separation of carbohydrates by HPLC using RID detector

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please i need a help
hi, i am new to HPLC and i am working on the detection of sugars in honey mainly fructose , glucose and sucrose,
i am using a specialized column for carbohydrates from Agilent, with RID detector.
the HPLC is new and automated one from Agilent,
so this is the first trial of the column and detector.

i used to prepare standards as the following:
2 g fructose with 25 ml methanol up to 100 ml water
1.5 g glucose with 25 ml methanol up to 100 ml water
0.250 g sucrose with 25 ml methanol up to 100 ml water


but the peak of fructose used to continue under the base line before returning back, so i prepared the same standards without methanol and it seems that the problem is fixed but not knowing why?.

i prepared 2 honey samples as 5 g honey up to 100 ml water.
these numbers are as the method of bogdanov.
i used 80:20 ACN:water as mobile phase, with flow rate 0.9 ml/min.

the result i got is as the following:

1_when using the fructose standard i got a good peak at about 3.4 min
2_the glucose standard showed exactly the same peak as the fructose!!!!!!!!!! and another small peak at about 6.9 min ,
3_sucrose standard showed a perfect peak at about 9.1 min , but still the fructose peak appearing..
Is this a fructose peak????????
and why it is appearing in the 3 standards, and if not where is the fructose peak????????

the surprise is that the standard mixture showed fructose peak , glucose peak but sucrose peak did not appear at all?????????????
this vial i prepared by taking 0.5 ml from each standard by micro-pipit.

i run the honey samples in the same run sequence but they did not show glucose at all and this seems not logic some how , but they show fructose and its calculation was very logic(about 32 g\100g).

i am mixed from these results,
please help
what shall i try or do to get a good results.

[WillyOne]

I have analyzed these sugars without problems as follows:
eluent from 75/25 to 80/20 ACN/H2O depending of the age of the column (Amino 5 microns 250 x 4,6 mm, Inertsyl from GL Sciences)
Column oven 35ºC
Flow 1,5ml/min
Detector RI (Waters) termostated to 40ºC
Standars from 4 to 40 g/l. I used 4, 8, 10, 20 and 40 g/l They were prepared mixed all the three in water containing 5% v/v of Ethanol as conservant
Injection volume 5 microliters.
The only problem is the column life: 3 to 6 month

[LabDog]

If you are using an Agilent Zorbax Carbohydrate column a few things to remember.

One is that retention time on the column of a given sugar will increase as the concentration of the organic increases in your mobile phase. This is due to high solubility of the given sugars in water which decreases the retention on the column as they interact more with the mobile phase then the stationary phase.

I would make sure that the temperature of your RI matches your column. The newer ones are not as sensitive as the older models but it can still make a difference especially with close eluting compounds.

On these columns fructose and glucose elute very close to one another. If both peaks show an exaggerated response due to large sample size the peaks overlap.

I would also recommend that you dissolve your standards and if possible your analyte in your mobile phase when working with Refractive index detectors. Sugars are very soluble in water and you will find no problem dissolving fructose, sucrose, and glucose in 75:25 ACN:DI mixtures. This will help eliminate your void volume peak.

There have been a lot of conversations here about Carbohydrates its a good idea to scroll through them there are a lot of people on here smarter than me (ie everyone) that have posted some real gems to help you into HPLC.
The nice thing is we have all been where you have been, or worse. I am sure there are some machines that came very close to exiting buildings through windows.

It also helps if you list the settings of your machine ie (column temp, flow rate, detector temp, and column dimensions and model). This gives everyone a good starting point to help you.

[forward]

thank you both LabDog and WillyOne, i will try these tips and sure i will let you know the results.

[forward]

hi, i want tell you that i tried the following and the results were perfect:

first i prepared the samples and the standard solution with the same mobile phase i used(80:20 ACN:water)

then i thermostat-ed the temperature of the column and the detector at 35 degree Celsius
the flow rate was 1.4 ml\min
sure i was using Zorbax Carbohydrate column from agilent with RID detector

the results were great, but the only difference was the long retention time based on the first trials
again , thank you all for help
and wish this would help others too for a good separation of carbohydrates