Chromatography Forum

Good evening, collegues!
I have a problem with separation of sugars (sweeteners).
Task: Identification of sugar in juice. Everything would have been understandable if in the laboratory installed a Agilent HPLC 1200 with simple configuration ,where is thermostate of column (TCC) can be heated until 80 degrees. But in my case, only Agilent HPLC 1120 Compact with limit for TCC - 60 degrees. It's a problem because starts anomer separation. And only two way for decrease separation of anomer: high temp and analysis under strong alkaline conditions.
I ask your advice on solving this problem, can anyone met with this?

Column - PL Hi-Plex Ca USP (250x4,0)
Detector-RID.

Thank you very much!
With best regards
Dmitry

you could also use a column like a 2nd or 3rd based amine like sugar-d or polar imidazole
the application work between 30-40 degrees using 70-85% ACn + water

generally done within 20 minutes for 6-7 compounds

you could also use a column like a 2nd or 3rd based amine like sugar-d or polar imidazole
the application work between 30-40 degrees using 70-85% ACn + water

generally done within 20 minutes for 6-7 compounds

Thank you, unmgvar!
This is very important and useful for me.

But now user have only current column and will wait new column is very long time. I will change flow rate and some percent of nonpolar solvent in eluent (ACN). Temperature will be - 55-60 degrees. May be in such a way I can find a solution.
I will be happy with your advice.
Good day!

With best regards
Dmitry

can you list your compounds?

can you list your compounds?

Thank you, unmgvar!

This is list of compound, which i want to determine in matrix (Juice): sucrose, glucose, fructose.

When i worked with similar HPLC 1200 Agilent, this is task was a simple. All compounds separated. But with "compact" i can see only standards separately in each analysis (high concentration). Yesterday, i tried to change flow rate, and added some drops of ACN in eluent. All my experiments was failed. I saw only one big split peak. Temperature of analysis was 58 degrees. (limit - 60).

It will be very interesting to learn out of this situation. Of course, I understand that the best thing that's another column, for example: Nucleosil NH2 column (250x4.6 mm) or Phenomenex AsahiPak 5 µm NH2P-50 Column. I understand correctly? But if you have different solutions, i will be very glad to listen them.

p.s. I am still very young in the development of methods of research, so any ideas would be helpful to me.

With best regards
Dmitry

What about buying an external column heater? I found one on eBay that will get to 100 C for $217 USD (current bid) plus shipping.

http://www.ebay.com/itm/Column-heater-m ... 35bbd52276

What about buying an external column heater? I found one on eBay that will get to 100 C for $217 USD (current bid) plus shipping.

http://www.ebay.com/itm/Column-heater-m ... 35bbd52276

Thank you! It's very interesting idea.

With best regards
Dmitry

the NH columns are a possibility, but actually they have not made it to be the standards for use
2 main reasons
1. the column production stability sucks for all vendors. it is very hard to validate and to get over time the same results for different batches
2. the samples are bad. they can kill the NH columns very fast. again the chemistry is very easily killed.

so the IEX columns with Ca or another type are is what is used the most often.
now that we know of the more stable 2nd and 3rd amine chemistries we have started using them and it is a lot simpler to get separations. and also faster
no need for 80 degrees. you can use more than one solvent composition. and if you have an ELSD or CAD you can use gradients and make real separation for sugars

the NH columns are a possibility, but actually they have not made it to be the standards for use
2 main reasons
1. the column production stability sucks for all vendors. it is very hard to validate and to get over time the same results for different batches
2. the samples are bad. they can kill the NH columns very fast. again the chemistry is very easily killed.

so the IEX columns with Ca or another type are is what is used the most often.
now that we know of the more stable 2nd and 3rd amine chemistries we have started using them and it is a lot simpler to get separations. and also faster
no need for 80 degrees. you can use more than one solvent composition. and if you have an ELSD or CAD you can use gradients and make real separation for sugars

Hi and thank you unmgvar!
I looked this type of column in Internet and my results, column which name: "HPLC Column for Saccharide Analysis COSMOSIL Sugar-D". do you agree with me? Are you also used the same column?
How i understood, this column have a stationary phase of secondary and tertiary Amine. It's Good, i think! Method will have a 30 degrees in TCC and all my modules pass for analysis.

Thank you for your time!
With best regards
Dmitry

dRima,
yes i know both the Sugar-D Cosmosil and the polar-imidazole from Sepax
both are good, in many cases they give the same results.
as for everything in life, for a certain case one is better that the other,
for your case of 3 sugars, both will be fine and easy to implement and use
70-75% Acn and the rest water.


one point is important for the use of those columns
as you dilute your samples, for the last step, use the mobile phase composition
right now you probably dissolve into water and that's it.
here you need to see that your mobile phase is mostly organic and the sample is mostly in water right now.

Good,
Of course, unmgvar, if in the eluent i have percent an organic solvent or the eluent compose from the different solvent, i must dilute substance for calibration in this composition of solvent (eluent). If not, i will see tailing peak, in many case. But, now, you are right, i was eluted with water and used to dilute the water.

Thank you unmgvar, that you help me solves this problem and task. It seems that very simple task, but real when has limit in the system, it's very interesting. I'll follow your tips!

With best regards!
Dmitry