YMC
Oligonucleotide
Columns
IP-RP
AEX
SEC
HILIC

YMC Oligonucleotide Columns

Nucleic acids

Different types of therapeutic nucleic acids
Antisense
oligonucleotide/
miRNA

siRNA

Aptamer

gRNA

mRNA

PlasmidType(s) of
nucleic acids

ssDNA, ssRNA

dsRNA

ss/dsRNA,
ss/dsDNAss/dsRNA

ssRNA

dsDNA

Length (mer)

11–24

19–23

20–100100

1,000–7,000

4,000–10,000

Mass range (kDa)

5

15

5–30

30

300–2,000

2,000-7,000

Modifications(phosphates,
ribose,nucleobase)

Yes

Yes

Yes

Yes

Nucleobases
only (5‘ cap,3‘ Poly(A) tail)No

Chemicalconjugates /carrier systems

Yes/
carrierpossibleYes/
carrierpossibleYes/
carrierpossibleNo/
carrierpossibleNo/
Lipid
nanoparticles

No/
carrierpossibleModeof action

mRNA
degradation

Genomeediting

Proteinproduction

Gene
expression

Examplestructure

2

mRNA
Protein function
degradation
modulation

Content

Page
Oligonucleotide Application Notes ..................................... 6–10
IP-RP Oligonucleotide Columns ........................................... 12–16
YMC Accura Bioinert Hardware.. ............................................ 17–20
AEX Oligonucleotide Columns.............................................. 22–24
SEC Oligonucleotide Columns............................................... 26–28
HILIC Oligonucleotide Columns........................................... 30–32
Ordering Information. . ................................................................... 34–38

3

YMC Oligonucleotide Columns

Phase selection guide

Oligonucleotides / Nucleic Acids
IP-RP
YMC-Triart
C18

BioProIEX QFSEC
YMC-Pack
Diol-120

YMC-Triart
Bio C18

YMC-Pack
Diol-200

Hydrosphere
C18

YMC-Pack
Diol-300

YMC-Triart
C8
YMC-Triart
Bio C4

4

AEX

HILIC
YMC-Triart
Diol-HILICOligonucleotide
Applications

5

YMC Oligonucleotide Columns

Oligonucleotide applications
Synthetic oligonucleotides
mAU

1. 5’-CAC UGA AUA CCA AU-3’ (14mer)
2. 5’-UCA CAC UGA AUA CCA AU-3’ (17mer)
3. 5’-UCA UCA CAC UGA AUA CCA AU-3’ (20mer)
4. 5’-GUC AUC ACA CUG AAU ACC AAU-3’ (21mer)

4

15

RP

3

10

1

2

5

0
0

Column:Part No.:
Eluent:Gradient:

2

4

6

8

YMC-Triart C18 (1.9 µm, 12 nm) 50 x 2.1 mm ID
TA12SP9-05Q1PT
A) 200 mM HFIP*-8 mM triethylamineB) methanol
10–20%B (0–10 min)10 min*Flow rate:
Temperature:
Detection:
Injection:

0.42 mL/min
65°C
UV at 260 nm
1 µL (2–4 nmol/mL)*hexafluoroisopropanol

LC/MS analysis of miRNARP

5’-pUGG AGU GUG ACA AUG GUG UUG-3’
5’-pUGG AGU GUG ACA AUG GUG UUG U-3’
Column:Part No.:
Eluent:Gradient:
Flow rate:(21mer, MW 6,890.1)(22mer, MW 7,196.3)

YMC-Triart C18 (3 µm, 12 nm) 150 x 2.0 mm ID
TA12S03-1502WTA) 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)
0.2 mL/min

Temperature:
Detection:
Injection:
System:Analysis of phosphoramidites
mAU
mAU

DMT-dT-CE phosphoramidite (dT)DMT-dT-CE phosphoramidite (dT)

400
400

Rs=3.01

5.0
5.0

10.0
10.0

15.0
15.0

min
min

DMT-dA(Bz)-CE phosphoramidite (dA)
DMT-dA(Bz)-CE phosphoramidite (dA)

Rs=2.07

Gradient:

6

5.0
5.0

10.0
10.0

15.0
15.0

min
min

DMT-dC(Bz)-CE phosphoramidite (dC)
DMT-dC(Bz)-CE phosphoramidite (dC)

Rs=3.10

750
750
500
500

200
200

Column:Part No.:
Eluent:

0
0 0.0
0.0
mAU
mAU

400
400

0
0 0.0
0.0

Rs=2.34

200
200

100
100

mAU
mAU

600

400
400

200
200

0
0 0.0
0.0

RP

DMT-dG(iBu)-CE phosphoramidite (dG)
DMT-dG(iBu)-CE phosphoramidite (dG)

mAU
mAU
600

300
300

30 ºC
A) UV at 260 nm
B) ESI-negative mode
4 µL (5 nmol/mL)LC) Shimadzu ProminenceMS) Shimadzu LCMS2020

250
250
5.0
5.0

10.0
10.0

15.0
15.0

YMC-Triart C18 (5 µm, 12 nm) 150 x 4.6 mm ID
TA12S05-1546PTH
A) 20 mM triethylammonium acetate (pH 7.0)/acetonitrile (90/10)
B) acetonitrile
45–95%B (0–12 min), 95%B (12–17 min)

min
min

0
0 0.0
0.0

Flow rate:
Temperature:
Detection:
Injection:

5.0
5.0

10.0
10.0

1.0 mL/min
30 °C
UV at 254 nm
4 µL (1.0 mg/mL)

15.0
15.0

min
min

Oligonucleotide applications
LC-HRMS analysis of the antisense oligonucleotide Mipomersen (Kynamro®)
Total ion chromatogram
100

80

90

70

80

Relative abundance

60
50
40
30
20

1

2

2.

70
60
50
40
30
10
0

0.0

1.0

1.0

2.0

2.0

3.0

3.0

4.0

4.0

5.0

5.0
Time
(min)

80

80

70
60
50
40

Relative abundanceRelative abundance

100 RT:100
4.60-5.00
RT: 4.60-5.00

44[M-4H]

50

88[M-8H][M-8H]
40
30

30

1793.0092

90 901793.0092

10

10

0

500

500

1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 m/z

Column:Part No.:
Eluent:Gradient:

Flow rate:
Temperature:
Injection:
System:2237.5110
2237.5110[M-3H][M-3H]3-3-

80 80
70 70
60 60

4- 4[M-4H][M-4H]

50 50

8[M-8H][M-8H]
40 40

30
20
10

1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 m/z

7.0100100
RT:RT:
3.80-4.20
3.80-4.20

896.007420
896.00742002390.9664
2390.9664

60[M-4H]

7.0

6.0

Mass
spectrum
Mass
spectrumofofLNA-mipomersen
LNA-mipomersen

33[M-3H][M-3H]

70

6.0Time (min)Relative abundance

0.0

MassMass
spectrumof mipomersen
spectrumof mipomersen
90

mipomersen

20

10

90

1. LNA- mipomersen (I.S.)
2.1. mipomersen
LNA- mipomersen (I.S.)

2

Relative abundanceRelative abundance

90

0

1

Total ion chromatogram

100

RP

0

8-

30

1677.9098

1677.9098

20

838.4558

838.4558

10
0

500

500

1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 m/z

1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 m/z

YMC-Triart C8 metal-free PEEK-lined (1.9 μm, 12 nm)*1 100 x 2.1 mm ID
TO12SP9-10Q1PTP
A) water/triethylamine/HFIP*2 (100/0.4/2; triethylamine 28.0 mM, HFIP 135.8 mM)B) methanol/triethylamine/HFIP (100/0.4/2)[Sample separation step]
10–40%B (0–5.0 min)[Column wash steps]
40–70%B (5.0–5.1 min), 70%B (5.1–7.0 min), 70–10%B (7.0–7.1 min), 10%B (7.1–8.0 min), 10–90%B (8.0–8.1 min), 90%B (8.1–9.0 min),
90–10%B (9.0–9.1 min), 10%B (9.1–10.0 min), 10–90%B (10.0–10.1 min), 90%B (10.1–11.0 min), 90–10%B (11.0–11.1 min)
0.3 mL/min
50°C
10 μL (1000 ng/mL)LC) Vanquish Binary Pump H system
HRMS) Orbitrap HRMS Q Exactive Plus

*1 Prewash the column prior to the first use with water/methanol/phosphoric acid (70/30/0.1) for 1 hour
*2 1,1,1,3,3,3-hexafluoro-2-propanol
Reference: Y. Sun et al, Development of a bioanalytical method for an antisense therapeutic using high-resolution mass spectrometry,
Bioanalysis, 2020 NOV 26, doi: 10.4155/bio-2020-0225.

