Chromatography Forum

Some background info,
We were analyzing e.g. sugars by Zorbax Carbohydrate column 4.6x150mm, 5um. We use Agilent 1100 series HPLC with a mobile phase of acetonitrile and water. Analytes are determined by PAD (Esa Coulochem III, 5040 analytical cell) after a post-column pH modification by NaOH. To improve our method we started using Zorbax Carbohydrate column 4.6x250mm, 5um., i.e. 10 cm longer one. After this change the peak area decreased clearly.

We have cleaned electrodes, we have changed fresh solutions, we have checked the same standards with both columns during one day to keep all the other parameters identical. With 150 mm column the peak area is about 3 x the peak area compared to the peak area that we get with 250 mm column. The decrease is the same when we look at the peak height.

I would be very grateful for any help to solve this problem.

What´s that 3x stuff suddenly?
Is the flow rate the same for both columns?

Or did you change the mobile phase composition to compensate for the longer retention on the 25 cm column?

Thank you for your comments. We have the same mobile phase composition and flow rate for both columns as well as all other parameters. At the moment the situation is improving after washing the column for whole weekend, i.e.. the decrease is not that big anymore with our new column (250mm) as it was on last friday.
Thus we are thinking the following question: is it possible that there is something ??? coming out from a new column, which could interfere our detector (PAD, Esa Coulochem III, 5040 analytical cell)? The longer we have used the longer column (now 1.5 weeks) the bigger the peak area is.
Marja-Leena

Is it possible that these sugar columns have sites which bind your analytes fairly strongly, those sites getting saturated slowly?
Actually, it seems more plausible that some mistake is playing "havok" here.

[quote="HW Mueller"]Is it possible that these sugar columns have sites which bind your analytes fairly strongly, those sites getting saturated slowly?
Good comment. Unfortunately I have to admit that I don't have knowledge on that. We are analyzing glucose and some cyclodextrins (CDs), e.g. alfa-CD and HP-beta-CD. Until now we have only used pure standards.

Both the glucose and cyclodextrins decreased, etc.?

Aaah yes indeed. I overlooked that you are using a PAD.

Amino columns do bleed quite drastically when they are new, and this can interfere with detection. The problem is simply that amino columns are not stable under the condition used for this analysis.

It does get better though, and now you will be able to use the column until it dies for other reasons. There are carbohydrate columns around, where this problem might be eliminated or at least minimized. The next time you need a column, you may want to try the High-Performance Carbohydrate Analysis column from Waters. We minimized the problem for this column.

Thank you for your comments. The decrease was detected for both glucose and for CDs.
Now we have got to the level that we had previously with our old shorter column. So it seems, that something really came out from the new longer column.
Surprise for me is that the carbohydrate column was "bleeding" eventhough we were using water and acetonitrile as solvents. We used a post-column pH modification by 0.5 M NaOH to protect the column from high pH.

You have an amine bonded to the surface. If you expose it to water, it will form a basic pH, which will result in some dissolution of the bonded phase until you have enough silanols on the surface to "neutralize" the remaining amine.

What does the bleed do? React with the carbohydrates, foul the electrodes, or produce such high background that the carbohydrates are partially obliterated?
malela, the baseline didn´t change with the longer column? (there was no indication that something was foul??).
(I have never worked with electr...., am very suspicious of them).

Hans,

Uwe's explanation is very reasonable considering the fact that the conditions used for detecting carbohydrates via pulsed amperometry are quite effective for detecting amines as well (typically giving even better signals than simple carbohydrates). Since the stationary phase degradation products will be amine fragments, it is to be expected that the column bleed fragments would have a potential for masking the signal for carbohydrates.

Malela,

There are a couple of potential solutions for your problem beside simply running the column for a long time including: using an amino phase which is hydrolytically stable (Shodex sells such a column, NH2P-50), optimize the waveform to minimize interference of an amine (more aggressive cleaning potential should help significantly) or switch to a separation chemistry which allows use of hydroxide in the eluent (e.g. the CarboPac PA1 from Dionex).