Running 3 mm ID columns on Standard LC Systems

[Tom Waeghe]

In order to get the best results for the 2.1 x 50 mm, 1.8 um column AND if you intend to have a UV detector upstream of the MS, you'd need to use the 2 uL flow cell for the DAD or better yet, the 500 nL flow cell (with appropriate changes to the input and output tubing to allow faster flow rates). At 0.21 mL/min the RT for a k value of 1 on this column is only 0.99 min. A column that size would run much better on an Agilent capillary system with a micro well plate autosampler installed. The tubing from autosampler to column (75 um x 500 mm) and column to 500 nL flow cell (75 um x 400 mm) would add about 58 bar for 70:30 MeOH/water at 25C at 0.21 mL/min. The column itself would give about 290 bar so you'd be at about 350 bar on a 400-bar system.

On the other hand, your separation at 0.4 mL/min on the 4.6 x 150 mm, 5 um column would translate directly to a flow rate of 0.083 mL/min on the 2.1 x 50 mm column. If you raised that flow rate to 0.15 mL/min to get to higher linear velocity of 1.4 mm/sec, your backpressure would be more like 235 bar at 25C. With the minimized extracolumn volume and at that flow rate you ought to get much better results.

With your current system, you will need the 2 uL flow cell for DAD or the 1 uL micro flow cell for VWD along with the 0.12 x 180 mm capillaries from ALS to column and column to flow cell. Changing the needle seat capillary to 0.12 mm ID will help a little too.

If you can eliminate the connection of the flow cell to the union, and connect the tubing from the flow cell directly to the column outlet, you'll fare better. If solubility permits, dilute your sample with water or aqueous you're using by 1:1, 1:2 or 1:3 and inject the respective larger injection volumes to account for the dilution. That can also have a significant effect on plates. If you can make the tubing from the flow cell to the mass spec be 75-100 um ID PEEK fused silica (SGE, Upchurch/IDEX) that would also help narrow your peaks in the MS chromatogram.

I hope this helps!

Tom Waeghe

[Anthony_Ng]

Thanks Tom and Neue.

I think I cannot go futher with the instrument. It is beause I am working in an organic synthetic lab in a university and Professor wants to put more resources in chemicals.

LC-MS is just one of the instruments for checking molecules and it is not worth to upgrade the parts she thought.

Maybe I can ask for a 3.0x75mm, 3.5um column instead.

Anyway, thanks again for telling me lots of knowledge.

[Tom Waeghe]

Dear Anthony:

Based on experiments I've done and calculations I've done, you should be fine with a 3 x 100, 1.8 um column with a 5 uL flow cell. A 3 x 75 mm size column also should be still fine for you too. Moreover, the 5 uL semi-micro flow cell can be bypassed with a short flexible SS connector from Agilent, such as a 0.12 x 70 mm (G1316-87303 SS Tubing, 70mm, 0.12mm id 0.00 USD 63.43 USD). You simply disconnect the heat exchanger from the flow path on the flow cell and replace it with this capillary above. This makes the 5 uL flow cell behave like a 1 uL micro flow cell (expt'l results by me). Combined with shortest 0.12 mm tubing between ALS and column and column to flow cell as I described above (don't use the built-in 3 uL heat exchanger if you use a TCC column compartment!), you should be able to get 12350 plates for peak at k = 0.1 (acetone, RT 2.3 min at 0.2 mL/min) and 21000 plates at k = 1.5 (RT 5.3 min at 0.2 mL/min). [NOTE: Those are the k values I obtained for acetone and xylene on your 4.6 x 150 mm, 5 um column at 0.4 mL/min.]

I'd use the fastest data rate you can and also compare it with the 2nd fastest data rate in terms of signal/noise ratio (Performance report style with calibration table). Of course, you'd be better off if you could drop the %MeOH to 65:35 or even 60:40 to get your peaks to be better retained. A k value of 0.1 is quite dangerous as everything in the world can elute at the void of a column depending on the mobile phase strength.

If you'd like to see the calculations/calculator for this, I'd be happy to send it to you.

Tom
email: twaeghe@mac-mod.com

[Anthony_Ng]

Hi Tom,

I found that G1316-87303 SS Tubing cannot connect to the flow cell's inlet directly because it has different fittings size.

What I found from Agilent's catalog is G1315-87325 (0.12mm ID, 290mm long) or use an outlet capillary instead G1315-87306 (0.12mm ID, 200mm long).

Are there any better choice?

Thanks

[Consumer Products Guy]

There are lots of companies that supply SS tubing of different lengths and inner diameters, just figure out what you need. PEEK tubing which can be readily cut is also available. We oftentimes put fingertight (universal) ferrules on Agilent SS tubing, one end is usually not swaged when received.

[Bryan Evans]

Where I work, no one wanted to be tied to a single manufacturer for all their columns, so we never bought UPLC. Curiously, if Waters had engaged with other column manufacturers to ensure alternative supplies of sub 3u particle columns, we might well have bought their instrument.

UPLC is here to stay and I anticipate the whole industry to shift eventually into it. Several manufacturers already offer such instruments and consumables (columns) and I would anticipate more of them to come up with relevant products this year...

Hi Kostas -

I (respectfully) disagree. Both U-HPLC and HPLC will have their place in the future.

Here is how I see things:

a) U-HPLC pressures will be used for (relatively) few niche applications.
b) HPLC pressures (< 25 MPa) will continue to be the standard for liquid
separations. This is because HPLC is a). more practical and b). more versatile

U-HPLC pressure applications may continue to "cannibalize" HPLC pressure
applications. But advances in the separation of solutes (in the liqid phase) will
continue to occur in the HPLC pressure region.

[Kostas Petritis]

Bryan,

I spoke more in terms of instrumentation than HPLC columns/consumables. UPLC hardware can be used to run lower pressure applications. At some point it will make more economic sense to maintain one (more versatile) line and that will probably be UPLC.

When I was in PITTCON I asked a couple of HPLC column manufacturers why they do not offer smaller particles and they told me that they haven't received enough requests to make it worthwhile for them... in terms of feasibility they do not anticipate any problems...

But we will see... some years ago, at the same forum, we were discussing if anybody will follow the trend that Waters started... now we are at the point where we say that UPLC will continue to cannibilize HPLC applications... let's see where we will be 5 years from now...

[Bryan Evans]

Kostas -

Thank you for your response.

Yes, we are seeing more people use Imtakt's 3um column on U-HPLC
systems because the sub 2um column did not work for their application.

I agree - it's an exciting time for HPLC!