Issues with HPLC-Refractive Index in analysis of Lactulose

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

6 posts Page 1 of 1
Hey there,

Posting in this forum to check if anyone of you could help! I am attempting the European Pharmacopeia method of analysis for assay and related substances of Lactulose (a dissaccharide). The following setup was used:

- Waters System 600-717 Plus-2414 (RID)
- Phenomenax 250 mm x 4.6 mm, 5 micron NH2 Column
- Column and Detector temp: 38C
- Sensitivity: 128
- Mobile Phase: ACN:Buffer(Sodium dihydrogen phosphate) = 80:20 (tried 82:18 too but worse)
- Flow rate: 1.0 ml/min
- Injection vol: 20 microlitre

However, from the chromatograms, I've observed:
- delayed retention time of Lactulose peak
- slight fronting of the lactulose peak
- unable to detect epilactose peak which is crucial for my system suitability of related substance analysis
- baseline noise, making it hard to detect other sugar impurities.
- high assay values (possibly due to merging of some impurities peak??)

Wondering if you guys have any idea on how to solve this. I have monitored the system under purge mode for 10% methanol and my mobile phase for about 30 mins and 1-2 hours respectively but seems like that didn't help much.

I've read that most sugar analysis does not use buffer in the mobile phase composition. Is it true and if so, why?

Your advice are much appreciated!

:)
What isour NeedleWash solvent?
Change it to meet your eluent, so ACN:water 80:20

And of course with RI youse premixed eluents, not online-mixing
Hi Jessxxoxo,

I agree with Hollow's suggestions and add a couple of things.

http://www.waters.com/waters/library.ht ... cale=en_US

Neue's HPLC Troubleshooting Guide, link above, has at least two Sections (17 and 33) devoted to the use of the aminopropyl phase to analyze sugars. From my own experience, it can take up to an overnight equilibration of a new column to obtain stable retention times (Section 17). Anomerization of sugars is discussed in Section 33--the role of the aminopropyl phase to increase anomerization of each sugar analyte to afford singly-eluted peaks is explained. Likely this is the reason that buffers are generally Not Used to analyze sugars via separation on aminopropyl phases with water/ACN (or /MeOH) eluents...unless perhaps the buffer is also basic--I'm curious, is this the case with the EP method for lactulose and epilactose?

Perhaps the easiest thing to do is to prepare separate lactulose and epilactose solutions and perform injections without a column (union in column's place) to see if the column may be the culprit here.
MattM
One of the most commonly overlooked items when using an amino column is that of proper washing and cleaning of the column. Amino columns quickly foul up and require well thought out washing procedure to remove retained material from the support. Failure to do so = worse and worse peak shape and loss of resolution over time (as if the support is bleeding off).

While hydrolysis can account for some loss of support over time (usually this only occurs at pH extremes, not under more neutral conditions), column fouling is more common in actual use because many novice users do not yet incorporate proper wash methods as follow-up to their methods (esp when isocratic runs are used). While I am unaware of any "standard" wash method specific to amino columns, a good wash method is: (1) always safe and compatible to us on the specific support and (2) includes solvents which will dissolve all of the sample types injected in the column. This is a good time to also mention that columns are consumable items. Insure you are using a new column for best results. Replace the column when washing no longer seems to help.

Now, a suggestion regarding a wash solution for your method. I asked our lab manager for advice on this and he suggested a solution of METHANOL and water. Maybe 80% methanol with water. Wash 5 column volumes through, then retest. *Ck w/the column supplier to see if that is safe first as Phenom is just a reseller of other company's products, but they will know where the real support comes from (supplier and brand).
Hi Again, Jessxxoxo,

If it is an issue, here is a link to Phenomenex' recommended column care guide for the Luna aminopropyl phases:

https://www.phenomenex.com/ViewDocument ... care+guide

Please refer to page 7 for cleaning of phases used for sugar determinations. Note: this stationary phase will always be "touchy" due to its makeup when water comprises a portion of the eluent use with it.
MattM
And also, I thought this was neat, though you (Jess) may not get to try this out:

http://mac-mod.com/pdf/application-pack ... Sugars.pdf
MattM
6 posts Page 1 of 1

Who is online

In total there is 1 user online :: 0 registered, 0 hidden and 1 guest (based on users active over the past 5 minutes)
Most users ever online was 1117 on Mon Jan 31, 2022 2:50 pm

Users browsing this forum: No registered users and 1 guest

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food & Beverage, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry