Chromatography Forum

Hi

I have almost developed a method of dissolution on LC.
My dissolution has two stages: one is Acid stage and other in buffer (pH 6.

Chromatorgaphic parameters:
Column: kinetex, 50*4.6, 2.6µm (POroshell)
Flow rate: 2.0 mL/Min
Column Temp: 35°C
Inj vol: 10µL for Acid stage
5µL for Buffer stage

Now, I am facing some problems.
In case of acid stage, I don't have any problem. but incase of buffer stage, after sucessive injections the column efficiency is going down very fast. (Almost USP plate count is near to 600 after 750 injection and USP tailing factor has gone down from 1.05 to 1.89).

1. Does it has to do with the column chemistry?
2. Is Buffer pH is very high for column?

Reply

This "poroshell" material looks like these tasty peanuts covered with a dough, flavored with chili or wasabi. Anyhow, if you switch between very acidic conditions and not so acidic conditions it is stress for every column. First of all we need more information what buffer you used for both stages, what organic solvent you use, gradient or isocratic. What is the plate number when you install a brand new column? You do dissolution, so do you filter your sample? Depending on sample preparation, chromatography conditions, different methods, a number of >500 injections on a 5cm length column is not so bad. What is your back pressure for both "stages"?

Buffer: 20mM (KH2PO4), pH 2.30 with OPA,
Mobile phase: Buffer: MeOH (60:40), Isocratic
Initial plate count: 2050
Filter: Teflon+ Glass
Back pressure: around 4200(Through out)

So your back pressure is around 290 bar, nothing special with your buffer. A plate count drop from 2050 to 600 is dramatic. Please do the same column test as stated on your certificate of analysis. Before doing that please do a washing procedure as stated in your column care guide. If that QC test show that the plate count is also dropped down than something happened with the packing in the column. In other words there is a void volume. Was it the first column you used with that method? Anyhow, unfortunately you need to order a new column - and please contact the technical support of the manufacturer.