Chromatography Forum

Hi,

I'm using an Hypersil SAX column with 0.33g/l phtalic acid, brought at pH 6.4.
Column temperature is 35°C.

When I injected yesterday morning my first reference, retention time was about 11.5 minutes.
After injecting 9 references (Na2SO4 in water), the retention time was already decreased to 9.7 minutes.
After several hours, the RT was about 6 minutes, and peaks are getting distorted/unseparated from other peaks in my sample.

I'm now backflushing my column with 100% acetonitrile to throughly clean the column, and hope to get back my retention.

Any ideas about this decrease, or how to get my retention back?

Thx

Ace

I actually don't have experience with that column, but I assume that your conditions won't be the reason for that shift.
How did you prepare the eluent? Would it be possible that the phthalic acid wasn't totally dissolved?
May be you check the eluent pH again.

eluent was prepared by dissolving 0.33g phtalic acid in about 900ml water. This was brought at pH 6.4 with KOH solution. After this, the solution was diluted to 1000ml with water and filtrated through a 0.45µm filter.

eluent pH after 24 hours: 6.4.

Thanks for your reply

Ace

Sound OK.

You may try to prepare the eluent from potassium hydrogen phthalate. This is better soluble than the acid.

But anyway think the effect is to big for just a solubility problem.

Was the background signal stable over time?

Potassium hydrogen phtalate sound nice, the acid is very slowly soluble.

The signal was stable.

Could it be that the pthalic acid is bonded to the surface of the column, so some kind of "endcapping" is formed? And hence the retention of sulphate is decreased?
Just some wild guesswork, as I have no experience with IC.

Ace

Actually at pH 6.4 all phthalic aicd is ionized (approx. 90 % in the phthalate2- form and the rest as hydrogenphthalate). There should be no problem with adsorption. You may add some methanol to the eluent (2 - 5 %). That finally should avoid adsorption.
We used to run other silica based columns with similar eluent and never have seen problems with the eluent.

Thanks for the tip with the methanol, I will try that, if I can get back my retention.

Any hints on how to regain my retention?

Ace

Update:
after running 4 gradient from 99% phosphoric acid 0.1% to 99% ACN in 30 minutes each, I got back my retention at 10.3 minutes.

Although my baseline is not stable yet after 30 minutes of equilibration before injection, this gives some hope .

Ace

Hello
I’m new at the forum. I wanted to post a comment here since I’m having the same problem of RTs getting smaller, but I’m using a 761 Compact IC and a Metrosep A Supp Column. I use NaCO3-NaHCO3 as eluent. The baseline is very stable and the conductivity of the eluent is within range. I have already replaced the eluent and purged all the line. I had even replaced the column but the RTs keeps getting smaller at a 0.01 min/hour rate. The peak shape is acceptable, but they are getting closer. Could you please give some ideas? Thank you all

Alma Hernandez,

did you find a solution for your problem?

Bart

I would also suspect the MP. Proper mixing and delivery.

I would also suspect the MP. Proper mixing and delivery.

Mixing was just one bottle, premixed mobile phase.
Delivery was fine by the HPLC pump, I don't suspect my HPLC to pump 1 ml at the beginning (RT 50 minutes), and then up to 6x more to get retention times of 8 minutes.

Would be nice if that was the problem.

Ace

Yeah sounds like you've ruled that out

Do you use a guard column?

Nope, no guard column.
As this is only Na2SO4 in water, I wouldn't suspect any clogging of my column.