor, peak height, and area (by same I mean within reason very similar to within 0.3% RSD easily)
So I have 0.002mg/ml with a 10uL injection for HPLC, that totals 0.00002mg going through the detector of each compound, which yields response A.
I also have 0.003mg/ml standards with a 2uL injection (sometimes 1.5) for UPLC which totals 0.000006mg total going through, which yields response B, and my heights are only a little off the HPLC's.
So relatively speaking, my UPLC peaks ARE taller, if I were to inject 2uL on the HPLC method the peaks would be tiny. But I was always under the impression UPLC peaks are always taller? Or is this salesmanship stating so and when practical comes into play, it isn't always textbook?
We have ruled out system malfunction band broadening, it is happening in the columns.
Usually the symptom is tailing, but could it be that the Excel Super 2 has a particular retentive capability on our compounds and thusly holds on and causes band broadening? We have a Excel 2 C18 (Not super) which from my knowledge just means it has less pH range and the peaks looked identical, so chances of having 2 columns with duff packing from 2 silica batches is...rare.
The Ultracore unfortunately does not give the required separation, but the peaks look much nicer, which, kind of answering my own question, suggests it's simply the Excel 2's chemistry doesn't play ball nicely with our compounds, while the ultracore 2.5 did.
The Ultracore 2.5 still only matched HPLC height of the peaks though, and the widths were slightly better but not to levels UPLC literature boasts about.
As another aside, 50mm columns have less theoretical plates, ergo the van deemter equaton the HETP will be lower, so more selectivity/resolution power, but surely less height, given the name of height equivalent of theoretical plates.
Have I misunderstood anything?
I sincerely apologise for not having pictures, I may be able to upload them later on. You'll have to take my descriptions for now, it's 3:35 am and don't have them to hand.
Overall I am happy with my method, I have fully resolved peaks and the method has been truncated from the usual HPLC half hour to 3.2 minutes. I just really expected these amazing super tall super thin 1-2 second wide peaks. My peaks at 0.6-1 minutes on the Excel are 0.1-0.15 mins wide, and the Ultracore at 0.6-1 minutes are around 0.05-0.1 minutes wide, so better than HPLC, but not by much.
Apologies for long post.
EDIT: I should point out this ISN'T a method transfer but a new method, it just so happens that 70% of the compounds of interest are in our original HPLC method so pretty much my first development phases were with similar columns but UPLC versions, and with most of the standards being the same compound.
I understand a method transfer would be a very easy set of equations simply taking into account column volume, I.D. and L/dp, and flowrate.
Also, I understand UPLC can run slower flowrates, and obviously, the longer the compound passes through the column, the more sensetivity, however, conversely, the slower the flowrate, the more time for B term (gradient/axial diffusion) to take place, so the peaks widen considerably, and the method ends up being 10 minutes long and the peaks become 0.5-0.6 minutes wide at the end, worse than HPLC. I use the water to slow down and separate the peaks, as our compounds are slightly hydrophobic - moderately hydrophobic. My method does have 20% more water than the HPLC method for better resolutions and separation. Could it be the water and hydrophobicity making the band broaden?
Also I need to use a 50mm column, or 75mm at worse. We're going fully UPLC and 10cm+ is HPLC. I have had advice of simply get a longer column but that really isn't an answer.
