You have to use ELSD detector or RI detector with amino column for sugars analysis.
No it is not really possible. RI detector and amino column is best. Maybe use Asahipak NH2 phase. I think this is a polymeric NH2 which has a slightly longer lifetime than conventional silica based NH2 columns.
Use aminopropyl column in HILIC mode (I use Thermo Biobasic AX). You can use ESI-MS/MS. You can also get selective conformation information that way. If you infuse an in-line reactor you can use derivatize the sugars and use DAD as well.
I have used RI detector and Rezex RCM-Monosaccharide C2+ (8%) (300x7.8 mm) column. I was able to analyze almost all type of sugars, however, polymeric NH2 column will work fine. Since I do not have ELSD detector, therefore, I did not try but it will work.
C-18 column and dad are not proper for detection of glucose. An amino column with ri detector or elsd are appropriate,
C18 columns and DAD detection are for sure the wrong choice.
A quick view at a column supplier catalog will show to use an NH2 column and RI detection.
I think ESI-MS/MS is a little bit 'oversized'.
Mode of sugar analysis depends on sample character and number of samples. Good choice is Aminex carbohydrate column (Pb) or equivalent column and water as mobile phase (temperature required is 80-85 °C). ELSD, RI are good detectors, bud in some cases UV (DAD) at 192-195 nm is possible to use (depend on glucose concentration).
Yes, you can. But it can't directory. You must need derivatives (e.g. 2-amnopyridine).
HPLC separations of carbohydrates
depend on differences in conformation, configuration, and bonding
mode, and are more difficult than analyses of other classes of compounds.
No single HPLC column or method is capable of separating all
carbohydrates. For this reason RI detector is the best though has limitations.The major advantage of RI detection is the universal response over a
fairly wide linear range of analyte concentration
Glucose is very short absorbs in the UV range for this method of detection.
You should use the RI detector or the ELSD with the Agilent Hi-Plex Na column. I personally prefer the RI detector though some say that it is not as sensitive as the ELSD. If your sugar is not too little, RI is a good go.
I think UV detection of sugar at 192...195 is not the best choice as trace oxygen levels in the mobile phase may produce serious interferences.
I completely agree with M. Kitagawa.
As explained elsewhere in an analytical forum, GC/MS is from my point of view the method of choice in order to detect C5, C6 and C12 sugars at very high sensitivity in complex matrices.
I agree with Peter Behrend , I work with sugar using subcritical water mobile phase. I used UV detector at 200 but it was not good...I needed to change for RI detector...
Differential refractive index detector or ELSD, can be used i have never tried it but it will work.
As said by precedent colleagues, two things
- separation: wich column and mobile phase ?
and which sort of technic (liquid of gas couple with MS) ?
- detection: which material and which wavelenght
As sugars are more " hydrophilics" than "lipophylic", using C18 obliges to "invert" the system; better to use a "direct" system with NH2 and not C18; but also depending on the size of sugar : more it's long less hydrophilic.
And so on ... so, perhaps different possibilities but you have to choose according to your possibility of material; and also the costs are different.
Regards
Didier J
Yes, it is possible. It is not prefered method, but at least you can do something.
First things to remember - you can't separate different sugars with C18 columns, because stationary phase is not chiral (ok, there are exceptions, like pentoses and hexoses which are not enantiomers, but completely different compounds).
If you don't need separation, you can still run into problems. Glucose doesn't have aromatic parts, so absorbtion is pretty low -> high limit of detection. And you have to mesaure it at wavelenghts close to 200 nm, which is very nonspecific. Combine that with previously mentioned high LOD, and you'll find out why everybody is suggesting you RI instead of UV-VIS.
But you can derivate glucose. With derivatisation, you avoid most of the previosly mentioned problems. Usually, the derivatisation is done by some amine.
There is quite good paper on this topic:
Fotini N Lamari, Reinhard Kuhn, Nikos K Karamanos, Derivatization of carbohydrates for chromatographic, electrophoretic and mass spectrometric structure analysis, Journal of Chromatography B, Volume 793, Issue 1, 5 August 2003, Pages 15-36, ISSN 1570-0232, 10.1016/S1570-0232(03)00362-3.
(http://www.sciencedirect.com/science/article/pii/S1570023203003623)
The only to use LC/DAD for this purpose is to derivatize glucose to make it UV active. But DAD is not a good choice, RI can be used. Better choice is to use GC-MS using the TMS derivative of glucose in standard and sample.