7

YMC Oligonucleotide Columns

Oligonucleotide applications
mRNA coupled to enhanced green fluorescent protein (EGFP)

RP

mAU

200

100

0

5

0
Column:Part No.
Eluent:Gradient:
Flow rate:

YMC Accura Triart Bio C4 (3 µm, 30 nm) 100 x 2.1 mm ID
TB30S03-10Q1PTC
A) 50 mM TEAA* (pH 7.0) / acetonitrile (95/5)
B) acetonitrile
5–10%B (0–10 min)
0.2 mL/min

Temperature:
Detection:
Injection:
Sample:min
80 °C
UV at 254 nm
2 µL (0.25 mg/mL)
CleanCap® EGFP mRNA (5 moU)
(TriLink Bio Technologies)*triethylammonium acetate

IEX analysis of EGFP mRNA (996mer)

AEX

mAU

20

10

0
0
Column:Part No.
Eluent:Gradient:
Flow rate:

8

BioPro IEX QF (5 µm) 100 x 4.6 mm ID
QF00S05-1046WPA) 10 mM NaOH (pH 12)
B) 10 mM NaOH (pH 12) containing 2 M NaCl
0–100%B (0–9 min), 100%B (9–13 min)
1.0 mL/min

5

10
Temperature:
Detection:
Injection:
Sample:15 °C
UV at 260 nm
5 µL (0.025 mg/mL)
CleanCap® EGFP mRNA (5 moU)
(TriLink Bio Technologies)minOligonucleotide applications
siRNA duplex under denaturing conditions

RP

1

mAU

1. siRNA duplex

40

30

antisense strand

sense strand

20

10

0
0
Column:Part No.:
Eluent:2

4

6

YMC Accura Triart Bio C18 (1.9 µm, 30 nm) 50 x 2.1 mm ID
TA30SP9-05Q1PTC
A) 15 mM TEAA* (pH 8)B) methanol
5%–20%B (0–15 min)Gradient:

8

10

12

Flow rate:
Temperature:
Detection:
Injection:
Sample:0.42 mL/min
65 °C
UV at 260 nm
1 µL (5 nmol/mL)
siRNA duplex

14

min

*triethylammonium acetate

siRNA duplex under denaturing conditions

AEX

1

mAU

1. siRNA duplex

10

sense strand

antisense strand

500Column:Part No.
Eluent:Gradient:

2

4

BioPro IEX QF (5 µm) 100 x 4.6 mm ID
QF00S05-1046WPA) 10 mM NaOH
B) 10 mM NaOH containing 1 M NaClO4
30%–37%B (0–15 min)

6

8

10
Flow rate:
Temperature:
Detection:
Injection:
Sample:

12

14

min

1.0 mL/min
25 °C
UV at 260 nm
4 µL (5 nmol/mL)
siRNA duplex

9

YMC Oligonucleotide Columns

Oligonucleotide applications
Challenging phosphorothioate oligonucleotides

RP5’-U^C^A^U^C^A^C^A^C^U^G^A^A^U^A^C^C^A^A^U-3’ (RNA 20mer)
5’-G^U^C^A^U^C^A^C^A^C^U^G^A^A^U^A^C^C^A^A^U-3’ (RNA 21mer)^=Phosphorothioated

1

mAU

2

5
4
3
2
1
0
0
Column:Part No.:
Eluent:Gradient:

1

2

3

YMC Accura Triart Bio C18 (1.9 µm, 30 nm) 50 x 2.1 mm ID
TA30SP9-05Q1PTC
A) 15 mM triethylamine - 400 mM HFIP*B) methanol
10–20%B (0–10 min)

4

5

6

min

Flow rate: 		 0.42 mL/min
Temperature: 65 º C
Detection:		 UV at 260 nm
Injection:		 1 µL (each 1.0 nmol/mL)*1,1,1,3,3,3-hexafluoro-2-propanol

AEX analysis of siRNA duplex under non-denaturing conditions

AEX

mAU

30

1. siRNA duplex

20

10

0
0
Column:Part No.:
Eluent:Gradient:

10

BioPro IEX QF (5 µm) 100 x 4.6 mm ID
QF00S05-1046WPA) 20 mM Tris-HCl (pH 8.1)
B) 20 mM Tris-HCl (pH 8.1) containing 1 M NaClO4
25%–40%B (0–15 min)

5

10
Flow rate:
Temperature:
Detection:
Injection:
Sample:

1.0 mL/min
25 °C
UV at 260 nm
4 µL (5 nmol/mL)
siRNA duplexminIP-RP

IP-RP

11

YMC Oligonucleotide Columns

IP-RP – UHPLC/HPLC Selectivities
Features• Selection of C18, C8 and C4 columns• For ion-pairing (IP-RP) UHPLC and HPLC
• pH- and temperature stable phases• Superior reproducibility• Bioinert coated YMC Accura column hardware
Selectivities for oligonucleotides
YMC-Triart
C18
Base particle
Modification

YMC-Triart
Bio C18

YMC-Triart
C8

YMC-Triart
Bio C4organic/inorganic hybrid silica

Hydrosphere
C18
silicaC18 (USP L1)

C18 (USP L1)

C8 (USP L7)

C4 (USP L26)

C18 (USP L1)

Particle size / µm

1.9, 3, 5

1.9, 3, 5

1.9, 3, 5

1.9, 3, 5

2, 3, 5

Pore size / nm

12

30

12

30

12

pH range

1.0 – 12.0

1.0–12.0

1.0–12.0

1.0–10.0

2.0–8.0

Temperature range

pH < 7: 90 °C
pH > 7: 50 °C

pH < 9: 90 °C
pH > 9: 50 °C

pH < 7: 90 °C
pH > 7: 50 °C

pH < 7: 90 °C
pH > 7: 50 °C

50 °C

Column hardware

bioinert coated YMC Accura

stainless-steel

Bioinert
hardwareavailable!
Bioinert YMC Accura Triart columns are available for improved sensitivity and peak shape of coordinating compounds
such as nucleotides or oligonucleotides.

