Frequently Asked Questions
Which document(s) contains shelf-life or expiration date information for a given product?
If available for a given product, the recommended re-test date or the expiration date can be found on the Certificate of Analysis.
How do I get lot-specific information or a Certificate of Analysis?
The lot specific COA document can be found by entering the lot number above under the "Documents" section.
What is special about Ascentis Express?
Ascentis Express columns provide a breakthrough in HPLC performance. Based on Fused-Core particle technology, Ascentis Express provides the benefits of sub-2 μm particles but at much lower backpressure.These benefits include the capability of providing fast HPLC and higher resolution chromatography.The Fused-Core particle consists of a 1.7 μm solid core and a 0.5 μm porous shell.A major benefit of the Fused-Core particle is the small diffusion path (0.5 μm) compared to conventional fully porous particles. The shorter diffusion path reduces axial dispersion of solutes and minimizes peak broadening.
When are additional phases expected?
Additional phases are being developed.The best way to track new products is to visitAscentis Express
for the latest updates.
Can I use Ascentis Express on any type of HPLC system?
Ascentis Express HPLC columns are capable of use on standard HPLC systems as well as UHPLC systems.Columns are packed in high pressure hardware capable of withstanding the pressures used in UHPLC systems.
Can I use Ascentis Express on a UHPLC system?
Yes.Ascentis Express columns are packed in a way making them suitable for these ultra high pressure instruments.In fact, Ascentis Express outperforms sub-2 μm micron columns on many applications since Ascentis Express provides the benefits of sub-2 μm particles but at much lower back pressure.These benefits include the capability of providing fast HPLC and higher resolution chromatography.The Fused-Core particle consists of a 1.7 μm solid core and a 0.5 μm porous shell.A major benefit of the Fused-Core particle is the small diffusion path (0.5 μm) compared to conventional fully porous particles. The shorter diffusion path reduces axial dispersion of solutes and minimizes peak broadening.
How do I find price and availability?
There are several ways to find pricing and availability for our products. Once you log onto our website, you will find the price and availability displayed on the product detail page. You can contact any of our Customer Sales and Service offices to receive a quote. USA customers: 1-800-325-3010 or view local office numbers
What is the Department of Transportation shipping information for this product?
Transportation information can be found in Section 14 of the product's (M)SDS.To access the shipping information for this material, use the link on the product detail page for the product.
Is there anything special I need to do to my HPLC system to use Ascentis Express?
Nothing special is required to use Ascentis Express HPLC columns. To obtain the full benefits of Ascentis Express, one should minimize dispersion or instrument bandwidth in the HPLC system (tubing, detector flow cell) as well as confirm the detector response system is set at a fast level. For more information, request Guidelines for Optimizing Systems for Ascentis Express Columns (T407102)
How can I measure my instrument bandwidth (IBW) and determine whatAscentis® Express HPLC Columns can be used with minimal efficiency loss created by too much internal instrument volume?
The Guide to Dispersion Measurement has simple instructions on how to measure IBW and can be found at sigma-aldrich.com/express.
Do I need special fittings and tubing to connect Ascentis® Express HPLC Columns?
While operating pressures may not exceed the 400 bar (6,000 psi) capability of your traditional instruments, sustained pressures of about 200 bar (3,000 psi) will exceed the recommended pressure for conventional PEEK tubing and fittings at the column inlet. We recommend changing to stainless steel fittings in all high pressure locations and have designed special High Performance HPLC Fittings/Interconnects that will stay tight at pressures of 1,000 bar (15,000 psi) or greater, even when elevated column temperatures are employed.
What flow rate should I use with Ascentis® ExpressHPLC Columns?
Based on the minimum in the van Deemter curves, higher flows than 5um particle columns are required in order to maximize Ascentis Express column efficiency. The suggested starting point for flow rate for Ascentis Express columns: 1.6 mL/min for 4.6 mm ID;0.8 mL/min for 3.0 mm ID; and 0.4mL/min for 2.1 mm ID.
Can Ascentis® Express HPLC Columns be used for LC-MS?
Express Fused-Core™ particles were designed with LC-MS in mind. Even extremely short column lengths exhibit sufficient plate counts to show high resolving power. The flat van Deemter plots permit resolution to be maintained at very high flow rates to maximize sample throughput. All Ascentis stationary phases have been evaluated for MS compatibility during their development, and the Express phases are no exception. You can expect extremely low column bleed and background while maintaining longest possible column lifetime. A bonus of Ascentis Express columns for high throughput UHPLC and LC-MS is that they are extremely rugged and highly resistant to plugging, a very common failure mode for competitor columns.
