Void volume when uracil and acetone are retained

[Brenton1]

Good day everyone,

I am trying to determine the void volume of an Alltima C18 column (25cm x 4.6mm, 5 um). My mobile phase is 0.05 M KH2PO4 buffer : MeOH (90:10). Unfortunately uracil and acetone are both being retained and elute at about 4 minutes. Does anyone know of any other compounds or methods which I can use? I have calculated the void to be about 2.65 minutes but now I need to find a way to prove it.

Regards
Brenton

[Gerhard Kratz]

What is your flow rate? In which solvent you have solved your uracil? In acetone?

[Brenton1]

My flow rate is 1.0 ml per minute and my solvent is mobile phase. I prepared a uracil solution in mobile phase and injected. When that didnt work I prepared an acetone solution in mobile phase. Both compounds eluted at roughly the 4 minute mark.

[tom jupille]

If this were my problem, the first thing I would do would be to check the actual flow rate ny timing collextion of 10 mL in a graduate cylinder.

If that checked out OK, I would re-measure t0 with 50/50 buffer/MeOH (with only 10% MeOH, you may be aeeing some retention).

[M Farooq]

In a strict sense, the determination of void volume is not a trivial matter. The conventional dead time markers don't seem to work in your case. You can try couple of things:
1. Deuterated water as a sample injection. It will give rise to a refractive index change peak at the dead time.
2. You could also try nitrate and bromide ions and see if they are not retained. Both of these ions absorb in the UV.
3. You can also see refractive index changes at very short wavelengths (~200 nm) during sample injection.

All values should closely agree.

[Consumer Products Guy]

If this were my problem, the first thing I would do would be to check the actual flow rate by timing collection of 10 mL in a graduate cylinder.

If that checked out OK, I would re-measure t0 with 50/50 buffer/MeOH (with only 10% MeOH, you may be aeeing some retention).

Both are good ideas. As to a non-retaining compound on that type of column, what about sodium xylene sulfonate, that aborbs in the UV. We see ionic hydrotropes/surfactants typically be non-retained on RP-18.

[Brenton1]

Hi everyone,

Thank you for your responses. I have managed to get the void volume figured out now. First I confirmed that the flow is 1.0 ml per minute using a 10 ml volumetric flask. Then I changed the mobile phase composition to 50 : 50 mob phase : MeOH. This caused the uracil to elute at 2.7 minutes but my manager didnt want a different mob phase for the void determination to the rest of the validation so I had to work with the initial mobile phase. During the specificity a peak eluted at 2.7 minutes for the 3 % peroxide solution as well as the 0.1 M HCl and 0.1 m NaOH so I injected each of these solutions. The peroxide yields a definite peak at 2.7 minutes while the acid and base also yield peaks at 2.7 minutes, but these are not as well defined as the peroxide. Next I injected a solution of sodium nitrite in water (3 mg to 100 ml). This yielded a clearly defined peak at 2.7 minutes.

Now I have 2 options. Either a 3 % peroxide solution (3 ml of a 30 % H2O2 solution into 100 ml) or the sodium nitrite solution. Does anyone have any suggestions as to which I should use or is there not really much of a difference?

Regards
Brenton

[HPLCaddict]

I'd say the most important thing to do would be to send your manager to a basic HPLC seminar. Sorry for being cynical.

I'd say all important thing's have already been mentioned. Just adding my 2 cents...
As already said, the exact determination of the void volume is far from trivial. There are heaps of articles in the scientific literature about this.
Concerning the "unretained solute approach" one should always be aware of the fact that no matter which test probe you are using (uracil, thiourea, nitrate,...) you are NOT doing a measurement of the void volume. You are determining the retention time of your test probe, nothing less, nothing more. If done correctly, this retention time can give you a pretty good estimation of the void volume. But it's still an estimation, nothing more. Although it should be enough for everyday purposes - for academic/highly scientific research it might be not enough.
How to do it correctly? Obviously you have to use conditions under which your "unretained test probe" actually is unretained. Using 90% aqueous on a C18, as you already found out, definitely is NOT correct. I've got the rule of thumb from somewhere that you should use at least 60% organic in the mobile phase for this and never got problems with this.
And remember (and tell your manager) that the void volume is a property of the column (plus the HPLC system, to be exactly). The mobile phase is NOT involved. Therefore relying on the method's exact mobile phase is nonsense in this case.

[M Farooq]

the void volume is a property of the column (plus the HPLC system, to be exactly). The mobile phase is NOT involved.

This is an extremely useful and a critical point for inorganic oxide based stationary phases like silica. However there are few but important exceptions. The mobile phase dependency for dead time is seen in certain polymeric columns bearing charged groups and ionic eluents. In ion chromatography, water inj is considered as a gold standard for dead time. However, what has not been reported before is that the water dip can change its position wrt to eluent. In that particular case, this had nothing to do with swelling or de-swelling of the polymer beads-the polymer substrate was a very highly cross linked polymer. Most likely, it was due the orientation of polymer brushes attached on the surface as a function of ionic strength of the eluent.

[tom jupille]

Some topics are perennial. Check this thread from tha archives:
http://www.lcresources.com/discus/messa ... 20041222pm