12

IP-RP – UHPLC/HPLC Selectivities

Column Selection Tool according to nucleotide length
Length (-mer)

10

20

50

100

150

300

500

1,000

YMC-Triart C18
12 nm
pore

YMC-Triart C8First choice es
otid
for oligonucle gths
nof various leHydrosphere C18

YMC-Triart Bio C18

30 nm
pore

YMC-Triart Bio C4

Appropriate oligonucleotide length range

Enhanced retention and resolution even at low buffer concentrations

100 mM Buffer

10 mM Buffer

mAU

mAU

15

Hydrosphere C18
3.0 µm

15

10

10

5

5

0

0
0

2

4

6

8

10

12

14

16

18

mAU

min*

15

YMC-Triart C18
1.9 µm

5

6

8

10

12

14

16

18

min*

tion
superior resolu
S& LC-M
compatibility

0
2

4

6

8

10

12

14

16

18

min*

mAU

mAU

15

15

10

10

5

5

0

2

4

6

8

10

12

14

16

18

min*

0

2

4

6

8

10

12

14

16

18

min*

0

2

4

6

8

10

12

14

16

18

min*

0
0

2

4

6

8

10

12

14

16

18

min*

mAU

mAU

15

15

10

10

5

5

0

0
0

Gradient:
Flow rate:

4

5

0

Column:Eluent:2

100Agilent AdvanceBio
Oligonucleotide
2.7 µm

8

15

10

0

Waters Acquity UPLC
Oligonucleotide BEH C18
1.7 µm

0

mAU

2

4

6

8

10

50 x 2.1 mm ID
A) 100 mM or 10 mM triethylamine-acetic acid (pH 6)
B) A/acetonitrile (80/20)
50–65%B (0–20 min)0.21 mL/min

12

14

16

18

Temperature:
Detection:
Injection:
Sample:

min*

35 °C
UV at 260 nm
2 µL (5 nmol/mL)
Oligodeoxythymidylic acid [d(pT)2-20]

The separation of oligo(deoxythymidylic acids), d(pT)2-20, was compared using 100 mM or 10 mM triethylammonium
acetate (TEAA) buffer, under the same gradient conditions. Both Hydrosphere C18 and YMC-Triart C18 showedenhanced retention and resolution compared to other commercially available C18 phases designed foroligonucleotide analysis, even at the low ion-pairing buffer concentration such as 10 mM. The higher concentration
provides stronger retention and superior resolution of oligonucleotides, although a lower concentration has theadvantages of increasing the signal intensity and reducing system contamination in HPLC-MS analysis.

13

YMC Oligonucleotide Columns

IP-RP – UHPLC/HPLC Selectivities
Optimum resolution for short and long poly(dT) oligonucleotides

Short poly(dT)

I.S.

30

60

100
80

120

40

15

30

30
40

20

10

25

20

10

25

Long poly(dT)

YMC-Triart Bio C18

60
40

100
80 120

15

XBridge Oligonucleotide BEH C18

I.S.

25 30

20

10

15
40

30
25

10

80 12060 100

30

40

20

40

15

DNAPac RP100
60 80 12060

100 120
80

30
I.S.

25

10
15

(Broadening)

0

40

20

5

10

15

20

25

min*

Columns:

YMC-Triart Bio C18 (3 µm, 30 nm) 50 x 2.1 mm ID
XBridge Oligonucleotide BEH C18 (2.5 μm, 13 nm) 50 x 2.1 mm ID
DNAPac RP (4 μm, proprietary) 50 x 2.1 mm ID
Part No.:
TA30S03-05Q1PTH
Eluent:A) 4 mM TEA* - 100 mM HFIP**
B) methanol
Gradient:
0.5%B/min, initial %B=5
Flow rate:
0.42 mL/min
Detection:
UV at 260 nm
Temperature: 65 °C
Injection:
1.0 µL
Sample:Poly(dT) oligonucleotides*trietylamine, **1,1,1,3,3,3-hexafluoro-2-propanol

The YMC-Triart Bio C18 column demonstrates a better resolution, higher recovery and reproducibility of poly(dT)oligonucleotides compared to the other two competitor columns. Longer poly(dT) oligonucleotides (60–120mer)
were separated poorly by the competitor’s hybrid silica based column, whereas YMC-Triart showed high resolution
for oligonucleotides of all sizes. Peak areas and therefore recoveries of shorter poly(dT) oligonucleotides (10–40mer) were much smaller when separated using the dedicated DNA competitor column. In addition, YMC-Triart
Bio C18 showed reproducible behaviour such as consistent peak areas. This makes YMC-Triart Bio C18 an ideal tool
for analysis of poly(dT) oligonucleotides.

14

IP-RP – Temperature and mobile phase effects
Versatile wide pH and temperature stability
15 mM TEA - 400 mM HFIP (pH 8, 90 °C)

Phosphate buffer (pH 11.5, 40 °C)

120%

YMC Accura Triart Bio C18

W0.5 per 1st run (%)

100%

YMC-Triart C18 ExRSYMC-Triart C18/C8
YMC-Triart Bio C4
Hybrid based C18
Hybrid based C18
Silica based C18

80%
60%
40%
20%
0%

0

20

40

60

80

100

Time (hr)Column:Eluent:Flow rate:
Temperature:Sample:Column:Eluent:YMC Accura Triart Bio C18 (1.9 μm, 30 nm), 50 x 2.1 mm ID
A) 15 mM TEA-400 mM HFIP (pH8)
B) methanol
Gradient:
6–16%B (0–10 min)Flowrate:
0.42 mL/min
Temperature: 90 °C
Sample:20mer RNA

5 µm, 150 x 4.6 mm ID
50 mM K 2HPO4-K3PO4 (pH 11.5) / methanol (90/10)
1.0 mL/min
40°C
benzyl alcohol

Effect of four different ion pair reagents on the denaturation of a siRNA duplex
at different temperatures
BA
initial=3%B
mAU

DenaturedRs(1,2)=2.63

1

2

65 °C

TEA
initial=7%B
mAU

60

DenaturedmAU

Denatured

1,2,3

40

40

20

20

20

20

2

4 min

4

4.4

0

4.8 min

2

4

3.6

min

20

3
0

2

4

6

8

10

12

14 min30Intact

2

4

6

mAU

3

2 1

20

8

2

4

6

8

10

12

14

16 18 min

10

6

8

10

YMC Accura Triart Bio C18 (1.9 µm, 30 nm), 50 x 2.1 mm ID
TA30SP9-05Q1PTC
A) 8 mM alkylamine - 100 mM HFIP* (pH 8)B) methanol
Gradient:
1%B/min
Flow rate:
0.42 mL/min
Temperature: 65 °C, 45 °C or 25 °C
Detection:
UV at 260 nm
Injection:
1 µL (5 nmol/mL)

12

mAU

14 min

4

0

0

2

4

6

8

min

8.6

9

min

min

10

15

2 1

20

2 1
5

4.8

Intact

12
4

4.4

1,2,3

20

mAU

3

2

4 min

10.4

Intact

30

0

9.6

3

2 1

10
0

9.2

1

0 2 4 6 8 10 min

min

20

10

2

10

IntactmAU

2Denatured

mAU
mAU

20

0

min

2 1

40

2 1

mAU

4

Partially

PartiallymAU

3.6

0 2 4 min

4 min

1,2,3

mAU

40

Intact25 °C

DenaturedRs(1,2)=1.98
2 1

DBA
initial=30%B

40

0

45 °C

HA
initial=38%B

3
20

25 min

3

10
0

5

10

15

min

1. sense strand
2. antisense strand
3. siRNA duplex

Column:Part No.:
Eluent:Sample:siRNA duplex, sense strand, antisense strand
5’-CGU ACG CGG AAU ACU UCG AdTdT-3’ sense strand
3’-dTdTGCA UGC GCC UUA UGA AGC U-5’ antisense strand*1,1,1,3,3,3-hexafluoro-2-propanol

15

YMC Oligonucleotide Columns

IP-RP – Expert Tips: Ion-pairing salts
100

100

Effect of composition and salt concentration of ion-pairing mobile phaseon the separation and signal intensity
50

