HFIP for Nucleic Acid Analysis

1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) is frequently used as a mobile phase additive for analysis of oligonucleotides below 100 nucleotides (nt) and their impurities, due to the high sensitivity and separation it enables. However, there are significant differences in LC-MS sensitivity and even detection of unidentified peaks depending on the manufacturer, product grade, and lot. This product is specifically tested using LC-MS for each batch, so researchers can be confident in their reagents.

Features

Suitable for analysis of oligonucleotides using HPLC and LC-MS
Suitable for analysis of oligonucleotide impurities (short-mers, long-mers, phosphodiester [PO] impurities in phosphorothioated [PS] products)
Each product lot tested using LC-MS

Performance comparison

Using the oligonucleotide (Poly(dT)19), LC-MS sensitivity was compared between this product and competitor HPLC and LC-MS grade products.

Nacalai Tesque product Company A product
(HPLC grade) Company B product (LC-MS grade)

LC conditions
Column	COSMOCORE 2.6C18　2.1 mm I.D. × 100 mm
Mobile phase	A : 100 mM HFIP-15 mM Triethylamine (TEA) B : Solvent A / Methanol = 1 / 1 (v / v) B conc. 20 → 50%（0 → 20 min）
Flow rate	0.2 mL/min
Temperature	40°C

MS conditions
Equipment	LCMS 2050 (Shimadzu)
Ionization	ESI/APCI (Negative), TIC
Mode	Scan
Mass range	550-2000
Nebulizing gas flow	2.0 L/min
Drying gas flow	5.0 L/min
Heating gas flow	7.0 L/min
DL temperature	200°C
Desolvation temperature	450°C
Interface voltage	-2.0 kV

Compared to the company A product (HPLC grade), our product showed about twice the sensitivity. Additionally, it had about the same sensitivity as the company B product (LC-MS grade).

Comparing 4 different lots, the difference in sensitivity was found to be within 10%. Furthermore, there was no significant difference in purity and metal content.

Analysis by LC-MS

Phosophorothioated oligonucleotides

By optimizing the analysis conditions, we were able to separate the target compound from impurities that are produced during synthesis.

Conditions
Column	COSMOCORE 2.6C18　2.1 mm I.D. × 100 mm
Mobile phase	A : 100 mM HFIP-15 mM Triethylamine (TEA) B : Solvent A / Acetonitrile / Methanol = 2 / 1 / 1 (v / v / v) B conc. 14 → 20%（0 → 20 min）
Flow rate	0.2 mL/min
Temperature	65°C
Ionization	ESI/APCI (Negative), SIM
Sample conc.	100 µM (FLP) and 25 µM (others)
Inj. Vol.	1 µL

DNA ladder

Because hydrophobicity increases with oligonucleotide length, longer strands are retained for longer.

Conditions
Column	COSMOCORE 2.6C18　2.1 mm I.D. × 100 mm
Mobile phase	A : 100 mM HFIP-15 mM Triethylamine (TEA) B : Solvent A / Acetonitrile / Methanol = 2 / 1 / 1 (v / v / v) B conc. 7.5 → 27.5%（0 → 20 min）
Flow rate	0.2 mL/min
Temperature	65°C
Ionization	ESI/APCI (Negative), TIC
Sample conc.	10 µM
Inj. Vol.	3 µL

ssDNA (PO and PS forms), ssRNA

Hydrophobicity increases in the order of RNA < DNA (PO) < DNA (PS), and the strands elute in the same order. Hydrophobicity also changes depending on the DNA sequence, so strands with similar length may have different retention times.

Conditions
Column	COSMOCORE 2.6C18　2.1 mm I.D. × 100 mm
Mobile phase	A : 100 mM HFIP-15 mM Triethylamine (TEA) B : Solvent A / Methanol = 1 / 1 (v / v) B conc. 20 → 60%（0 → 20 min）
Flow rate	0.2 mL/min
Temperature	40°C
Ionization	ESI/APCI (Negative), SIM
Sample conc.	Sample conc.
Inj. Vol.	3 µL

References

Separation and purification of short-, medium-, and long-stranded RNAs by RP-HPLC using different mobile phases and C18 columns with various pore sizes
Ozaki M, et al. Anal. Methods. 2024;16:1948-1956.
https://doi.org/10.1039/D4AY00114A
* Featured on Front Cover

Separation of long-stranded RNAs by RP-HPLC using an octadecyl-based column with super-wide pores
Kuwayama T, et al. Anal. Sci. 2023;39:417-425.
https://doi.org/10.1007/s44211-022-00253-w
* Selected as Hot Articles 2023

Ozaki M, et al. Medical Science Digest December Special Issue. 2023, 49, p.40-43.