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.
53972-U
Ascentis® Express 90 Å HILIC (2.7 μm) HPLC Columns
L × I.D. 15 cm × 3 mm, HPLC Column
Synonym(s):
Core-shell (SPP) Fused Core Si HPLC column
About This Item
Recommended Products
Product Name
Ascentis® Express HILIC, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 15 cm × 3 mm
material
stainless steel column
Quality Level
Agency
suitable for USP L3
product line
Ascentis®
feature
endcapped: no
manufacturer/tradename
Ascentis®
packaging
1 ea of
parameter
≤100 °C temp. range
600 bar max. pressure (9000 psi)
technique(s)
HPLC: suitable
LC/MS: suitable
UHPLC-MS: suitable
UHPLC: suitable
L × I.D.
15 cm × 3 mm
surface area
135 m2/g
impurities
<5 ppm metals
matrix
Fused-Core particle platform
superficially porous particle
matrix active group
silica phase
particle size
2.7 μm
pore size
90 Å
operating pH
1-8
application(s)
food and beverages
separation technique
hydrophilic interaction (HILIC)
normal phase
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General description
Visit the Ascentis Express home page for more information on this new column technology.
Legal Information
guard cartridge
related product
required but not provided
Storage Class Code
11 - Combustible Solids
WGK
WGK 3
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
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Articles
For separation of polar compounds including polar neutrals, polar acids, and polar and non-polar basic amines use our Ascentis® Express HILIC column.
For separation of polar compounds including polar neutrals, polar acids, and polar and non-polar basic amines use our Ascentis® Express HILIC column.
For separation of polar compounds including polar neutrals, polar acids, and polar and non-polar basic amines use our Ascentis® Express HILIC column.
For separation of polar compounds including polar neutrals, polar acids, and polar and non-polar basic amines use our Ascentis® Express HILIC column.
Protocols
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Why is it recommended to run isocratically for HILIC methods?
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Do I need special fittings and tubing to connect Ascentis® Express HPLC Columns?
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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.
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What is the Department of Transportation shipping information for this product?
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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.
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In HILIC separations, what happens if the sample is an aqueous matrix? Does it always have a negative effect?
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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.
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How does the flow rate influence the water layer on the column?
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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.
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Can I use Ascentis Express on any type of HPLC system?
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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.
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How can I measure my instrument bandwidth (IBW) and determine what Ascentis® Express HPLC Columns can be used with minimal efficiency loss created by too much internal instrument volume?
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The Guide to Dispersion Measurement has simple instructions on how to measure IBW and can be found at sigma-aldrich.com/express.
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Can Ascentis® Express HPLC Columns be used for LC-MS?
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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.
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Is there anything special I need to do to my HPLC system to use Ascentis Express?
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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)
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When you recommend only changing one parameter at a time, does this also refer to the total ionic strength?
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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.
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