0

0

5

10

15

50

20

Comparison of separation and ESI-MS signal intensity using different ion-pairing buffers
and organic solvents

methanol
10 mM TEAA (pH 6.0)

100

0

0

0

0

0

15

10

15

50

25 min

10

15

20
20

15

15

20

20

20

15
15

20
20

15
15

20
20

15

20

0 25 min
0 0
0

15

20

16

0

25 min

5

5

10

10

15

15

20

20

0

25 min

100

50

50
50

0

0

25 min

100

50

5
5
5

5

10
10
10

10

15
15
15

15

20
20
20

20

25 min25 min

0

00
0

5
5
5

5

10
10
10

10

15
15
15

15

20
20
20

20

25 min25 min33.6–56% methanol (0–20 min)

15
15

20

20

20
20

20
20

0

0 25 min
0 025 min
0

10

50

0

100

50

50
50

0

25 min25 min0
25 min25 min025 min

0
0 0
0

0
0
0
0
0 25 min
0
100
100

5

5

5

10

10

10

15

15

15

20

20

20

25 min25 min

100

100
100

0

0

5
5

5

10
10

10

15
15

15

20
20

20

25 min25 min

5
5
5

5

10
10
10

10

15
15
15

15

0

20
20
20

20

25 min25 min00 25 min00
25 min
0
100
0
100
100
100
0

0
0 0
0

5
5
5

5

10
10
10

10

15
15
15

15

20
20
20

20

25 min25 min0 25 min
0 25
0 min
0

0
0 0
0

15–37.5% acetonitrile (0–20 min)

5

5

5

10

10

10

15

15

15

20

20

20

25 min25 min25 min

0

5
5

5

10
10

10

15
15

15

20
20

20

25 min25 min25 min7–35% methanol (0–20 min)

50
50

0
0

0
0

5
5

10
10

15
15

20
20

25 min25 min

5
5

10
10

15
15

20
20

25 min
min
25

100
100

Note: The data for HFIP-TEA/acetonitrile was             not obtained because of its immiscibility

50
50

0
0

0
0

Hydrosphere C18 (3.0 µm, 12 nm) 50 x 2.0 mm ID
HS12S03-0502WT
0.2 mL/min
35 ºC
UV at 269 nm (red), ESI negative mode (orange)
5 µL (25 pmol/mL)
Oligo(deoxythymidylic acid) [dp (dT) 2-20]

5
5
5

5

10
10
10

10

50

50

0

10
10

50
50
50

0
0
0
0
0 25 min
0
100
100

0
0 min
0 25
0

10

10
10

50

50

0
0 025 min
0

5
5

10

100

100

50

50

5

5
5

100

100

50

5

50

100

50

5

0 0
0
100
100

25 min50

100

he mobile phase composition has different effects on
the separation and signal intensity in electrospray ionisation mass spectrometry (ESI-MS) of oligonucleotides. Using different gradient conditions, acceptable retention andresolution can be achieved (upper UV chromatograms; redtrace) for each separation by optimising the gradient slopeof the organic solvent regardless of the type of mobile
phase. The ESI-MS intensity is significantly influenced by
25 min

15

15
15

0

50

Column:Part No.:
Flow rate:
Temperature:
Detection:
Injection:
Sample:25 min

10
10

15

10

50

50

100

50

50

25 min
min
25

10

10
10

5

100

50

0

50
50

50

25 min25 min55510

15–37.5% acetonitrile (0–20 min)00
0 25 min00
25 min
0
100
0
100
100
100

100

25 min

5

0

100

100

50

50

50

25 min

0
0 0
0

5

5

100

100

0

25 min50

100

33.6–56% methanol (0–20 min)20

10–13% acetonitrile (0–20 min)

0
0 0
0

100
100

25 min

15

50

100

50

25 min

T

20

50

100

25 min

20

20

10

50

50

25 min5 mM DBAA (pH 6.0)

15

15

15

acetonitrile

5

50

0
0
0 25 min
0
0 0
0
100
0
100
100
100

50
50

400 mM HFIP-TEA (pH 7.0)

15

15

50

50

100

10

10

0

50

50

20
20

100

100

20
20

15

10 mM DBAA (pH 6.0)

100

15

10

50

50

100

10

5

100

100

0

5

50

100

50

0

0

50

100

100

50

50

100

0

22–25% methanol (0–20 min)100

0

25 min100

eluent abbreviations
TEAA: triethylammonium acetate
DBAA: di-n-butylammonium acetate
HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol
TEA:
triethylamine

the type and concentration of ion-pairing buffer as shownin the lower MS chromatograms (orange trace). HFIP-TEA
buffer/methanol systems provide the maximum MS intensity. Enhanced retention and MS intensity are obtained using 10 mM DBAA buffer compared to 10 mM TEAA buffer,and the lower DBAA concentration results in approximately 1.5–3 times increase in the intensity without any change
in the concentration of organic solvent.

5
5
5

5

10
10
10

10

YMC Accura bioinert hardware
Bioinert columns for bioseparations and coordinating compounds• Exceptional peak shapes with high sensitivities• Excellent recoveries without column preconditioning• Superior reproducibility and no carry-over effects• Ideal for highly sensitive LC/MS analyses

Specifications
YMC Accura Triart

YMC Accura BioPro IEXModifications

C18, Bio C18, C8, Bio C4, Diol-HILIC

QF

Particle Size

1.9, 3 and 5 µm

3 and 5 µm

Column hardware

stainless-steel with bioinert coating

Frit hardware

stainless-steel with bioinert coating
1.9 µm: 100 MPa (15,000 psi)
3/5 µm: 45 MPa (6,525 psi)

Pressure limit

3 µm: 15–20 MPa (2,175-2,900 psi)
5 µm: 10–30 MPa (1,450-4,350 psi)

Improved sensitivity for coordination compounds
1. ATP

3. AMP

2. ADP
N
O

O
HO

P
OH

O

O

OH

P

O

O

N

N

N

O

P

O
HO

N

O

P

O

N

O

N

N

O

P
OH

OH
OH

NH2

NH2

NH2

OH

O

N

N

OH

OH

Standard column

3

2

O

OH

YMC Accura Triart C18
ｍAU

P
OH

OH

OH

N

O
HO

N

ｍAU

1
10

10

3

2

5

5

1
0

0
0

Column:2

4min

YMC Accura Triart C18 (1.9 µm, 12 nm) 50 x 2.1 mm ID (bioinert hardware)
YMC-Triart C18 (1.9 µm, 12 nm) 50 x 2.1 mm ID (standard hardware)Part Nos.: TA12SP9-05Q1PTC
TA12SP9-05Q1PT
Eluent:5 mM HCOONH40

Flow rate:
Temperature:
Detection:
Injection:
System:2

4

min

0.21 mL/min
25 °C
UV at 265 nm
1 µL (10 µg/mL)
bioinert/”metal-free” HPLC system

Metal coordinating compounds, which have a phosphate group in their structure, tend to show poor peak shapedue to interactions with metals, such as the stainless-steel in column bodies and frits. By using a bioinert column
hardware, better peak shapes can be expected.
Nucleotides with phosphate groups also show better peak shapes when compared to the regular column hardware.
The bioinert coated YMC Accura column hardware is ideal for highly sensitive analyses using LC/MS.

17

YMC Oligonucleotide Columns

YMC Accura bioinert hardware
YMC Accura: durable bioinert coating
The robust bioinert coating used on YMC Accura hardware is 130 to
320-fold thicker, making it more durable than other similar hardware concepts. A long-term inertness against sensitive substancesis ensured. In order to demonstrate its robustness, a YMC Accura
column was packed multiple times. Even though this process
is hard on the column surface, the coating remains unaffected(SEM* picture: top area is bare steel for comparison).