PAD detector also possible, UV is not the best choice. I normally use APCI-MS (triple Q)
I would use HILIC (with an HILIC NH2 column) to get some retention. Glucose will not be retained by C18 (unless you have a verry long column). NH2 columns work best.
as a conclusion of the above comments, your question
"Is it possible to quantify glucose using HPLC with a C18 column and DAD detector? "
can be answered "No, you will waste your time".
I still recommend HN2-HPLC and IR detection.
However, if you have access to capillary GC equipment, switch to GC or eve GC/MS.
The eralier the better....
As othters said amino column and Refractive index detector is the best option available, abudantly used and a standard method. The best response of the query "Is it possible to quantify glucose using HPLC with a C18 column and DAD detector? " is given by Peter Behrend ·
"No, you will waste your time".
Either the person who raised this question knows nothing about the separation science or he is just kidding here.
You should use a HILIC method. Reverse phase analysis is not suitable for analysis of hydrophilic compound such as sugars. Best.A
In our lab we perform sugar profile testing which utilizes C18 column and RI detector.
You can quantify glucose with DAD using at least two alternatives. The problem is glucose does not absorb UV-Vis radiation, however it ccan be conveniently derivatized to compound able to absorb in this region.
Other alternative is the use of indirect photometric detection, thus a dye capable to absorb in the UV or mainly Vis region is incorporated in a low concentration to the mobile phase, whenthe analyte (glucose) reach the detector a decrease in the absorbace is produced as a concequence of the presence of the analyte. The decrease in the absorbance value is proportional to the non absrobing analyte concentration.
I agree with Andre. Use Thermo GOLD NH2 MP: 75:25 (ACN:H2O), GOLD version is more stable than regular. Ref to Acarbose USP testing.
As mentioned above glucose can be quantified using an Amino (NH2) column and RI detection, the point to note with RI detectors is that they have to be used isocratically. If you have to use a gradient due to the composition of the sample, the a light scattering detector will work.
with DAD, free solution capillary electrophoresis is the best to me experience (see paper from P. Gareil's group last year in Analytical Chemistry).
Depending upon seperation technique and instrument used in the lab; the detection varies., Mostly C18 column cannot be used, instead try some other short columns.,
usually sugar compounds can be detected by RI detector and by using amino bonded column (NH2).
You probably will not separate Glc from other hexoses on C18, look at the Dionex system using high pH and a PAD detector
It would be good if you give us an idea on what are the other components beside glucose in your solution. But, in general there are two parts in this to focus on:
1) Will the c18 column septate the glucose from the components? Answer is NO, this is a silica column it will not be able to capture polysaccharides and retain them subsequently. You will need a gel column (preferably Amide based). To decide which column will work best for you, you need to read the specification sheet of the column to make sure it can separate all the contents of your solution, a couple of trail runs will be good as well if you already have the column.
2) Will the DAD detector detect glucose? The answer is no, glucose dose not absorb near UV-Visible radiation without prior treatment. Your best hit would be a refractive index, or if DAD is the only available detector you might want to try a colouring dye like Dinitrosalicylic acid to allow glucose detection, I am not sure though if this would affect the other components in your solution.
A side hint: Is HPLC the only option you have? You can quantify reducing sugars using any UV spectroscopy commonly available in labs. The method is called DNS Method, a quick google search will give you lots on information on it.
Good luck!
yes, you can do it but you need to derivatize your sample and standard before analysis. As glucose do not have any chromophore or auxophore.
Dear Willy,
it is possible , however it is quite difficult to do and possibly offer questionable results.I would agree with Wahib, please do also refer on manuals such as Demain and Davies,1999 or Ganestos and Baker, as well as look on the relevant literature in scientif journals such as Journal of Chromatography a by Elsevier.
×
Rozilawati mohamad achil：usually sugar compounds can be detected by RI detector and by using amino bonded column (NH2). Good answer
I think u can use an amide bonded column for this purpose. Secondly If you want to use the same column, then u will have to derivatise glucose bcoz it does not possess a chromophoric group. Better will be to go with an amide linked column.
HPLC retention: The C18 coluimn you have is very poor at retaining sugars and will not enable resolution of multiple sugars at all. If you wish to achieve separation between sugars then an amino or a HILIC column will be neccessary. It is possible (eg Carbopac) to purchase specific columns designed for carbohydrates also.
