Highresolution
resolutionanalysis
analysisof
ofOligonucleotides
Oligonucleotideson
onreversed
reversedphase
phasechromatography
chromatography
HighF121018AE

Oligonucleotides analysis on reversed-phase ion-pair chromatography (RP-IPC)
Comparison of retention and separation under various mobile phase conditions
Retention mechanism of

Oligodeoxythymidylic acid, [d(T) 2-20]HC
3(F050218A)

O

P

H3C

・・・

OO

HN

separation even by one-nucleotide difference is achieved. OnOOOCH
3CH
3

Phosphate buffer (pH 6.0)d(pT)2-20 is well retained under TEAA buffer system, and goodO+NH: 1.0 mL/min: 35ºC: UV at 269 nm: 5 µL (5 nmol/mL)

NNOOHNPOOO

O・・・Acetate buffer (pH 6.0)

Flow rate
Temperature
Detection
Injection

three different buffer systems are compared.

OO(F050126E)

Eluent: Hydrosphere C18 (3 µm, 12 nm)
50 X 4.6 mmI.D.
: A) 100 mM bufferB) 100 mM buffer/acetonitrile (80/20)
50-65%B (0-20 min)Separations of oligodeoxythymidylic acid [d(pT) 2-20] underCH3CH
3oligonucleotide on RP-IPC
Triethylamine-Acetic acid buffer (TEAA) (pH 6.0) (Image)

ColumnCH3

the other hand, retention and separation of d(pT)2 -20 underacetate buffer and phosphate buffer are insufficient.

+NHCH
3Ion-pairing reagents that have both positive charge andhydrophobic moiety in molecule, such as triethylamine (TEA)
or dibutylamine (DBA), form ion pair with negatively charged(F050210B)

02468

10

12

14

16

18

oligonucleotides. This interaction contributes to long retention

Surface ofpacking materialminand improvement on resolution.

Applicability to LC/MS analysis
Impact of concentration and types of ion-paring reagent on resolution and signal intensity
ColumnEluent: Hydrosphere C18 (3 µm, 12 nm), 50 X 2.0 mmI.D.
: A) 10 mM triethylamine-acetic acid (pH 6.0)
B) 10 mM triethylamine-acetic acid (pH 6.0)/acetonitrile (80/20)
50-65%B (0-20 min)A) 10 mM di-n-butylamine-acetic acid (pH 6.0)
B) 10 mM di-n-butylamine-acetic acid (pH 6.0)/acetonitrile (50/50)
30-75%B (0-20 min)A) 5 mM di-n-butylamine-acetic acid (pH 6.0)
B) 5 mM di-n-butylamine-acetic acid (pH 6.0)/acetonitrile (50/50)
30-75%B (0-20 min)Flow rate: 0.2 mL/min
Temperature : 35ºCDetection: ESI-negative modeInjection: 5 µL (5 nmol/mL)

Oligodeoxythymidylic acid, [d(T) 2-20]
Scan ESTIC
7.50e7100%

10 mM TEAA 10.0-13.0% CH3CN (0-20 min)
T2T10T20

F060120C-03

010 mM DBAA 15.0-37.5% CH3CN (0-20 min)100

TEA and DBA are both volatile ion -pairing reagents, and applicable toT10%T20T2LC/MS analysis. W hen comparing the separation characteristics ofd(pT)2-20 with those reagents under the same buffer concentration, signal

F060119D-02

intensity and retention with DBA is superior to that with TEA.

05 mM DBAA 15.0-37.5% CH3CN (0-20 min)100

At 5 mM dibutylamine-acetic acid buffer (DBAA) condition, higher signal

T10

intensity of oligonucleotides is achieved even though retention

T20T2%andresolution is slightly decreased. On LC/MS analysis of oligonucl eotides,
5-10 mM DBAA/acetonitrile (or methanol) is recommended to be used asF060118B-020 051015mina standard mobile phase. And then, it is effective to optimize t he types oforganic solvent and gradient condition.