YMC Accura Triart hardware
Coating thickness: 400–1600 nm*Scanning Electron Microscope

Other coated columns can lose their inertness over time. This will again
lead to adsorption of sensitive compounds on the uncovered metallic surfaces. Peak tailing, loss of recovery and sample carry-over are typical results of the delamination of the coating. On a competitor’s column, mostof the coating was already delaminated after merely unpacking the column. (The dark spots in this picture are the remaining coating.)

Other coated hardware
Coating thickness: 3–5 nm

High surface inertness without any adsorption

1. TEA-HFIP / methanol

2. TEAA / methanol

Recovery of Oligonucleotide

125%

125%

YMC Accura column

YMC Accura column
100%

100%

86%

75%

lly
Substantia
r
e
high
!
recoveries

75%Standard column
50%

50%

34%

No sampleg
conditioninrequired!

25%

Standard column

25%

0%

0%
0

5

10

15

20

0

Number of injections
Empty YMC Accura (without stationary phase)
1) 8 mM TEA 200 mM HFIP / methanol (82/18)
2) 100 mM TEAA / methanol (82/18)
Flow rate:
0.42 mL/min
Detection:
UV at 260 nm
Temperature: 65 º C
Injection:
1 µL
Sample:All PS RNA 20mer (1) (5’-U^C^A^U^C^A^C^A^C^U^G^A^A^U^A^C^C^A^A^U-3’)

5

10

15

20

Number of injections

Column:Eluent:^=Phosphorothioate

The YMC Accura hardware with its inert surface area prevents adsorption of oligonucleotides using a rangeof different buffers. No sample conditioning is required.
YMC Accura columns further provide significantly higher recoveries and sensitivities that cannot be achieved withregular stainless-steel columns – even after conditioning with 20 sample injections. These ready-to-use columns
ensure high recovery and reproducibility from the very first use.

18

YMC Accura bioinert hardware
Significantly higher sensitivity and recovery
Ideal choice for challenging analytes such as phosphorothioate oligonucleotides
YMC Accura Triart Bio C18

mAU

6

1

6

2

d
Increase yitsensitiv
h
ig
and h
!
recovery42Standard column

mAU

4

2

1
2

0

0

7

8

min

7

8

min

Column:Part No.:
Eluent:YMC Accura Triart Bio C18 (1.9 µm, 30 nm) 50 x 2.1 mm ID
TA30SP9-05Q1PTC
A) 15 mM triethylamine - 400 mM HFIP*B) methanol
Gradient:
8–18%B (0–10 min)Flow rate:
0.42 mL/min
Temperature: 65 ºC
Detection:
UV at 260 nm
Injection:
1 µL
Sample:All PS RNA 20mer (1) (5’-U^C^A^U^C^A^C^A^C^U^G^A^A^U^A^C^C^A^A^U-3’)
All PS RNA 21mer (2) (5’-G^U^C^A^U^C^A^C^A^C^U^G^A^A^U^A^C^C^A^A^U-3’)^=Phosphorothioate*1,1,1,3,3,3-hexafluoro-2-propanolHigh sensitivity and recovery
Peak Height

Peak Area

Peak 2

Peak 2

Peak 1

Peak 1

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.0

5.0

10.0

[mAU]

15.0

20.0

25.0

30.0

35.0

[mAU*s]YMC Accura Triart Bio C18 column

Standard column

YMC Accura Triart Bio C18 column

Standard column

ght and area!
Doubled peak hei

The YMC Accura Triart Bio C18 column provides double peak heights and peak areas for the oligonucleotides
compared to those for regular stainless-steel columns. YMC Accura Triart columns enhance the analytical sensitivity
significantly, thereby avoiding the loss of high-value samples.

19

YMC Oligonucleotide Columns

YMC Accura bioinert hardware
Reliable results from the first injection
No preconditioning required for reliable results
YMC Accura Triart Bio C4
mAU

0b
10

0b
10
0b
20

0
Column:Part No.:
Eluent:Gradient:

Standard column

0b
20

b
0 b 00 b 00 b 50 b 000
1
5
30
7
4

5

10th

10th

8th

8th

6th

6th

4th

4th

2nd

2nd

1st

1st

10 minYMC Accura Triart Bio C4 (3 μm, 30 nm) 100 x 2.1 mm ID
TB30S03-10Q1PTC
A) 50 mM TEAA* (pH 7.0)/acetonitrile (95/5)
B) 50 mM TEAA (pH 7.0)/acetonitrile (50/50)
9–14%B (0–10 min), 80%B (10–15 min)b0 b 00 b 00 b 50 b 000
7
4
5
1
30

0

5

Flow rate:
Temperature:
Detection:
Injection:
Sample:10 min0.2 mL/min
80 ºC
UV at 254 nm
1 μL (0.25 mg/mL)
100–1,000 bases (CenturyTM-Plus RNA Markers)* Triethylammonium acetate

Constantly higher peak areas and therefore recoveries[mAU*s]YMC Accura Triart Bio C4

Peak area of 300 b

200
150

100

Standard column
50

0
1

2

3

4

5

6

7

8

9

10

Number of injections

The YMC Accura Triart Bio C4 column shows stable peak areas from the first injection, while the standard
stainless-steel column provides only 10% of the peak area (for the 300 base marker) with the first injection.
Even after the tenth injection, the peak areas of the stainless-steel column are considerably less than those of the
YMC Accura Triart column.

20

AEX

AEX

21

YMC Oligonucleotide Columns

AEX – HPLC Selectivities
Features• Non-porous hydrophilic polymers• High recovery and high resolution• Low nonspecific adsorption• Excellent reproducibility• Bioinert coated YMC Accura column hardware

BioPro IEX
QF

H

CH3

C

N

CH3

H

CH3

+ strong anion exchanger

BioPro IEX QF
Matrixhydrophilic polymer(polymethacrylate)Particle size / μm

3, 5Pore size / nm

non-porous

Charged group-CH2N+(CH3)3Counter ion

Cl-

Available pH range

2.0–12.0

Temperature range

4–80°C

Pressure limit
Column hardwareY

3 μm: 15–20 MPa (2,175–2,900 psi)
5 µm: 10–30 MPa (1,450–4,350 psi)bioinert coated YMC Accura

Non-porous polymer beads

MC´s anion exchanger (AEX) columns of the BioPro IEX series are available with strong exchanger modification, basedon 3 or 5 μm non-porous (QF columns) hydrophilic polymer beads. The non-porous particles offer high efficiency,
very high resolution and low operating pressures.

