Hi,
I'm trying to pack a 2.5 cm Merck Self-packer HPLC column with Phenomenex Luna(3) C18 10 µm sorbent. Until now all trials result in bad peak symmetry (several pre-eluting steps) and a low number of plates.
Has anyone experiences with such small diameter self-pack columns and can provide me helpful hints? Which slurry solvent do you use? How much slurry solvent? How do you prepare the slurry? Do you fill some slurry solvent into the column before adding the slurry? How do you suck or blow out the solvent? Which hydraulic pressure do you apply (slow/fast)?
I would be grateful for every hint and help!
Thanks a lot.
Hi Mattihas Korling
Column packing is an art. it need skills and expertise. For good packing you have to optimize your packing parameters such as pressure, slurry solvent, slurry concentration, Packing design, column dimension etc. As we are packing various ID columns (1mm, 1.8 mm, 2.1 and 4.8 mm having various lengths, 15 cm, 10 cm, 5 cm)
I will provide you some basic steps for column packing, I hope it will help you
1) Column dimension: Which ID column you want to pack? You have mentioned only length ( ID= ? length= ? ) calculate the volume of your column by using formula for the calculation of volume of cylinder.
2) Wt of stationary phase: Weight stationary phase equal to the volume of column and dry at 60-70 degree centigrade.
3) Slurry concentration: Usually we use 10 % slurry,
4) Slurry solvent: For C18 phase methanol, 2-propanol, chloroform etc are used as slurry solvent.
5) Packing solvent: Methanol is commonly used solvent for packing C18 phase, 2-propanol can also be used.
6) Check the volume of your reservoir column (The column to which solvent line is attached from top and the column which you want to pack attach at the bottom.
7) Attach the column and pout the slurry from the top and connect solvent line and start pressure.
8) Packing pressure can be used 8000 psi- 15000 psi, your particles are quite big so use around 10,000 psi.
9) Close the gas after 30 minutes and wait until the solvent pressure reach zero.
10) Detach your column and attach necessary union/connectors etc and attach to your HPLC.
For good column packing you have to optimized packing conditions, pressure, time, slury solvent etc.
So practice more and more to find best packing conditions.
I hope it will help you, may be other scholars give you more detail and best way of packing.
Best of luck
You can use ultrasonic bath to produce the slurry. A good practice is to decant few volumes of solvent prior to use the slurry to remove the dust and "under-particles" (much smaller particles) from your slurry.
Regards
G
Thanks for your advice.
I have to pack a 2.5 cm I.D. column with various lengths (20-35 cm) in order to estimate the critical amount of theoretical plates for a difficult peptide purification.
Why do you need to dry the sorbent prior to usage? How long?
I do not really get what you mean by reservoir column and how you apply the pressure?
Here is the link of the column I'm using:
https://de.vwr.com/store/product/2106013/praeparative-hplc-saeulen-selfpacker
So I need to suck the slurry solvent through the bottom of the column and I feel that there is some kind of channel formation during this step.
A 10% slurry is very low concentrated, isn't it? For a 26 cm column I would need 76 g sorbent and 680 mL slurry solvent. I would expect removing the slurry solvent to last 2 hours at least. Phenomenex recommends much less solvent.
How do you apply the packing pressure?
Matthias we are packing narrow bored analytical HPLC columns, the ID of our column are in the range of (1 mm- 4.6 mm). I saw your column which is preparative HPLC column. For our columns we are using alltech slurry packer. I think you can also used this slurry packer as it will be more useful than the piston packing. Yes 10 % slurry will be very diluted in your case, so make a slurry according to your stationary phase and column dimension. No need to inject the slurry from the bottom, you can pored it from the top. For this packing you will need proper setup, including Alltech slurry packer, packing solvent, tubing which push packing solvent to your column, reservoir column (an additional empty column having same ID or little bigger than your column, the tubing from slurry packer will be attach to the top of this reservoir column and your column (the column which you want to pack) should be attach to the bottom end of reservoir column by unions, nuts, bolts etc. N2 gas or air will be used to push the solvent from alltech packer to your column. Adjust the packing pressure (10000 psi-15000 pis) and then put your slurry into the top end of reservoir column which will reach to the column you have attached (your column) close the tubing and start the gas flow. Wait for 20-30 minutes and then close the gas and wait until pressure of the packer get down to make a tight bed in your column. untie your column and your column is ready now for use.
Dear Mattias,
there are 2 critical points for a good performance here.
Luna has to be packed with high pressure and the packing process has to be very smooth.
Did you get fronting or tailing in your performance check?
If you can provide a chromatogram i can for sure give you some advice.
Kind regards
Johannes
Hallo.
Sorry for answering so late.
In fact both. The fronting is due to the autosampler I recognized. Hints for how to prevent tailing are highly welcome.
Thanks for any further advice.
Matthias
Dear Matthias,
i don t believe that your fronting is resulting from your autosampler.
There is a rule of thumb:
Fronting results from less media in the column (not perfectly compressed which means to low packing pressure)
Maybe there are regions in the bed which are not perfectly packed because you had to pump manually during the packing.
Please feel free to contact me if you need more advice.
I fully agree with Ashraf Ali that the MODcol Multipacker system packs the media easily and reproducible with a very good performance and symmetry.
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