Need help interpreting SEC chromatogram

[Ge_D]

Please help me solving the problem (see photo attached). I've calculated the column volume but have no idea what to do next (

[tom jupille]

No photo attached; it just says "image". When I tried to access the URL directly (I'm the admin and I can do that), I got "unauthorized".

[Ge_D]

Oh I'm so sorry about that.

Here it is, I hope it works this time.

[tom jupille]

Since this is a quiz, presumably you have studied the procedure. A few hints:

Hint #1: the calculation you have penciled in is for an empty column. Your column is not empty.

Hint #2: If a molecule is too large to enter the pores, at what time (or volume) will it elute from an SEC column?

Hint #3: If a molecule is small enough to enter all the pores, at what time (or volume) will it elute from an SEC column?

[Ge_D]

Since this is a quiz, presumably you have studied the procedure. A few hints:

Hint #1: the calculation you have penciled in is for an empty column. Your column is not empty.

Hint #2: If a molecule is too large to enter the pores, at what time (or volume) will it elute from an SEC column?

Hint #3: If a molecule is small enough to enter all the pores, at what time (or volume) will it elute from an SEC column?

Thanks for your reply.

I know that in a SEC chromatography, the larger molecules should be eluted first, then the intermediate sized ones, and the smaller sized ones at last, so the first peak should be the large molecules and the last peak is for the small ones, but how do I relate it to the calculation of the void volume and the volume of mobile phase trapped in the pores of resin? And I'm confused by the unit of the flow rate, it's mL/mL, but shouldn't it be mL/min? Do you think there's an error?

[tom jupille]

Flow rate should be mL/min.

You're on the right track, just take your logic further (or look at your notes!). The void volume of the column is the total volume of liquid it contains (i.e., the volume of liquid inside the pores plus the volume of liquid in between the particles, soooo:

Hint #4: *Why* do big molecules come out first? (and why do small molecules come out last?)

[Ge_D]

Flow rate should be mL/min.

You're on the right track, just take your logic further (or look at your notes!). The void volume of the column is the total volume of liquid it contains (i.e., the volume of liquid inside the pores plus the volume of liquid in between the particles, soooo:

Hint #4: *Why* do big molecules come out first? (and why do small molecules come out last?)

Big molecules come out first since they are exposed to a smaller amount of mobile phase (since they cannot pass through the pores of the resin), small molecules are the last ones to be eluted for the column because they are dealing with a large amount of mobile phase since they pass through the pores. I calculated the void volume of the column to be 2.40min * 1mL/min = 2.40mL since the first peak only start to show up at 2.40min, (or do I use 2.55min which is the centre of the first peak??) not sure this is correct though...:s

Since the smaller molecules have to deal with the mobile phase trapped inside the pores, my guess is that the volume of mobile phase trapped in the pores of the resin is equal to 4.20min * 1mL/min - 2.40mL = 1.8mL, but doesn't that contradict the column volume (which I calculated to be 2.49mL)?

[tom jupille]

You got it! The time to use is the retention time (highest point) of the last peak.

Re your earlier estimate, that was for an *empty* column. The terminology is a bit confusing, but "column volume" in LC is defined as "the volume of mobile phase in the column" (i.e., the volume between the particles plus the volume in the pores).

[Ge_D]

You got it! The time to use is the retention time (highest point) of the last peak.

Re your earlier estimate, that was for an *empty* column. The terminology is a bit confusing, but "column volume" in LC is defined as "the volume of mobile phase in the column" (i.e., the volume between the particles plus the volume in the pores).

Alright! Thank you for your hints and patience! :p

One last question, is it correct to use 2.40min to calculate for the void volume, or is it also the highest point of the first peak (~2.55min)?

[mattmullaney]

I apologize in advance for stepping on Mr. Jupille's shoes a bit...yes, please use the time at the peak maximum of the last eluting peak. Nice job on both questions, by the way.

[tom jupille]

is it also the highest point of the first peak (~2.55min)

Actually, I'd use the little "blip" at about 2.45 minutes as representing the first thing to elute from the column, but that's splitting hairs.

[mattmullaney]

UGH!! I meant...Highest Point of the First Eluting Peak! It's been a long day!