Chromatography Forum

Yes, Carls is right. The vsicosity is aound 1.1 cp comparing to 100% ACN at 0.5 cp and 100% water at 1 cp (25C).

The v/v% viscosity maximum for ACN/H2O is ~24% ACN. What is the intial %ACN in your mobile phase?

So this is a HILIC method. What column are you using?

With HILIC you'll need to reduce the water and remove the salts in your sample. The sample solvent strength (% ACN) and inj volume tolerated depend on the column dimensions (50x4.6mm ID?).

Can you afford to dilute the sample 4:1 with ACN (i.e. to 80% ACN)? If so, filter the diluted sample. Hopefully you wont lose any analyte thru co-precipitation.

Also, you can start/end your gradient at 85% ACN instead of 95% to speed up re-equlibration and to reduce run time and precipitation problems.

The column is Agilent Luna NH2 100A, 5 um, 4.6x250mm.

I can not dilute the sample, since it was fomulated, I have to test it as it was. But I would like to try start from 80-85% ACN.

This is first time to hear HILIC, good to learn. Thanks

So this is a HILIC method. What column are you using?

With HILIC you'll need to reduce the water and remove the salts in your sample. The sample solvent strength (% ACN) and inj volume tolerated depend on the column dimensions (50x4.6mm ID?).

Can you afford to dilute the sample 4:1 with ACN (i.e. to 80% ACN)? If so, filter the diluted sample. Hopefully you wont lose any analyte thru co-precipitation.

Also, you can start/end your gradient at 85% ACN instead of 95% to speed up re-equlibration and to reduce run time and precipitation problems.

The column is Agilent Luna NH2 100A, 5 um, 4.6x250mm.

You're welcome! I'm happy to help a fellow chromatographer.

What is your flow rate? One column volume on a 250x4.6mm ID column is ~2.7mL. Typical minimum re-equilibration in HILIC is 10 column volumes or 27mL but this varies with the sorbent and the mobile phase. You may be able to get away with less but you'll have to try it and see.

At 1mL/min flow rate you'll likely need to allow at least 25min for re-equilibration at 85% ACN. Otherwise, the retention time of the second and all subsequent injections in an automated sequence will be shorter than the first injection. If using an autosampler, every injection after the first will have reproducible retention times but they will be shorter than the first injection.

You can reduce the re-equilibration time by ramping the flow rate during re-equilibration at 85% ACN.

Standard amino columns bleed like crazy during the initial runs, and this can be the cause of clogged outlet frits. To get around this problem, you should consider purchasing a preconditioned column such as the Waters Carbohydrate Analysis column.

Standard amino columns bleed like crazy during the initial runs, and this can be the cause of clogged outlet frits.

Dr. Neue, that's an interesting point to consider. Luna Amino is not standard amino propyl chemistry and does not share their bleed charateristics.

That did remind me of another potential issue.

Flora,

Have you flushed this column with IPA? New amino columns are shipped with normal phase solvent (hexane/IPA) which must be purged with 100% IPA (10 column volumes,CV) prior to use in HILIC mode. Please perform this purge/flush prior to doing any further work. Be sure to reduce the flow rate so as to not over pressuize the column. Inadequate purging will cause a host of different problems including unpredictable and poor chromatographic performance as well as very slow equilibration. After purging with IPA equilibrate the column with 20 CV of your initial mobile phase.

Carl
can you say what is the difference?
in the phenomenex web-site it states that it is a NH2 chemistry

If you get a very short RT at high % of Water it`s perhaps best to change from RP to HILIC mode, if possible.

From the website, I do not see anything special about the Luna NH2. My comment stands!!!

Luna Amino's proprietary bonding and manufacturing process provides enhanced stability and reduced bleed.

flora0975 - Any luck so far?

Carl, maybe instead of repeating meaningless marketing statements by the manufacturer who pays your salary, you could brush up on the technology difficulties with amino phases reading my book about "HPLC Columns". If you want to discuss this privately in detail (and maybe learn something), feel free to contact me directly.

It's easy to create durability data for NH2 phase - just pump water through through the column.

Imtakt compared durability of Unison UK-Amino vs. standard NH2 phases:
http://www.imtaktusa.com/site_media/fil ... TI304E.pdf

Dr. Neue,

Since the bonding chemistry is proprietary I, unfortunately, cannot elaborate any further.

Can you provide the details of the pre-conditioning process you imply is used with the Waters Carbohydrate Analysis Column?

Thank you for your offer to discuss amino chemistry as I do not propose to be an expert in this area. I have read your book and keep it handy for reference.

XXXX columns are packed with 3µm silica particles containing 100Å pores. A novel bonding strategy was adopted to improve chemical stability of the bonded phase. First the silica is reacted with a trimethylsilane endcapping reagent at a low stoichiometric ratio, before reacting residual and accessible silanol groups with trifunctional alkylaminosilane reagent. The resulting bonded phase provides a better safeguard against hydrolysis of the underlying silica.

Carls, other manufactuerers have also developed new NH2 phases and give some information about the bonding. Also your company is cooking only with water. Major customers expect more than just nice marketing flyers and brochures. Propriarity is not equal to Quality.
All columns on the market are good columns, but we want to see the differences to help end users to select the right column for their application.
Hope you have also the first book from Uwe on your desk. Check out the pages about HILIC columns. If not, I`m sure Uwe will copy and send you the 5 pages.
Some years ago only 5 HILIC columns were on the market, now about 150 different columns claim to be HILIC columns.
I would recommend to every user in the lab to ask for a free test column to check with his samples the right HILIC column.

is this HILIC?