Detection: You will have poor sensitivity even at 200nm with a UV detector, as well as contending with a reising baseline if you are running gradient conditions. Refrective index is useful foe purely isoctatic conditions only. The best choice is an amperometric detector -eg pulsed amperometric detection - for the best sensitivity. If absolute sensitivity is not an issue then use of ELSD is an alternative, but you must optimise the heat/gas flow levels to achieve good reproducibility and sensitivity.
Alternatively if you derivatise the sugars you can use the DAD.
If you want to quantify the glucose, DNS will not be feasible because it quantifies the total reducing sugar. I would suggest you go for the GOPOD test. This will be specific to glucose.
Glc can also be detected by ELSD (Evaporative light scattering detector).
If you want to derivatize Glc, you have to pay attention to the derivatization reaction which must be quantitative to accurately quantify the sugar.
As noticed by Yong, the DNS method is not specific to Glc, so make sure that there is no other reducing sugar in your medium, or try to make a blank test to be sure that nothing else that Glc could give a positive response.
surely not, i myself use ELSD to detect sugars and their derivatives
Friends you can call me Joseph.. Yong in itself is not a name, but Yong Kuen put together will be my first name where as Ho is my surname! Chinese culture..
Yes, but with high purity of solvent such as ACN and Water at lower level wavelength about 195 nm or Ion paring reagent at higher wavelength using DAD or UV and L1 column (C18) with or without derivatisation.
The sugar can be analyzed by GC-MS. We have to prepared derivative of sugar by using derivatization agent. The prepared sample can be injected in GC-MS to identify compound present in it with the help of NIST & NBS library. EI source use for ionization.
Why does it take more than 50 comments to answer the simple question:
How to analyse sugars with RP-18 and UV-Detection.
The answer will be the same - NO.
And as Mr. Yee mentioned, he has no RI detector - what are all the hints for HPLC with RI detection good for ???
Glucose does not have a useful UV spectrum. You need derivatization or to change your detector. Use CAD, RI. or MS.
It seems hard to realize, that any suggestion about the analysis using HPLC / UHPLC with a DAD detector is is not accepted .
Glucose may be analyzed by HPLC with Ion Exchange columns or better via GC (even with a low-price detector after HMDS/TFA-Derivatisation.)
Please, is it possible to focus again to important questions form other users?.
If you are interested, I can send yu a copy for the analysis of glcose via HPLC/ RI oder GC, that really works.
Best of regards,
Peter Behrend
You can use LC-DAD detection but NOT using a C18 column - there is NO resolution with a C!8 and your peak will be buried in all the noise at the start of a run! You need to use an ion exchange column to get some retention then hopefully by looking at 190-200nm you might be lucky and get a very small peak on a rising background. Stop this topic it is dragging on with no end in sight!
Yes you can detect sugars by UV or PDA with pre or post chromatographic derivatization. But you need amino, amide or Hilic column for the same. As sugar molecules have no interaction with C18 stationary phase column.
For sugar analysis Ion chromatography is the best technique. As it is dedicated on sugar analysis only.
again my suggestion, reading the question more carefully hay help to reduce meaningful answers to about one dozen instead of 63 answers, a lot of them not considering the question:
Is it possible to quantify glucose using HPLC with a C18 column and DAD detector?
Simply NO !
Useful alternatives as RI detection ion exchange / Hilic columns of GC with silylation.
This was explained several time - so, why all those answers with nearly the same content?
I want to ask you all, if I use GC , is it possible the glucose can covert to any compound, I mean Glucose derivative such as HMF or else. because GC need high tempertaure for analyze. Thank You
As mentioned earlier, silylation with eg. 500µl STOX / 450µl HMDS / 50µl TFA
works fine without any problem for three decades and has been the the method of choice in routine and reseach project for C5 / C6 / C12 sugars.
I never observed problems due to typical GC temperatures for GC/FID and GC/MS.
Peter Behrend, Can you give the paper which using GC for Glucose analysis to me? [email protected]. Thank you
You will need to use ACN as organic solvent and your detection will lack sentitivity. There are methods far more suitable to achieve your goal
I agree with Peter Behrend. I just want to add, that there is also the possibility to work with radiactive labeling, PAD (pulsed amperometric detection) or with fluorescent labelling (e.g. 2-AB).
Without any labelling, sugar detection is not possible, because all sugars have a UV maxima at around 195 - 230 nm.
Good luck.
