non-gradient for Reversed phase HPLC

[superbenben]

I was using a C18 column to separate a water soluable polymer based on polarity. When I used constatnt 5% ACN in water or 50% ACN in water, the retention time were both 30 seconds. Should there be a difference? Thanks.

[tom jupille]

Two possibilities here:

1. If it's unretained with 5% ACN, it will probably not be retained with 50% ACN either. In that case, you need either a different column or (more likely) an entirely different separation mode (depends on the chemistry of your polymer).

2. If it's totally retained with 50% ACN it will also be totally retained with 5% ACN. That means that what you are seeing at 30 seconds is "t0 noise" at the system dead time and not your polymer at all. In that case, try increasing the ACN concentration to 80% and see what happens.

[superbenben]

Thanks for your reply.

your 1) is exactly right, the the water soluable polymer ( has polar and non-polar components) is unretained in both 5% and 50% ACN without gradient. However, when i use gradient from 5% ACN to 62% ACN within 6 minutes, the polymer have good separated peaks at 5~8 minutes, more polar polymer eluted earlier.

This is my confusion, by the principle of reversed phase HPLC, if I use 5% ACN straight ( without increasing the solvent hydrophobicity ), the retention time should be later than 5~8 minutes if it is not totally retained. why it was not even retained?

Thanks.

[unmgvar]

what are the sizes of your polymers and what type of C-18 are you using? not all columns C-18 are equal
what is your HPLC system? what is your flow rate?
old system can have 2.5 minutes of dwell volume and a 250mmX4.6 column at a flow rate of 1ml/min has a further volume of 2.5min or so. if 30 seconds is your t0 then it is a very small column.

the retention of some of your compounds is very poor and you should probably look out for a another type of separation, not c-18

why do you not try a 100% aqueous state and "re-wet" with organics to prevent phase collapse? this should provide you with a better separation as well, especially if you use a gradient
you can use a buffer to better retain them as well

these days there are columns with polar groups to retain polar compounds
you can try HILIC
or a mixed mode column with SAX or SCX type with C-18