analysis of glucose and fructose using RI detector

Basic questions from students; resources for projects and reports.

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i am currently doing my undergrad project glucose isomerization by glucose isomerase

sample = glucose + fructose in phosphate buffer (pH 6-8)
mobile phase = 75% ACN + 25% water
column = kromasil
flow rate = 1 mL/min
temperature = 40 C

for my standard curve, retention time for glucose is 5.2 while for fructose is 5.1 ( i dont know whether the retention time is logic or not)
for my samples, all graph only show 1 peak of retention time but have two curves (small one for fructose while the large one for glucose) where HPLC cant fully separate it (maybe because the retention time difference is too small or maybe effect from the phosphate buffer in my sample.)

i already tried using 60% ACN + 40% water, 100% water, and flow rate around 0.5-1 mL/min but the result still same with the previous one and really difficult for me to identify area of fructose and glucose. can anybody help me to find the solution?
OK, let's start from the beginning:

Were the separation conditions suggested by your advisor or professor? If they were, then you should approach him/her for advice.

If you came up with them on your own, then a bit more detail would be helpful:
- column dimensions and actual flow rate
- exactly what column ("Kromasil" is a brand name; they sell a wide range of columns, kind of like "Ford" does for autos)? The order of elution can be different depending on the column chemistry. Sucrose/glucose is not a difficult separation if you have the right column.

For now, with only a 2% difference in retention times for the standards, I'm surprised that you see two peaks. As a (grossly oversimplified) rule of thumb, you need about a 10% difference in retention to get baseline separation.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
thanks Tom for the reply. really appreciate it.
nope i came up with my own idea.
mobile phase = ACN 75% water 25%
F= 1mL/min
T= 40
column kromasil 100-5 NH2
dimension 25cm x 4.6mm
pore diameter 100 A
http://www.kromasil.com/publications_do ... d=PUBBwwfo


but below are the graph for operating conditions
mobile phase = water 75% ACN 25%
flowrate = 1ml/min
T = 40 C
inj volume 20 µL



Image
Image
Image
Image

ret time for glucose 5.49 and fructose 5.22 and for my sample ( glucose + fructose + phosphate buffer ) is 5.48. for the third image,my lab assistance who in-charge this HPLC told me that glucose and fructose did not fully separate it ( as we can see there are two curve ) and thats why the HPLC did not detect the fructose

like i said earlier i tried several operating conditions like ACN 75% water 25%, water 25%, water 100%, flow rate 0.5ml/min and 0.6 mL/min but i still got graph like the third image but in difference retention time. do you have any idea?
Bad news: your column is probably dead.

Your peaks are *extremely* wide, and both of the big peaks show a similar tailing and a shoulder on the back side. This may be due to too much "extra column volume" (too large/long tubing or poorly assembled fittings), but is more likely symptomatic of a void space at the head of the column. The latter can sometimes be fixed by opening up the column and repacking the void, but it's not a procedure for the faint of heart. You really need to get your prof or another student with some HPLC experience to help you out.

Here's a link to a short tutorial on peak shape problems that will give you a bit more background:
http://tinyurl.com/2a5sqa9

Since you will probably need a new column anyway, I would suggest that you take the opportunity to look for a "turnkey" solution. Contact HPLC column vendors and ask if they have an application note showing the conditions for separating sucrose and glucose. There's no point "reinventing the wheel".
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
More bad news:
The Amino columns when used with an eluent containing water, dies in about 3 months. I suffer that problem at my work.
I have read somewhere that water creates an acidic environement that dissolves silica and strippes off the amino groups. The result is that the column deactivates rapidly.
I used at the lab Inertsil NH2, 250 x 4 mm 5 microns, when new I obtain very good separation between Fructose and Glucose, later elutes Sacarose and near that Maltose and Lactose. ( I do not have the numbers but ask me for them if you need). Conditions are 75/25 ACN/H2O and flow 1.5 ml
As the column is ageing I should increase the ACN % in order to keep the separation, but when 80-82% ACN is reached the only way is changing the column, because tailing becomes unacceptable.
Now I'm trying to work with Carbopack columns (Dionex) where the eluent is NaOH 10 to 50 mM with electrochemical detection.
But (Murphy's Law) eluent is cheap but these columns are very expensive and so are the HPLC equipements
Waters have a very interesting-looking application note for a hilic method for oligosaccharides, not using an amino column (for reasons of stability). Someone here must have seen it.
tertianary amine columns in rings are generally doing a good job for sugars and are alot more stable then NH2 columns