22

AEX – Loadability
Virtually
No Adsorption
on Oligonucleotide
Analysis
Virtually
No Adsorption
on Oligonucleotide
Analysis
Sharp peaks and reliable recovery with different column dimensions
PEEK column
StandardStandard
PEEK column

YMC-Accura
BioProYMC-Accura
BioPro IEXQF IEX QF
mV

mV

20

20

15

15

10

10

5

5

YMC Accura BioPro IEX QF

mV
20

mV
20

15

15

10

10

5

5

Standard PEEK column

Adsorption Adsorption
4th

3

3rd

2nd

2nd

0

1st

0
0

0

5 10

5

10 15

15
min

SampleSample: 21mer RNA

1st

0

0

0

0

min

Column5mmI.D.
μm,
100
4.6 mmI.D.
Column YMC Accura: 5 μm, BioPro
100 X 4.6:IEX
Columns:
QF(5Xµm)
100 x 4.6 mm ID
Eluent: A) 20
Eluent: A) 20 mM Tris-HCl(pHmM8.1)Tris-HCl (pH 8.1)
(bioinert
coated hardware)B) 20(pH1.08.1)McontainingB) 20 mM Tris-HCl(pHmM8.1)Tris-HCl
containing
NaClO4 1.0 M NaClO4
BioPro IEX QF (5 µm) 100 x 4.6 mm
ID
25-40%B(0-15min),
40%B (15-20 min)25-40%B (0-15 min),40%B(15-20min)(standardhardware)Flow: 1.0 mL/min
Flow rate: 1.0rate
mL/minPart Nos.: QF00S05-1046PTC: 60℃
Temperature Temperature: 60℃
QF00S05-1046WP
Detection: UV at 260 nm : UV at 260 nm
Eluent: DetectionA) 20 mM Tris-HCl(pH 8.1)
Injection: 4 µL (5 nmol/mL)
Injection: 4 µL (5 nmol/mL)B) 20 mM Tris-HCl (pH 8.1) containing 1.0 M NaClO4

4th
rd

5 10

5

min
15

10 15

Gradient:

25–40%B (0–15 min), 40%B (15–20 min)

System:bioinert HPLCmin

Oligonucleotides
maypeakshowshape/recovery
poor peak shape/recovery
on IEX
analysis
Oligonucleotides
may showon IEX analysis
mainlydue mainly due
Flow
rate:
1.0poormL/minto theironto
adsorption
onto hardware.
the columnAccura
hardware.
AccuraBioProIEX gives goodto their adsorption
the
BioProIEX gives
good
Temperature:
60°Ccolumn
UVpeakat 260nmandshape
the first
injection.
Thereproducible.
results are reproducible.
recovery Detection:
andrecovery
peak shapefromthefirstfrominjection.
The
results are
Injection:µL
AccuraIEX(5isnmol/mL)
ideal offoroligonucleotides.
analysis of oligonucleotides.
It doesany
not require any
Accura BioPro
IEX isBioPro
ideal4 foranalysis
It does not require
Sample: preconditioning
21mer RNA
preconditioning
process
before analysis.
process before
analysis.

: 21mer RNA

Oligonucleotides generally exhibit poor peak shape and therefore low recovery in AEX analysis, mainly due toadsorption onto the column hardware. YMC Accura BioPro IEX columns provide high recovery and very goodpeak shapes from the first injection. This makes YMC Accura BioPro IEX QF columns ideal for the analysis ofoligonucleotides with reproducible results. The columns show stable peak areas from the 1st injection, so that no
preconditioning is required.

150 mm Length Column for High Resolution Analysis

Different column lengths for each separation purpose

1. antisense strand
2. siRNA duplex

2

mV
80

50 x 4.6 mm ID

60

25–40%B (0–10 min)40

1

Rs(1,2) = 3.2

20
0

0

10

Shortest
run time
ColumnEluentFlow rate
Temperature
Detection
Injection

30 min20: Accura BioPro IEX QF (5 μm): A) 20 mM Tris-HCl (pH 8.1)
B) 20 mM Tris-HCl (pH 8.1) containing 1.0 M NaClO4: 0.5 mL/min: 60℃: UV at 260 nm: 8 µL (each 5 nmol/mL)

mV

100 x 4.6 mm ID
25–40%B (0–20 min)

2

80
60

1

40

Rs(1,2) = 4.6

20
0

0

10

20

Best
ise BioPro IEX has 150 mm length option fcompromAccurahigh resolution analyses.

30 minmV

150 x 4.6 mm ID
25–40%B (0–30 min)

80

2

60
40

1

20

Rs(1,2) = 5.1

Single-stranded RNA (antisense) and Doub
stranded RNA are separated with Accura BioP
IEX QF. As the column length becomes longresolution of two peaks becomes greater. Using
longer column can be one of the solutions
improve resolution.

Highestresolution00Columns:
Part Nos.:
Eluent:10

YMC Accura BioPro IEX QF (5 µm)
QF00S05-0546PTC
QF00S05-1046PTC
QF00S05-1546PTC
A) 20 mM Tris-HCl (pH 8.1)
B) 20 mM Tris-HCl (pH 8.1) containing 1.0 M NaClO4

20

30 minFlow rate:
Temperature:
Detection:
Injection:
System:

0.5 mL/min
60 °C
UV at 260 nm
8 µL (each 5 nmol/mL)
bioinert HPLC

23

YMC Oligonucleotide Columns

AEX – Stability and Reproducibility
Excellent stability using temperatures up to 80 °C

Stable nioretent ciencyfiand ef

uV
50000

40000

75
50

100 hr
30000

25
0

75 hr

20000

% of initial retention time

%
100

0

50 hr

25

50

75

100 hr% of initial theoretical plate number

%
100

10000

25 hr

75
50

initial

0

25
0

0

5

10

15

20

0

min

Column: YMC Accura BioPro IEX QF (5 μm) 100 x 4.6 mm ID
Part No.: QF00S05-1046PTC
Eluent:A) 20 mM Tris-HCl (pH 8.1)
B) 20 mM Tris-HCl (pH 8.1) containing 1.0 M NaCl
Gradient: 40–90%B (0–25 min), 90%B (25–35 min), 40%B (35–60 min)

25

50

75

100 hrFlow rate:		
0.5 mL/min
Temperature: 80 ºC
Detection:		UV at 260 nm
Injection:
4 μL (5 nmol/mL)
Sample:		
21mer RNA

Excellent batch-to-batch reproducibility

Column lot E

Column lot D

Column lot C

Column lot B

Column lot A
0
Column:Part No.:
Eluent:Gradient:

5

10

15

BioPro IEX SF (5 μm) 100 x 4.6 mm ID
SF00S05-1046WPA) 20 mM NaH2PO4-Na 2HPO4 (pH 6.5)
B) 20 mM NaH2PO4-Na 2HPO4 (pH 6.5) containing 0.2 M NaCl
0–50%B (0.5–30 min)20
Flow rate:
Temperature:
Detection:
Injection:
Sample:

25

30

min

0.5 mL/min (180 cm/hr)
25 °C
UV at 215 nm
10 μL
monoclonal antibody (IgG1)

BioPro IEX columns exhibit excellent batch-to-batch reproducibility. All gel batches are inspected by rigorous
quality control tests, and must meet the required criteria before release. BioPro IEX columns are the best choicefor the quality control of biopharmaceuticals such as oligonucleotides or mAbs as in this example.

24

SEC

SEC

25

YMC Oligonucleotide Columns

SEC – UHPLC / HPLC Selectivities
Features• Excellent reproducibility with minimal secondary interactions• 2 μm for UHPLC• Cost-effective
YMC-Pack
Diol-120

YMC-Pack
Diol-200

YMC-Pack
Diol-300For
short
oligonucleotides

For
intermediateoligonucleotides

Forlongeroligonucleotides

silicaBase particle
Particle size / µm

3, 52, 3, 52, 3, 5

Pore size / nm

12

20

30

dihydroxypropyl

Modification

40 °C

Temperature range

2 µm: 45 MPa (6,525 psi);
3/5 µm: 20 MPa (3,000 psi)

Pressure limit

Column Selection Tool according to nucleotide length
Length (-mer)

20

40

60

80

100

150

YMC-Pack Diol-120

YMC-Pack Diol-200

YMC-Pack Diol-300

Most appropriate oligonucleotide length range
Appropriate oligonucleotide length range

26

200

250

300

SEC – Pore size selection
SEC analysis using YMC-Pack Diol columns with different pore sizes
Analysis of ssDNA of 10–120mer length
mAU
125

120

100

YMC-Pack
Diol-120(Pore size: 12 nm)

