Chromatography Forum

Hello,

I'm having a bit of a problem with a separation I've developed. I have developed a gradient, using the scouting method from Tom Jupille, to separate the following compounds:

• maleic acid
• phthalic acid
• o-toluic acid
• benzoic acid
• citraconic anhydride
• phthalide

I was able to develop a good baseline separation for the compounds, but I've also developed a method with a very poor baseline. Here is the gradient:

A = 15mM KH2PO4, adj to pH 2.45 w/ phosphoric acid
B = ACN
flow rate = 2.0 mL/min

Time %A %B
0 100 0
1 100 0
9 65 35
12 65 35

Post time: 5 min

Column: Waters XBridge 4x150mm C18 3.5u @ 30°C
Detection: UV @ 210nm

and here is a 25uL injection of water on an Agilent 1100:



I replaced the eluents, containers and inlet frits for both eluents. Since I was using a previously used guard column, I removed it to see if that was the problem. Same results.

I've read here and there in this forum about problems with acidic phosphate buffers and acetonitrile. Should I try a gradient with methanol instead?

PS - Pardon my poor gradient table formatting. I'm not sure how to make a nice orderly table.

We do gradients like this also for organic acids. You probably need to condition the column with 60:40 buffer:MeCN until the baseline stabilizes. Then be very suspicious of your water quality. Have you tried an alternate source of water?

I haven't tried an alternate source for water, but when I obtained a full-range UV spectrum for the prepared buffer, the spectrum looked clean.

The trouble is that junk in the aqueous mobile phase will accumulate on the column and elute later in a more concentrated lump. Whenever you work at 210 nm, the requirements for clean solvents and reagents become more rigorous. Even a dirty pH electrode can sabotage you.

randy, I'd also say that it looks like garbage building up on the column. The standard diagnostic is to run a set of three "dummy" (no injection) gradients with different pre-gradient equilibration times:
1. any equilibration time (this is just to clean junk off the column)
2. your regular equilibration time
3. longer than your regular (2x or 3x)

Compare the traces from runs 2 and 3. If the problem is coming from contamination in the A reservoir, the long-equilibration run will have significantly larger peaks, as in this example:


John Dolan wrote this up a few years ago in one of his LC Troubleshooting columns: LC-GC 16(11) 992 (1998)

OK, I performed the suggested experiment, and it appears that the contamination is coming from the A (buffer) reservoir. So in order to determine if the contamination was indeed coming from the buffer, I decided to perform the same experiment with the same gradient program using just water from a separate reservoir. I got similar results!

The contamination peaks don't match up completely, but a majority of the stuff is in the water also. It looks as though I need to try a different source of water. We don't have an in-house filtration system, so we have to order our water. Up until now, the water has been satisfactory for our IC and LC work.

I am also going to obtain a 190-400nm UV spectrum for each compound to determine if I can increase the detector wavelength and maybe minimize the problem.

Comments?

The quality of water is the largest source of analytical error while your buffer introduces additional variability- My suggestion for you is:

1. Buy commercially available water, B& J-
2. Use highest HPLC grade ACN from Fishers
3. If (1) and (2) are not sufficient, you can filter your mobile phase A offline using 3 M Empore carbon Disks and use the filtered mobile phase.

Minor change to ^ - Use Empore extraction disks to filter your water, then prepare your A-phase. Having anything else in the water seems to help get the stuff you don't want to pass through the Extraction disk, which defeats the purpose of using it - it is most effective if used on pure water.

Also - I'd try the Empore disks first, especially if you have more time than $ (B&J, Optima et al brands of water get expensive and frequently don't solve the problem at <215nm).

LC grade Phosphates are usually pretty clean, but it wouldn't hurt anything to filter your A phase through a nylon filter after dissolving the PO4.

Also - isn't a pH <3 a bit low for PO4 to have any buffering capacity?

Not according to this http://www.mac-mod.com/tr/pbprep-tr.html

Also, I tried the gradient at 240nm and am still getting garbage out around 7-10 min.

Is there a way to start a gradient on an Agilent 1100 controlled by Chemstation without making an injection? I haven't yet found a way. I had to make injections when performing the suggested experiment above, so i guess I can't eliminate the injector as a contamination source.

Just inject 0 μL

Best Regards

I found another way as well. In the Sample Info option in the Run Control menu, leave the Sample Location blank and then click Run Method.

Well, I've been busy in the lab but finally found the solution to the problem a few weeks ago. Turns out the problem was in the water. Once I used HPLC-grade water the junk peaks were drastically reduced and in most cases eliminated completely. Luckily the few remaining peaks did not interfere with my target analytes.

Glad you got the problem solved, and thanks for letting everyone know the outcome.