Chromatography Forum

I have the following gradient program:

Time Water% ACN% 20mM kH2PO4 (pH3)
0 50 50 0
11 0 100 0
25 0 100 0
30 50 50 0
30.01 0 20 80
35 0 20 80

Stop 35min
post time 5min

initial condition
Time Water% ACN% 20mM kH2PO4 (pH3)
0 50 50 0

at 30.01 min, I switch from 50:50 water/ACN system to 20: 80 Buffer/ACN system and at 35 min, I change it back to 50:50 water/ACN system.

That is unusual gradient program for me. I am curious, why do you need such switching? Is there any particular reason for this?

I did compose all kind of gradients so far and I do not think it is unusual. The only concern I do have about this is the combination AcN-Phosphate, can you eliminate the possibility that there will be no precipitation of phosphate? I mean, the phosphate concentration is low but I saw some precipitation on that level to. However, the gradient can help providing some specificity in separation.
regards

I did compose all kind of gradients so far and I do not think it is unusual.

Ok, then please give me reasonable explanation to me what is the role of 5 min change of buffer instead of water (when the mobile phase of the whole run is consisted of ACN and water)?

Well, I suggest to perform some experiments on column re-equilibration effects when using water and phosphate buffer. My observation is that the column re-equilibration is significantly faster when using inorganic part of high ionic strength instead of (acidified) water. I do not know why there is a change from water to AcN here, but the re-equilibartion can be a reason.
regards

My observation is that the column re-equilibration is significantly faster when using inorganic part of high ionic strength instead of (acidified) water.

Ok, I haven't done such experiments so I am not sure whether this is true or not (I'm kind a suspicious about that). Maybe other members can give their opinions regarding this issue. All I know, regarding the time needed for column re-equilibration, is that the greater difference between the mobile phase composition at the end of the run and the initial mobile phase (at the beginning of the run) the longer the re-equilibration time. The duration of the column re-equilibration process is affected also by the value of the gradient dwell volume and column void volume.

Regards

I do agree that the column equilibration is affected by the facts you wrote. However, I will take care to post some publications describing these effects. However, give me some time since I am quite busy now. Many tips about this topic can be found in LCGC.

Regards

Thanks for your replies.
The reason is because I would like to flush a retained component out at the end of the run (after 30min). I do not care about the sepeartion of that component. I only care about stuff coming out before 25 min. And I do not want the mobile phase changes to affect my separation before 25min, which means I hope the re-equilibration can be done in 5min (post run 5min).

I understand another choice would be to use a guard column.

By the way, no instant precipitation was observed, but I am not sure if there is slow effect.

Please give me some suggestions about the following issue.

About my retained components, I have 3 options:
1) flush it out for each single injection (I wasted 5min to flush out retained components, and I do not know if 5min is enough to equilibrate between mobile phase change)
2) flush it out for every N injections (I can save some time, and re-equilibration time can be set longer than 5min. But is this practical? I might have ghost peaks)
3) use a guard column (question: what about those components I am looking at? Retention times of them will change, and maybe I cannot save much time.)

Moonchips,

You should post the column dimensions and flow rate in order to expect any suggestion regarding equilibration time.
One thing you can be sure of: A guard column will not speed up the equilibration. On the contrary!

Best Regards

Rule of thumb for re-equilibration is 3X system volume + 5 X column volume. Unless you have a very short column or very high flow rate it is probably longer than 5 minutes.

Dwell volume is 0.8mL. Column is 150mm*4.6mm*5um. I am not sure about Column volumn (maybe about 70%?)

What is system volume?

Moonchips,

The described column has approximate volume of 1.75 mL. (refer to equation 1)
If the flow rate is 1 mL/min, it’ll need 11 – 12 min equilibration time.

(1) Volume (column) = D^2 x 3.14 x L x f/4

D = column diameter in cm
L = column length in cm
f = approx. 0.7


Best Regards