Yes you can detect sugars by UV or PDA with pre or post chromatographic derivatization. But you need amino, amide or Hilic column for the same. As sugar molecules have no interaction with C18 stationary phase column.
For sugar analysis Ion chromatography is the best technique. As it is dedicated on sugar analysis only.
You can detect glucose by HPLC using:
1) UV or PDA with pre or post chromatographic derivatization
or
2) Refractive index detector
3) Use HILIC or aminde column
Hi Hassan,
in nearly every Industry lab, R&D lab as well as in universities there are GC instruments available, performing this task with ease.
You simply repeated Sanjay's hints - not an ultimative help, I think
Peter
Sugar can be analyzed by GC-MS. Sample preparation is needed for it. You have to derivatize sugar with derivative zing agent. That is injected in GC-MS.
For many years I routinely analysed glucose concentrations using a Waters pulsed amperometric detector (PAD) with no real problems, but with a special ion chromatography column - C18 has no real retention on sugars. Dionex (Thermo now) are more conversant with this as they deal with ion chromatography. You can use RI but the sensitivity is not very good and the detector really needs to be stable for a good baseline. Also it is not possible to use a gradient with RI - the detection window closes very rapidly. HILIC is a very versatile way to retain sugars provided they can sustain the high ACN concentration in most mobile phases.
Dear Doctor Peter Behrend, I am a Research Scholar and I am also struggling with the unavailability of RI detector for monosaccharide analysis. I read your suggestion about GC for sugar analysis. Can you kindly give an idea of the procedure or some publication related to it ?
Dear Batul Diwan
One of my published paper regarding quantification of glucose using GC-MS is attached herewith.
Dear Batul,
there are several methods for analysing sugars from the ppb up to the percent range.
However, most of them produce several peaks by silylation.
In order to avoid these problems, me and my team used a quite simple method since the early 80th for research and routin analysis by GC/FID ans since the middle 80th by GC/MS.
Hydroxylamine hydrochloride is used for preparing oximes of sugars prior to silylation. It is a pyridine solution containing 25 mg/ml hydroxylamine
hydrochloride .
Sugars containing multiple hydroxyls and, if they exist as enols, there is potential for confusing results during analysis of derivatized sugars if
the oximes are not formed prior to derivatization.
Principle:
Stox™ Reagent is used primarily with sugars. This reagent is a pyridine solution containing 25 mg/ml hydroxylamine
hydrochloride.
This method is designed to assist in the routine determination of sugars in food products and syrup. Sugars are treated with hydroxylamine hydrochloride. The resulting oximes are converted to TMS ethers, and oximes are silylated directly. The results are quantitative and reproducible
and multiple peaks from the tautomeric forms of reducing sugars are eliminated.
1. Combine 10-15 mg sugar mixture and 0.5 ml Hydroxylaminchloride in a 1.5 ml GC vial.
2 Cap vial and heat at 70-75°C for 30 minutes.
3. Cool to room temperature. Add 0.45 ml HMDS
4. Add 0.05 ml Trifluoroacetic acid (TFA). Cap vial and shake for 30 seconds. Allow the white precipitate to settle for 30 minutes at room
temperature.
5. Analyze by gas chromatography using a GC/MS with helium as the carrier gas.
Typical GC condition:
28m * 250µ * 0,25µ HP-5ms / 1 µl Splitless (best with a TDS injector or standard injector @ 250 °C.
Attached a sample chromatogram .
If you have further questions, do not hesitate to contact me.
Best regards,
Peter
peak order
13,20 Arabinose
13,70 Ribose
16,20 Manitol
16,50 Fructose
17,20 Galactose
17,39 Glucose
26,80 Saccharose
28,60 Trehalose
Best regards,
Peter
Dear Ms. Batul
C18 columns are not suitable for separation of glucose and monosaccharides in general by HPLC using DAD detector. Usually amino bonded columns are used and usually with refractive index detector.
However the most senetive method for analysis of these carbohydrates are by GC-MS after derivation as described by Dr. Peter Behrend.
Dear Ms. Batul,
Amino bonded phase HPLC columns with RI detector are generally used for analysis of sugars. Gradient elution cannot be carried out with RI detector. Sugars cannot be detected using UV or fluorescence detector without derivatization. On the other hand, sugar analysis using electrochemical detector (ECD) does not require any derivatization and the sensitivity is thousand folds higher as compared to RI detector.
I did reply previously to this question. The reply of Asit Ray is very good too.
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