Chromatography Forum

Hi there,

I am attempting an analysis of Lactulose (a dissaccharide) using with the following setup:

- Waters System 600-717 Plus-2414 (RID)
- 150 mm x 4.6 mm, 3 micron NH2 Column
- Column and Detector temp: 38C
- Sensitivity: 128
- Mobile Phase: ACN:Buffer(Sodium dihydrogen phosphate) = 80:20
- Flow rate: 1.0 ml/min
- Injection vol: 20 microlitre

However I observed a retention time that is drifting in the forward direction (becoming earlier) between injections (19 mins, 18 mins, 17 mins...) of same sample.

Have anyone of you come across such cases? Is there a particular reason for the forward drift of the injections?

Your advice is much appreciated!

Dear jessxxoxo

What is your sample matrix?

Typical cases for reduced retention time is loss of column capacity. This may come from loading the column with matrix components.

Also a change in the eluent composition over time may lead to RT changes.

You are using ACN at a high pH. Under these condictions ACN is not very stable. Eluents need to be exchanges frequently.

best regards

Dear jessxxoxo

What is your sample matrix?

Typical cases for reduced retention time is loss of column capacity. This may come from loading the column with matrix components.

Also a change in the eluent composition over time may lead to RT changes.

You are using ACN at a high pH. Under these condictions ACN is not very stable. Eluents need to be exchanges frequently.

best regards

Dear Dr Markus,

Thanks for your rely.
My sample matrix is Liquid Lactulose in diluent ACN:Water 50:50.
I have performed the test using a brand new column (in fact it is my first time using it).
I am performing the analysis following the parameters as outlined in the British Pharmacopeia, though I did not use a "pre-column" as suggested by BP.
By change of eluent composition over time, do you mean components of mobile phase that have evaporated off?
I believe normally for sugar analysis the composition of ACN is typically at 60-80%.
Is there a way to overcome this?

Thanks.

Dear jessxxoxo

I actually do not have experience with your type of analysis.

I also just realized that your buffer is dihydrogenphosphate which is amost neutral. Therefore ACN stability is no problem. Also evaporation of e.g. ACN will be minimal.

With the pure sample solution also reduction of capacity should be no problem.

Actually my points were thought as hint for possible changes in the retention time.

Right now I only could think about an equilibration effect. I have seen column / eluent combination requireing very long equilibration time. This can reflect in shifting retention times. Anyway, as I do not know your system, this is only a guess.

best regards
Markus

What is the concentration of sodium phosphate in your mobile phase? I mean the concentration overall (i.e., after addition of the acetonitrile), not just the concentration in the 20% of the mobile phase that's aqueous.

Generally lower temp, for example, room temp is used for amino columns. Buffer is probably not necessary for sugar analysis. Try it tand see if it makes any difference. Did you have this problem before?

Dear jessxxoxo

I actually do not have experience with your type of analysis.

I also just realized that your buffer is dihydrogenphosphate which is amost neutral. Therefore ACN stability is no problem. Also evaporation of e.g. ACN will be minimal.

With the pure sample solution also reduction of capacity should be no problem.

Actually my points were thought as hint for possible changes in the retention time.

Right now I only could think about an equilibration effect. I have seen column / eluent combination requireing very long equilibration time. This can reflect in shifting retention times. Anyway, as I do not know your system, this is only a guess.

best regards
Markus

Dear Dr Markus,

Thanks for your input. I will attempt to lengthen the equilibration in the next run to see if there is any improvement.

Regards
Jess

What is the concentration of sodium phosphate in your mobile phase? I mean the concentration overall (i.e., after addition of the acetonitrile), not just the concentration in the 20% of the mobile phase that's aqueous.

Dear Andy,

My mobile phase is prepared by dissolving 0.253g of sodium dihydrogen phosphate in 200ml of water before making up to 1000ml. Im assuming you are referring to 0.253g/1000ml*100% = 0.0253%w/v of concentration? Do correct me if I'm wrong.

Regards,
Jess.

Generally lower temp, for example, room temp is used for amino columns. Buffer is probably not necessary for sugar analysis. Try it tand see if it makes any difference. Did you have this problem before?

Dear JI2002,

I'm afraid the temperature and the mphase composition are outlined as per BP monograph hence i wont be able to do much adjustment for that if the product is claiming BP.

Regards
Jess

What is the concentration of sodium phosphate in your mobile phase? I mean the concentration overall (i.e., after addition of the acetonitrile), not just the concentration in the 20% of the mobile phase that's aqueous.

Dear Andy,

My mobile phase is prepared by dissolving 0.253g of sodium dihydrogen phosphate in 200ml of water before making up to 1000ml. Im assuming you are referring to 0.253g/1000ml*100% = 0.0253%w/v of concentration? Do correct me if I'm wrong.

Regards,
Jess.

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I was referring to the molar concentration. You didn't specify if this "sodium phosphate" was the anhydrous salt (NaH2PO4) or NaH2PO4.H20. Let's assume that it's the anhydrous salt. Its molecular weight is 120. Your solution contains 0.253/120 = 0.0021, or 2.1 mM overall. A saturated solution of sodium phosphate in 80% ACN is about 5 mM. I suggest that you try doubling the concentration of sodium phosphate and see if that affects the retention time drifting. While the sugars that you're analyzing aren't ionized and so wouldn't be involved in any ion-exchange mechanism, the concentration of counterions available to the amine groups on your stationary phase surface will affect its polarity. It takes at least 20 mM salt in the mobile phase to form a reasonably complete electrical double layer of ions on the surface. You can't get there with sodium phosphate (although you could with triethylammonium phosphate...) in 80% ACN, but 4 mM salt would be better than 2 mM salt.

Hi Jessxxoxo,

Amino-phase columns can be tricky to use, buffer or no. Here's a link that may help:

http://www.waters.com/webassets/cms/lib ... a20769.pdf

Neue notes on p. 22 that some care in the choice and handling of these columns is warranted. The amino bonded phase does seem to require patience in use as I've seen in my experience with regard to its initial equilibration when just taken out of the box. Page 40 of the same document holds some info regarding other observations of amino bonded phase use and saccharide separations. Best wishes!

You did not say if you had this problem with a different column and that info is important. For example, the solvent for a new amino column from Agilent is hexane, which means that the column needs to be flushed with IPA first prior to put into use with water acn mix. Don't know if that's the case here, but at least you can rule out some of the possibilities.

Just one comment : most silica based amino columns are non stable with aqueous buffers. There is an hydrolysis of the stationnary phase that can explain retention problems. So first check the column you use : silica base or not ?

If you have an evaporative detector (ELSD or CAD) in the lab, it's easy to verify the stability of the column. With RID, not sure you can see it.

There are some good amino column makers that provide stable amino columns (silica base or polymeric based).