Hi all, i came here looking for some advice about the separation of a few µg ssRNA in range from 1000bp – 4000bp on HPLC.
I know that IP-HPLC with 0.1M TEAA buffer and MeCN allows such separations if you have the right setup. I have a agilent 1260 system and analytic fraction collector available, but only silica-based RP columns – my best setup so far was using a Agilent Poroshell 120 C18 150x4.6mm 4µm. I already figured out that a non-porous PS/DVB column should be better, so i think about buying one. I saw the following suitable options: GE Resource RPC, Machery-Nagel Nucleogel RP 300A, Dionex DNAPac RP, Transgenomic DNASep or RNASep
Has someone made good experience and can recommend me a column? Also I’m very open for other suggestions.
Cheers,
Paul
Hi Paul,
we use for analysis and purification of oligonucleotides (30-50 bp) the following setup:
Columns: Analytical: XBridge C18 5µm, 2.1 x 100 mm
Preparative: XBridge BEH C18 OBD Prep Column, 10 x 50 mm Eluents: A: Water / Acetonitrile (95/5) (V/V), 15 mM Hexylamine, 50 mM
Hexafluoroisopropanol
B: Water / Acetonitrile (50/50) (V/V), 15 mM Hexylamine, 50 mM
Hexafluoroisopropanol
Injection volume: PreAnalytical: 0.1 µL
Preparative: 20.0 µL
QC: 2.0 µL
Temperature: 60°C
Flow: Analytical/QC: 1.5 mL/min
Preparative: 3.5 mL/min
Gradient: Analytical: Time/min %A %B
0.00 74 26
15.00 55 45
20.00 55 45
20.01 0 100
25.00 0 100
25.01 74 26
30.00 74 26
Preparative: Time/min %A %B
0.00 74 26
51.40 55 45
60.00 55 45
60.01 0 100
74.00 0 100
74.01 74 26
85.50 74 26
Hardware: G1316C Infinity TCC (column oven)
G7129B 1290 Vialsampler (autosampler)
G4204A 1290 Quat Pump (pump)
G4212B 1260 DAD (DAD)
Wavelength: 260 nm, 2.5 Hz Scanrate
G1364C Analyt FC (analytical fraction collector)
G6135B LC/MSD XT (MS)
Scanrange: 600-3000 Da, 1.0 Hz Scanrate
Water: Water for RNA work, DEPC-treated and Nuclease free
Autoclave Vials: Nalgene Cryogenic Vials
Software: OpenLab CDS, Chem Station Edition, Rev. C.01.08
[216] Agilent MassHunter Walkup-System Vers.
C.03.01 [B3185.0]
Maybe you can use these settings as a starting point for you.
Best regards
Markus
Hi Markus Christ , thank you for your answer. I manage to separation of small oligonucleodites on the Agilent Poroshell 120 C18 150x4.6mm 4µm using a gradient of 90%(A) TEAA 0.1 10%(B)MeCN to 80%(A) 20%(B) over 30 min, flow 0.7ml/min, T = 60°C
However, this approach does not seem to be suitable for larger RNA eg. 1000-4000 bp. I get no to very poor seperation even when i extend my run to 60 min.
Hello, Paul,
it looks like your gradient is way too shallow. Try 50% acetontrile. 20 % are too little. I would also increase the flow. You have 0.7 ml/min with a 4.6 column. We use 1.5 ml/min with a 2.1 column. Think about changing the column. We have had very good experiences with the XBridge. Especially columns for oligonucleotides from different manufacturers (Agilent, Waters, Phenomenex etc.) died relatively fast (after 200-300 injections). The XBridge seems to be very robust.
Regards
Markus
Thanks Markus,
i will try again with a different gradient and maybe also try your buffer system and see how it goes. I'll be posting here about any progress I make.
Cheers,
Paul
Markus, just wanted to kniw what back pressure you get with a 1.5 ml/min flow ratr with a 2.1 mm diameter column?
Markus, aldo i would like to know your mobile ohase compisition and pH if you can share.
Hi Navid,
the back pressure in our system is in the range of 300-400 bar.
Regards
Markus
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