LC/MS analysis of miRNA
5’-pUGG AGU GUG ACA AUG GUG UUG-3’5’-pUGG AGU GUG ACA AUG GUG UUG U-3’A) UV(21 nt, MW 6890.1)(22 nt, MW 7196.3)

mAU
20Column
Eluent

21 nt

22 nt

15.0

17.5

10B) MS

00.0
Int.( x 100000)
52.5

5.0

7.5

10.0

12.5minMIC(m/z 1721.5, m/z 1798.1)
21 nt

22 nt

: YMC-Triart C18 (3 µm, 12 nm), 150 X 2.0 mmI.D.
: A) 10 mM di-n-butylamine-acetic acid (pH 7.5)
B) 10 mM di-n-butylamine-acetic acid (pH 7.5)/acetonitrile (50/50)
62-72%B (0-20 min)Flow rate: 0.2 mL/min
Temperature : 30ºCDetection: A) UV at 260 nm
B) ESI-negative mode
Injection: 4 µL (5 nmol/mL)
System: LC) Shimadzu ProminenceMS) Shimadzu LCMS2020

Mixture of miRNA of 21 nt and 22 nt is separated by using 10mM
DBAA/acetonitrile as a mobile phase and detected with MS.

00.02.55.0

7.5

10.0

12.5

15.0

17.5minCourtesy of M. Yamada, SHIMADZU CORPORATIONYMC Co., Ltd.

http://www.ymc.co.jp

High temperature analysis of oligonucleotides with YMC-Triart C18Effect of mobile phase and column temperature on separation of siRNA duplex
Crude synthetic siRNA duplex (19 bp) :5’-CGU ACG CGG AAU ACU UCG AdTdT-3’3’-dTdTGCA UGC GCC UUA UGA AGC U-5’Condition A : 10 mM DBAA buffer (pH 6.0)/methanolCondition B : 20 mM TEAA buffer (pH 7.0)/acetonitrilesiRNA Duplex

30ºC

siRNA Duplex¥201203010001003.D¥201206220000008.D

Single-stranded siRNA
40ºC¥201203010001006.D¥201206220000014.D¥201203010001009.D¥201206220000017.D

50ºC

60ºC
High resolution

High resolution¥201206220000020.D¥201203010001012.D

024681012Column: YMC-Triart C18 (1.9 µm, 12 nm)
100 X 2.0 mmI.D.
Flow rate : 0.2 mL/min
Detection : UV at 269 nm
Injection : 1 µL (5 nmol/mL)
System : Agilent 1290

14min

024681012141618min

Condition A Eluent : A) 10 mM di-n-butylamine-acetic acid (pH 6.0)
B) methanol
35-60%B (0-15 min)Condition B Eluent : A) 20 mM triethylamine-acetic acid (pH 7.0)
B) acetonitrile
5-12%B (0-20 min)Separation of siRNA duplex under different mobile phase conditio ns at various temperatures with YMC-Triart C18 is shown.
Under both condition A and condition B, peak shape and resolutio n between immediate peaks is improved by increasing the column
temperature.
Due to the improvement of dispersion and distribution velocity w hen increasing column temperature, bio-macro molecules such as RNA andDNA generally exhibit sharper peak shape and improved resolution .
Under condition B at 40 ℃ or higher temperature, two peaks of single -stranded RNA that is generated by denaturation of siRNA duplex a reobserved. This HPLC technique that is utilizing high temperature to generate single-stranded RNA is called “Denaturing HPLC”, and widelyused in the field of gene mutation analysis.
As shown above, denaturation of duplex DNA or RNA is also influe nced by ionic strength (type and concentration), pH and polarity as well astemperature. Those analysis conditions (temperature and mobile p hase) are recommended to be optimized depending on characteristi cs oftarget analyte and purpose of analysis.

% of initial theoretical plate of d(pT)12Durability at pH 6.0 (DBAA buffer) and 65℃
120%

YMC-Triart C18(1.9 µm)

100%
80%
60%
40%

Silica based C18(2.0 µm)

20%
0%0

Test condition

ColumnEluent: 1.9 µm or 2.0 µm, 12 nm, 50 X 2.0 mmI.D.
: A) 10 mM di-n-butylamine-acetic acid (pH 6.0)
B) methanol
30-50%B (0-20 min)Flow rate: 0.4 mL/min
Detection: UV at 269 nm
Temperature : 65ºCSample: Oligodeoxythymidylic acid, [d(T)2-20]
Injection: 1 µL (5 nmol/mL)
System: Agilent 1290

Combination of neutral buffer containing amino ion -paring reagent and high
100

200

300

400

500

600

700

Injection

temperature is useful for high -throughput analysis of oligonucleotides ordenaturing HPLC. However, conventional silica -based reverse-phasecolumn can hardly used with such condition due to the poor durab ility.
YMC-Triart C18 using inorganic/organic hybrid silica with thorough surface
modification offers excellent durability at elevated temperature and pH.
YMC-Triart C18 is ideal for oligonucleotides analysis.

YMC Co., Ltd.

http://www.ymc.co.jp