Very Large Nucleic Acids on a DNAPac RP Column
Posted: Wed Mar 27, 2019 12:35 pm
I'm having trouble separating and resolving an RNA 9k nucleotide ladder. 9k meaning it contains 10 transcriptions ranging from 300 nucleotides to 9000. The first three transcriptions elute with good separation and resolution. After the third peak, the other transcriptions come out all at once. There are "dips" in the large peak, meaning it's trying to separate, but the larger nucleotides just aren't being retained on the column. Here are my parameters:
I'm using a DNAPac column, 4um, 2.1x100 mm.
MPA: 0.1M TEAA, pH7
MPB: 80/20 0.1M TEAA, pH7/ACN
I have also tried the following mobile phases. Separation and resolution are only slightly better.
MPA: 25mM HAAc
MPB: 25mM HAAc, 50% ACN
Column Temperature is 50 deg C. Flow rate is 0.2 ml/min. I'm running this on a UHPLC system.
Should I try a stronger ion pair? Increase the concentration of these pairs? Try mixing them? Maybe use TFA instead of acetic acid to create the ion pair?
Another possibility that's I'm considering is the column. TF's DNAPac column touts the ability to resolve double stranded DNA and RNA of up to 10K base pairs, and single stranded DNA and RNA of over 10K nucleotides. That's what I need. However, I can't find any example chromatogram in their application methods that demonstrate this. I highest they go is 1500 bp's and 1000 nucleotides. Their application methods is where I got the TEAA mobile phase from.
Any ideas? Any other experiences with the DNAPAc column for very long nucleic acids? I appreciate all feedback, and thank you in advance.
I'm using a DNAPac column, 4um, 2.1x100 mm.
MPA: 0.1M TEAA, pH7
MPB: 80/20 0.1M TEAA, pH7/ACN
I have also tried the following mobile phases. Separation and resolution are only slightly better.
MPA: 25mM HAAc
MPB: 25mM HAAc, 50% ACN
Column Temperature is 50 deg C. Flow rate is 0.2 ml/min. I'm running this on a UHPLC system.
Should I try a stronger ion pair? Increase the concentration of these pairs? Try mixing them? Maybe use TFA instead of acetic acid to create the ion pair?
Another possibility that's I'm considering is the column. TF's DNAPac column touts the ability to resolve double stranded DNA and RNA of up to 10K base pairs, and single stranded DNA and RNA of over 10K nucleotides. That's what I need. However, I can't find any example chromatogram in their application methods that demonstrate this. I highest they go is 1500 bp's and 1000 nucleotides. Their application methods is where I got the TEAA mobile phase from.
Any ideas? Any other experiences with the DNAPAc column for very long nucleic acids? I appreciate all feedback, and thank you in advance.