they work in HILIC mode but need a higher concentration of ACN
sugar-D from nacalai is a good solution
we have used it well for sugars in wine applications
To unmgvar:
Separation of sugars in wines, liquors and foods is my deal. Can you e-mail me or at this forum a further description of the columns thatn you suggest?
For WillyOne -- IF you need to separate sucrose/maltose/lactose from one another that is most commonly done with the amino column. Fructose/galactose/glucose can be done on the amino or various resin columns including Ca++ and Pb++ form. Sucrose on H+ form columns will invert (to fru/glu) on column depending on acid concentration and column temperature. Maltose, on the other hand, does not break down to glu/glu.
For many food and beverage monitoring, glycerol methanol, ethanol might be important. Ion exchangers (made most famous by BioRad HPX-87H but many mfrs. and variations of cation exist) do the best job there and prove durable for thousands of injections when reasonable sample prep is done.
...and for Mr Glucose/Fructose
Amino columns do degrade in use, continuously, and this is one reason why we use RI detection rather than Evap.Light Scattering which experiences very high noise due to dissolved packing material as a background in the detector.
The very poor peak shape of your separation suggestion a column too old or too abused to pursue. That said, if you want to try and salvage it and make it last longer, lower the temperature to 25-30C and cut the water down (suggested by someone earlier, too) to 20 or even 15%. If you only look for Fructose and glucose 85% ACN would be my first choice, at the lower temperature. Best keep temp down on these columns to extend the life. I have some more than a few years old that still work okay. Not Kromasil but this is not a vendor specific problem -- it is a problem of all silica-based amino columns used in ACN/water conditions. Go to web sites of a few main vendors and search for "Carbohydrate Analysis Column". Many of us have specialized amino columns tested for this sugar work.
LCguy
WillyOne

sorry for not seing your request
the column is called sugar-D
http://www.nacalai.co.jp/cosmosil/data/AppSample.cfm?D=12&lc=JE
Good day everyone.
Read with great interest your disskus, since the sugars did not work much.
1) protection from degradation of the column can be precolumn.
2) Use a buffer to achieve stable values ​​of protonation aminofazy. This will improve the peak shape.
3) You do not share the glucose and fructose, and their peaks overlapped at 80% height. Therefore, for mixtures of the device issues a "cross" between them.
4) Generally speaking, the RI detectors require a greater flow - 3 ml per minute and above. Y series refractometers are very large internal volume of the cell compared with UV detectors.

PS) There is an old way of TLC chromatography on silica gel glucose impregnated with silver nitrate.

PPS - See book Chromatography fundamentals and appications of chromatographic and electroforetic method editad by E Heftmann Western RRC Elsevuer Scientific publish company 1986 part II table 7.1. ( HPLC sugars on m-bondapack)
The old horse did not spoil the furrow (russian prowerb , translate)
To Mr Mwoodman:
Many thanks per your advice.
I will try lowering the Column Oven temp, but this is Spain means we can have 30ºC at the evening when the air conditioner is Off, in the Lab. To get a stable temp I've worked at 35ºC. (the equippements works overnight).
About ionic columns, I don't have too much experience, but it seems that their capacity is really low (Dionex Carbohidrate columns). If I need analyze a sweet drink having 400 g/l total sugar content I should dilute 1:20000, and this a lot to have accurate numbers.
About ACN/H20 ratio, I like it, I will work over composition in the near future
To DSP007
The effect of precolumns over amino degradation is nothing. I thought as you but it didn't work.
Fructose and glucose can be easily separated in a new amino column.
To RI, I work quite well at flows between 1.0 and 1.5 /min but I think it should be very dependent of Manufacturers.
Apologize my english, is really bad
You will always have problem with amino columns and water/acetonitrile system without any acid/buffer in the mobile phase. Amino column creates high pH inside pores due to the presence of amino groups and it undergoes hydrolysis. You are better off with HILIC approach. Modern HILIC columns are more stable in my opinion than amino columns, although there are few amino columns on the market which have a better stability.

I have few methods for various sugars and can send you a file with our method (fructose, glucose, maltose, sucrose, and lactose).
here is another application for sugars. This is HILIC cation-exchange column operated in HILIC mode:
http://www.sielc.com/upload/file/pdf/Su ... column.pdf
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
www.helixchrom.com
Vlad Orlovsky wrote:
You will always have problem with amino columns and water/acetonitrile system without any acid/buffer in the mobile phase. Amino column creates high pH inside pores due to the presence of amino groups and it undergoes hydrolysis. You are better off with HILIC approach. Modern HILIC columns are more stable in my opinion than amino columns, although there are few amino columns on the market which have a better stability.

I have few methods for various sugars and can send you a file with our method (fructose, glucose, maltose, sucrose, and lactose).
here is another application for sugars. This is HILIC cation-exchange column operated in HILIC mode:
http://www.sielc.com/upload/file/pdf/Su ... column.pdf



I would be curious to see your method. Do you happen to have one which also includes galactose?
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