SB column issue

Record: 369

Category: Column Problem
Product: SB
Sample:

Question
Attached is the file containing different GPC trace I got. Second slide is the BSA standard and third slide shows the comparison between 2 different trace of the same sample taken 8 months apart.

Please let me know your thoughts.

Would organic solvent be more effective to clean the column rather than doing an aqeous clean up? The running buffer we have is 150 mM sodium acetate, pH 4.4.

Answer
I understand your target substance is cationic polymer but also include methacrylate.
We think there is a possibility that methacrylate is adsorbed on the packing material.
So could you wash the column with a basic condition like 20mM Na2HPO4?
Methacrylate must be eluted with the alkaline condition.
If there is no effect, could you also wash the column with 10% of acetonitrile?

Additional Comments

Translation:

Regarding your inquiry, the component that eluted at about 10 minutes 8 months ago (red LS chromatography, RI = blue chromatography was not detected because the component was too small) is now eluting at 14 minutes, around the time when the target component is eluted, which is why the molecular weight value, Mw/Mn, is so different.

The RI chromatogram (blue) has not changed much, so I don’t think the separation state has changed.

Also, there has been little change in the LS response intensity, so it can be inferred that the polymer is not decomposing and eluting late.

In addition, I don’t think it is caused by gel debris. The elution of gel debris is not delayed, and since you are using a three-angle LS, the sensitivity should not be that high.

The component that eluted at 10 minutes 8 months ago is now eluting late due to accumulation of adsorption of the components that have been analyzed so far? or something? This is the cause, so the column will be cleaned to prevent this component from being delayed.

Polylysine is indeed a cationic polymer, so it is good to apply the conditions for cationic polymers. However, in the case of water-soluble methacrylate, it is easier to elute if the eluent is basic, so I would recommend washing under basic conditions (such as 20mM Na2HPO4). After that, you may want to consider washing with a cleaning solution that contains an organic solvent. (Alternatively, you can use a mixed solution such as 50mM Na2HPO4 aq./CH3CN=90/10 to do both at the same time. Please check the solubility of your sample in this cleaning solution beforehand.

Original
お問い合わせの件ですが、8ヶ月前には10minくらいの位置に溶出していた成分（赤
いLSのクロマト、RI=青クロマトでは成分が少なすぎで検出されていません）が、最
近は目的成分が溶出する辺りの14分に溶出しているのが分子量値、Mw/Mnの値が大き
く異なる理由です。
RIで得られているクロマト（青）はあまり変化がないので、分離状態は変化してい
ないと思います。
また、LSの応答強度にもあまり変化がないので、高分子が分解して遅れて溶出して
いるわけではない、ということも推測できます。
（加えて、ゲルくず由来でもないのではないかと思います。ゲルくずの溶出は遅れ
ませんし、お使いいただいているのが3角度のLSなのでそんなに感度は出ないはず）

8ヶ月前に10minに溶出していたものが、今は今まで分析した成分の吸着の蓄積？か
なにか？で遅れて溶出しているのが原因ですので、この成分が遅れないようにカラ
ムを洗浄する、ということになります。
ポリリジンは確かにカチオン系高分子の一種なので、カチオン系ポリマのー条件の
適用は良いとは思いますが、水溶性メタクリレートの場合には、溶離液を塩基性に
したほうが溶出しやすいので、塩基性条件（20ｍM Na2HPO4など。）で洗浄してみて
いただいてはいかがでしょうか。
その後、有機溶媒を含む洗浄液での洗浄をお考えいただければと思います。
（あるいは、50mM Na2HPO4 aq./CH3CN=90/10　など混合溶液を使用して同時にやっ
てしまう、という方法もあります。この洗浄液に対する試料の溶解性は事前にご確
認いただいてください）

Download or Print PDF