15

40

75

100

50

80

25

30 25

8

10

12

14

16

18

20

40

100

(Pore size: 20 nm)

dTMP

0
125

YMC-Pack
Diol-200

10

20

60

120100

75

30
80

22

26

min

24

26

min

24

26

min

15
25 20

10

dTMP

60

50

24

25
0
8

10

12

14

16

18

20

22

125
100

YMC-Pack
Diol-300(Pore size: 30 nm)

4075

120 100

50

30 25

15

20

80

10

60

25

dTMP

0
8

10

12

14

16

18

20

22

Analysis of dsDNA of 10–300mer length
mAU
30

YMC-Pack
Diol-120(Pore size: 12 nm)

300~75

20

50

10

10

20
35

*

0
8

10

12

14

16

18

20

22

24

26

min

24

26

min

26

min

20

100

16

YMC-Pack
Diol-200

12(Pore size: 20 nm)

4

300 150

8

*

10

200

20
75

50

35

0
8

10

12

14

16

18

20

22

20
16

YMC-Pack
Diol-300(Pore size: 30 nm)

10

100
20

12
8

300

200 150

75

4

50

*

35

0
8

Columns:
Part Nos.:
Eluent:

10

12

14

YMC-Pack Diol-120 (5 µm, 12 nm) 300 x 4.6 mm ID
YMC-Pack Diol-200 (5 µm, 20 nm) 300 x 4.6 mm ID
YMC-Pack Diol-300 (5 µm, 30 nm) 300 x 4.6 mm ID
DL12S05-3046WTDL20S05-3046WT
DL30S05-3046WT
0.1 M KH2PO4-K 2HPO4 (pH 7.0) containing 0.2 M NaCl

16

18

Flow rate:
Detection:
Temperature:
Injection:
Samples:

20

22

24

* Impurity peak derived from the sample

0.17 mL/min
UV at 260 nm
25 °C
1.0 µL (each 5 nmol/mL)
ssDNA (10–120 mer +dTMP)
dsDNA (10–300 mer)

YMC-Pack Diol-120 is best suited for shorter oligonucleotides (10–40mer), whereas YMC-Pack Diol-200 shows
the best resolution for oligonucleotides of medium size (30–80mer). Longer oligonucleotides of 60–120mer inlength are separated most effectively by YMC-Pack Diol-300.
Similar results are obtained when analysing dsDNA. Small oligonucleotides are separated with higher resolution
when smaller pore sizes of 12 and 20 nm are used. Above a length of 50mer, oligonucleotides are unable topenetrate the small pores and elute at the same time. YMC-Pack Diol-200 can resolve oligonucleotides up toa size of 100mer. dsDNA of 150–300mer are only separated by YMC-Pack Diol-300 with the largest pore size of
30 nm. This column also shows the best resolution over a wide range of oligonucleotide lengths.

27

YMC Oligonucleotide Columns

SEC – Elution volumes
Comparison of the elution volume of ssDNA and dsDNA
Selected corresponding ssDNA and dsDNA pairs of the same number of bases are marked

1000

× ssDNA

Number of bases

●

dsDNA
100mer

100

YMC-Pack
Diol-300

40mer
20mer

YMC-Pack
Diol-120

10

1
0.35

0.40

0.45

0.50

0.55

YMC-Pack
Diol-200

0.60

0.65

0.70

0.75

0.80

0.85

Elution volume / Column volume

Columns:

YMC-Pack Diol-120 (5 µm, 12 nm) 300 x 4.6 mm ID
YMC-Pack Diol-200 (5 µm, 20 nm) 300 x 4.6 mm ID
YMC-Pack Diol-300 (5 µm, 30 nm) 300 x 4.6 mm ID
Part Nos.:
DL12S05-3046WTDL20S05-3046WT
DL30S05-3046WT
Eluent:0.1 M KH2PO4-K 2HPO4 (pH 7.0) containing 0.2 M NaCl
Flow rate:
0.17 mL/min
Detection:
UV at 260 nm
Temperature: 25 °C
Injection:
1.0 µL (each 5 nmol/mL)
Samples:
ssDNA (10–120 mer +dTMP)
dsDNA (10–300 mer)

Although dsDNA has the same length as its single-stranded counterpart, the dsDNA elutes at lower elution
volumes when separated by SEC. This behaviour is most probably due to the larger hydrodynamic radius of
dsDNA compared to ssDNA, which results in faster diffusion through the stationary phase.

28

HILIC

HILIC

29

YMC Oligonucleotide Columns

HILIC – UHPLC/HPLC selectivity
Features• pH- and temperature stable• Superior reproducibility• Bioinert coated YMC Accura column hardware
Base particle

Modification Particle Size/ Pore Size/ pH Temperature
µm
nm
range
range

YMC-Triart organic/inorganic
Diol (USP L20)
Diol-HILIC
hybrid silica

1.9, 3, 5

12

2–10

Columnhardwarebioinert coated
YMC Accura

50 °C

Pre-conditioning of a stainless-steel and a bioinert coated column
with short DNA mixture
YMC-Triart Diol-HILIC(standard column)

YMC Accura Triart Diol-HILIC

150% of inital peak area

% of inital peak area

140

800

130
120
110
100
90

80

1

3

5

7

9
11
13
Injection Number

15

17

19

80

1

2

3

4
5
Injection Number

6

7

8

Columns:

YMC-Triart Diol-HILIC (1.9 µm, 12 nm) 150 x 2.1 mm ID (standard hardware)
YMC Accura Triart Diol-HILIC (1.9 µm, 12 nm) 150 x 2.1 mm ID (bioinert hardware)
TDH12SP9-15Q1PT
TDH12SP9-15Q1PTC
Eluent:A) 50 mM ammonium acetate (pH 6.9)
B) acetonitrile
Gradient
75–45%B (0–30 min)Flow rate:
0.3 mL/min
Temperature: 40 °C
Detection:
UV at 260 nm
Injection:
2 µL
Sample:deoxythymidine oligonucleotides: dT15-35 (2 µM)
System:bioinert system
Part Nos.:

Pre-conditioning is a typical procedure when working with stainless-steel columns. Using a bioinert column
such as YMC Accura Triart usually achieves great performance from the first injection when working with an
IP-RP phase. HILIC phases still need some pre-conditioning when a bioinert column is used; however, thenumber of injections is remarkably reduced. While 20 injections are necessary for the stainless-steel column,
the YMC Accura column is already conditioned after 8 injections, with very little difference (less than 10 %)between initial and final peak areas.

