Problem in eluting the sample with Amide-80 column in NP

[sboopathi]

I am using normal phase HPLC method with TSK Gel Amide80 column for glycosyltransferase enzyme assay with fluorescent detection. The sugar standard was made with PA-Maltooseoligosaccharides. After making the calibration curve, I injected the first sample (unpurified sample obtained after the enzyme reaction with proteins in Tris HCL buffer with pH 7.3) and got the expected peaks. But after using that sample, there is no retention of sample or standard by the column. I checked if any blockage due to proteins blockage in the guard column and the column, but the pump pressure is stable and there is no blockage. The solvent peaks are eluted but not the sample or standard peak. What I want to know is how to check whether the stationary phase material has been altered in the column, or what will be the reason for no retention of sample in the column. Whether column backflush can help in this regard? Please help me to sort out this problem....

[tom jupille]

If the problem is due to precipitated or adsorbed protein, it will probably be stuck on the head of the guard column. If this were my problem, the first thing I would do it to change the guard and try re-injecting a standard. If that looks OK, then you have confirmed the problem. Backflushing the guard with something that's a good solvent for your proteins would be the next step (given that you are using a guard, backflushing the analytical column is not likely to help).

[sboopathi]

I changed the guard column and injected the standard, still I am getting only the solvent peak and there is no retention of the standard. Is there any other solution which can solve this problem.

[HW Mueller]

Using fluorescence as detection it should be relative easy to determine whether your enzyme gets stuck on the column or elutes at tm. You say that solvent peaks came through, I don´t see how your solvent produces peaks (maybe a few wiggles), do you get some protein /enzyme in all the runs out front? What is PA-Maltooseoligosacharides? Am I misunderstanding the post, you are not analysing the enzyme, but rather products which were derivatized?

[sboopathi]

Yes Meuller, you are right, I am not analysing the enzyme, only the products formed by the enzyme by using the derivatized substrate. I am using Maltobiose, Maltotetraose and Maltohexaose as sugar standard for our analysis. The derivatized substrate is a tetramer and the product formed will be a pentamer or hexamer. I told about solvent peak is the mobile phase blank run. Now only get peak which is also coming in blank run even if I run the PA-Maltose.

[Andy Alpert]

What is the mobile phase, and what is your sample and the standards dissolved in when you inject them?

[HW Mueller]

So your "solvent peak" is not solvent but unretained(?) derivatization product. The PA is a fluorescing moiety? Still don´t know what it is. Anyway, is the PA-maltose a commercial standard or did you derivatize maltose?
By the way for the newer people, my name is Hans.

[sboopathi]

The mobile consists of Acetonitrile, Water and Triethylammonium acetate (pH 7.3) run in gradient method with the ratio between Acetonitrile and Water is 75:15 in buffer A and 50:40 in buffer B. The sample and the standards were dissolved in mobile phase buffer A.

[Andy Alpert]

I see. First, please note that 75 + 15 = 90. The components of your mobile phase should add up to 100 here. I will assume you mean 75% acetonitrile, 15% water, and 10% ammonium acetate buffer solution. Same goes for mobile phase B. Now, what is the concentration of ammonium acetate? Please specify if you mean in the 10% of the mobile phase that it represents or in the concentration of the mobile phase overall (e.g., if that 10% is 100 mM, then the overall concentration is 10 mM). Next, you don't need a gradient to elute oligomaltosides up to the 6th member of the series. With 75% ACN the 6-mer (maltohexaose) should elute isocratically in about 4-6 column volumes. For an analogous series, see: A.J. Alpert, J. Chromatogr. 499 (1990) 177 [figure 10]. Anyway, try running isocratically with 80% ACN, not 75%, and see if your retention increases.
Another thing to check: is the pump for mobile phase A working? Are both check valves working properly? It might be that you're actually in 100% mobile phase B without knowing it.

Hans: PA- stands for pyridyl 2-amino. One attaches it as a Schiff base to the reducing end of sugars and oligosaccharides, then reduces it to yield a stable secondary amine. The resulting compounds are fluorescent.

[sboopathi]

10% Triethylammonium (TEAA) acetate in both buffer A and buffer B. 500mM TEAA is prepared by equimolar concentrations of Triethylamine and Acetic acid. 10% TEAA is the overall concentration of mobil phase.
I will try the isocratic condition with 80% ACN. Pump A works fine and the valves are also working properly. Thanks for sending the reference.

[sboopathi]

Hans, we derivatize Maltose by using 2-Aminopyridine and Borane dimethylamine complex.

[Andy Alpert]

Here's another speculation. You're running at pH 7.3. At so high a pH, Amide-80 has a significant degree of negative charge (ref: A.J. Alpert, Anal. Chem. 80 (2008) 62). PA- tags have a (+) charge and so would be electrostatically attracted to the surface. They wouldn't elute unless you have enough salt in the mobile phase. Now, if you're preparing a mobile phase that contains 10% (500 mM TEAA), then the concentration in the final mobile phase would be 50 mM. Maybe that's not high enough for the purpose. Test this by preparing a mobile phase containing triethylamine formate instead, at pH 3.0. That pH is low enough to uncharge most of the ionized groups in Amide-80.

[HW Mueller]

My questions go in the direction of the thought that you got the derivatization to work initially and then not again. Or, maybe the derivative´s stability is not that great and deteriorated ( I obviously have no experience with this derivatization, though).

Andy, you are talking about SiO- in the Amide 80?

[Andy Alpert]

Hans -
In the past 5 years there have been about 5-6 papers that demonstrate that Amide-80 has a significant degree of negative charge above pH 4, but that could be due to either silanol or carboxyl groups. It's possible that some of the amide groups in the polymer used to make the Amide-80 coating have been hydrolyzed to carboxyl- groups. Since Tosoh hasn't described their material in any but the most general terms, it's not possible to say.

[HW Mueller]

That reminds me of my attempt, which is partially documented here, to find out what happened with my silica based SEC columnn from Tosoh after exposing it to aqu. TFA. This TSK Gel SW 3000 (if I emember correctly) had a super performance before it "saw" the TFA.