Can peptide or protein samples be analyzed using HILIC columns?
Polar peptides are quite amenable to HILIC separations; however, our experience with larger peptides has been only minimally successful - mainly due to solubility issues. Proteins are even more difficult due to the same issue. An additional problem with proteins is that they are often multiply charged. When IEX is performed on multiply charged analytes, you often get what is referred to as a rolling effect where the analyte interacts with ionic sites on the surface in many different ways as it 'rolls' down the column; this produces broad and misshapen peaks.
Would you advise addition of a buffer when using diol or amide stationary phase?
Yes. if possible you should always have at least a small amount of buffer in a HILIC system to help mediate/control IEX and other polar interactions that are bound to be present (even if at a low level). It is not so much the 'buffering capacity" that is important, but the presence of the competing ions. We have found that their presence helps with day to day and column to column reproducibility. There are times when you need to eliminate the buffer, but aside from special circumstances, our recommendation is to include them.
How does the flow rate influence the water layer on the column?
We are not aware of any systematic studies with respect to the impact of flow rate on HILIC separations. Our concern would be that as you move to higher flow rates, you might observe peak shape issues due to the slow kinetics of IEX and adsorption mechanisms. If the retention mechanisms for a given system are partition dominated, this should be of less concern. It will be a case by case cause and effect.
Why is it recommended to run isocratically for HILIC methods?
When running in HILIC mode, both isocratic and gradient practices result in instability. If you keep the re-equilibration times constant, gradient should not be a problem, but changing this parameter can have a significant impact. It is not so much that it is bad as it is different than we are used to in reversed phase. Usually, we assume that once equilibrated (5, 10, 15 min, etc.), we can leave the system for any time period and come back to the same results. This does not appear to be the case in our studies of HILIC. Knowing that the re-equilibration time has an impact, you should get in the habit of making several injections with known re-equilibration times prior to making any development decisions. To get around this, isocratic runs are recommended.Attached are two posters; the first was presented at HPLC 2013 (Amsterdam) and the second was presented at Balaton Symposium on High Performance Separation Methods 2013 (Hungary). Both show 'reproducibility' at any set re-equilibration time is good but both show that if you change the re-equilibration time; then retention, peak shape and selectivity can change especially where ionic interactions are prevalent.
When you recommend only changing one parameter at a time, does this also refer to the total ionic strength?
If you change organic, try to keep the overall buffer concentration (and all other parameters, for that matter) constant. There are times when you will want to change both and perhaps pH/temp/etc. simultaneously, but that drastically complicates the system and thus should be avoided, if possible.
When referring to the pH of the mobile phase (pH 3, pH 4, etc.), does that refer to the aqueous part of the mobile phase?
Typically when we refer to pH in HILIC, we use the effective pH or the pH as measured after the addition of organic. The point is that we should always define what pH we are stating. The common way to distinguish is using notation of w/w pH or s/w pH (usually superscript/subscript). The notations mean superscript = solvent the pH is measured in (s would indicate some mixture of aqueous:organic) and the subscript = the solvent the pH meter is calibrated in (typically water (or w) as we readily have calibration standards).
What column do you recommend for an anionic compound?
If the acids are hydrophilic or you can adjust the pH to make them hydrophilic enough, any of the phases that exhibit HILIC partitioning are possible (bare silica, OH5, diol, Zwitterionic, amide). We typically go with the OH5 first to try and avoid any negative impacts on the like charge.
In HILIC separations, what happens if the sample is an aqueous matrix? Does it always have a negative effect?
Yes, it would be highly preferential (especially in this case where you want partitioning to dominate) to inject in high organic. That said, you can 'get away' with it if the injection volume can be kept small - much like we can inject low volumes of stronger solvents in RP mode, if needed. What you will want to do to minimize impact is to get as much retention on the analytes of interest as you can, this helps give the sample solvent some time to dissipate and negate the effects.
How should I store the Ascentis Express HILIC column?
Long-term storage of silica-based columns is best in 100% acetonitrile. Columns may be safely stored for short periods (up to 3 or 4 days) in most common mobile phases. However, when using buffers, it is best to remove the salts to protect both the column and the HPLC equipment by first flushing the column with the same mobile phase without the buffer (e.g., when using 90/10 ACN/buffer, flush the column with 90/10 ACN/H2O) to eliminate any concern about salt precipitation or corrosion from the salts then flush the column with 100% acetonitrile for storage.Before storing the column, the end-fittings should be tightly sealed with the endplugs that came with the column to prevent the packing from drying.
My question is not addressed here, how can I contact Technical Service for assistance?