30

HILIC
Improved chromatographic results using bioinert coated YMC Accura Triart column
YMC-Triart Diol-HILIC(standard column)

0,05

1

YMC Accura Triart Diol-HILIC
0,05

0,04

0,04

0,03

0,03

5

1) dT152) dT203) dT254) dT305) dT35

2
1

0,02

2

1

3

0,02

4

5
0,01

0,01

0

0

8

2

3

4

10

12

14

16

8

18

0,09

0,09

0,08

0,08

0,07

0,07

0,06

0,06

3

0,05

0,04

1

0,03

2

10

12

14

3

18

4
1) dT402) dT603) dT804) dT100

2
1

0,05

4

16

0,04

0,03

0,02

0,02

0,01

0,01

0

0

8

10

12

14

16

8

18

3,00E-02

10

12

14

16

18

0,03

1) rU152) rU203) ru30

3
2,00E-02

0,02

1

3

2
1

1,00E-02

0,01

2

3

0,00E+00

0

8

10

12

14

16

18

8

10

12

14

Columns:

16

18

YMC-Triart Diol-HILIC (1.9 µm, 12 nm) 150 x 2.1 mm ID (standard hardware)
YMC Accura Triart Diol-HILIC (1.9 µm, 12 nm) 150 x 2.1 mm ID (bioinert hardware)
dT15–35
TDH12SP9-15Q1PT
TDH12SP9-15Q1PTC
dT40–100
Eluent:A) 50 mM ammonium acetate (pH 6.9)
B) acetonitrile
rU15–30
Gradient
75–45%B (0–30 min)Flow rate:
0.3 mL/min
Temperature: 40 °C
Detection:
UV at 260 nm
Injection:
2 µL
By courtesy of Honorine Lardeux,
Sample:deoxythymidine oligonucleotides: dT15-35 (2 µM) and dT40-100 (2µM)
Institute of Pharmaceutical Sciences of Western Switzerland
RNA oligonucleotides: rU15-30 (2µM)(University of Geneva), Geneva, Switzerland.
System:bioinert system
Part Nos.:

1
2
3

Reference: H. Lardeux, A. Goyon, K. Zhang, J.M. Nguyen, M.A. Lauber, D. Guillarme, V. D’Atri, The impact of low adsorption surfaces
for the analysis of DNA and RNA oligonucleotides, J. Chromatogr. A 1677 (2022) 463324.

After conditioning and analysing the short DNA oligonucleotide mixture of dT15-35, longer DNA oligonucleotides dT40-100 and short RNA oligonucleotides rU15-30 are analysed. Higher sensitivities, peak areas andless tailing are achieved using the bioinert YMC Accura Triart Diol-HILIC column. Non-specific adsorption does
not vary according to length, even though the adsorption is usually higher for longer oligonucleotides in IP-RP.

31

YMC Oligonucleotide Columns

HILIC Expert Tips
Influence of sample solvent
20mer

mAU

Sample solvent
water/acetonitrile(60/40)

6

15mer

4

dT 10mer

2
0

2

4

6

8

10

12

14

16

18

min

2

4

6

8

10

12

14

16

18

min

0

mAU
6

water/acetonitrile(20/80)4
2
0

0Column:Part.No.:
Eluent:YMC Accura Triart Diol-HILIC (3μm, 12 nm), 150 X 2.1 mm ID
TDH12S03-15Q1PTC
A) 50mM HCOONH4 (pH 6.5)/acetonitrile (30/70)
B) 50mM HCOONH4 (pH 6.5)
Gradient: 0–43%B (0–20 min), 43%B (20–25 min)((70–40% acetonitrile (0–20 min), 40% acetonitrile (20–25 min))Flow rate:
Temperature:Sample:Injection:

0.21 mL/min
40 °C
Oligo DNA (poly(dT) 10, 15, 20mer)
2 µL (each 2.0 nmol/mL)T

he influence of the sample solvent on the peak shape is significant. The organic composition of the sample solvent
must be equal to or higher than the initial gradient composition. A higher water content in the sample solvent leads tomassive peak deformation.

Influence of mobile phase pH
I.S.(Caffeine)mAU

Eluent pH
Acidic (pH 3.6)
Neutral (pH 6.5)
Basic (pH 9.5)

14

12
10

21mer

8

20mer

6

17mer
14mer

4
2
0

Column:Part.No.:
Eluent:Gradient:

T

0

2

4

6

8

YMC Accura Triart Diol-HILIC (3μm, 12 nm), 150 x 2.1 mm ID
TDH12S03-15Q1PTC
A) 50 mM ammonium formate buffer/acetonitrile (30/70)
B) 50 mM ammonium formate buffer
7.1–21.4%B (0–20 min), 21.4%B (20–25 min)(65–55% acetonitrile (0–20 min), 55% acetonitrile (20–25 min)

10

12

Flow rate:
Temperature:Sample:Injection:

14

16

18

min

0.21 mL/min
40 °C
All PO RNA (14, 17, 20, 21mer) + I.S.(Caffeine)
1 µL (1.8 nmol/mL-3.6 nmol/mL)

he pH of the mobile phase has a massive effect on the recovery and retention of oligonucleotides. Acidic pH results inhigher retention, but at the cost of drastically reduced recovery. A neutral-to-basic pH is recommended for the mobile
phase. When using a basic pH, a shorter retention time is observed, but also the highest recovery.

32

Ordering information

Ordering
information

33

YMC Oligonucleotide Columns

IP-RP – Ordering information
1.9 µm bioinert coated UHPLC columns (max. pressure 100 MPa)
Phase

ColumnID[mm]

YMC Accura Triart
C18
YMC Accura Triart
Bio C18
YMC Accura Triart
C8
YMC Accura Triart
Bio C4

Column length
[mm]
50

100

150

2.1

TA12SP9-05Q1PTC

TA12SP9-100Q1PTC

TA12SP9-15Q1PTC

2.1

TA30SP9-05Q1PTC

TA30SP9-10Q1PTC

TA30SP9-15Q1PTC

2.1

TO12SP9-05Q1PTC

TO12SP9-10Q1PTC

TO12SP9-15Q1PTC

2.1

TB30SP9-05Q1PTC

TB30SP9-10Q1PTC

TB30SP9-15Q1PTC

3 µm bioinert coated HPLC columns (max. pressure 45 MPa)
Phase

ColumnID[mm]

YMC Accura Triart
C18
YMC Accura Triart
Bio C18
YMC Accura Triart
C8
YMC Accura Triart
Bio C4

2.1

Column length
[mm]

4.6
2.1
4.6
2.1
4.6
2.1
4.6

50

100

150

TA12S03-05Q1PTC
TA12S03-0546PTC

TA12S03-10Q1PTC
TA12S03-1046PTC

TA12S03-15Q1PTC
TA12S03-1546PTC

TA30S03-05Q1PTC
TA30S03-0546PTC

TA30S03-10Q1PTC
TA30S03-1046PTC

TA30S03-15Q1PTC
TA30S03-1546PTC

TO12S03-05Q1PTC
TO12S03-0546PTC

TO12S03-10Q1PTC
TO12S03-1046PTC

TO12S03-15Q1PTC
TO12S03-1546PTC

TB30S03-05Q1PTC
TB30S03-0546PTC

TB30S03-10Q1PTC
TB30S03-1046PTC

TB30S03-15Q1PTC
TB30S03-1546PTC

3 µm HPLC columns (max. pressure 25 MPa)
Phase

ColumnID[mm]

Column length
[mm]
30/33

Hydrosphere
C18

2.1
3.0
4.6

50

75

100

Guard cartridges*
with 10 mm
length150

250

HS12S03-03Q1WT HS12S03-05Q1WT HS12S03-L5Q1WT HS12S03-10Q1WT HS12S03-15Q1WT HS12S03-25Q1WT
HS12S03-0303WT HS12S03-0503WT HS12S03-L503WT HS12S03-1003WT HS12S03-1503WT HS12S03-2503WT
HS12S03-0346WT HS12S03-0546WT HS12S03-L546WT HS12S03-1046WT HS12S03-1546WT HS12S03-2546WT(pack of 5)
HS12S03-01Q1GC
HS12S03-0103GC
HS12S03-0104GC*Guard cartridge holder required, part no. XPGCH-Q1 (for EMEA)/XPGCHP1 (outside EMEA)

Further dimensions and guard cartridges available in regular stainless-steel hardware.

34

IP-RP – Ordering information
5 µm bioinert coated HPLC columns (max. pressure 45 MPa)
Phase

ColumnID[mm]

Column length
[mm]
50

100

150

YMC Accura Triart
C18

2.1
4.6
10

TA12S05-05Q1PTC
TA12S05-0546PTC–

TA12S05-10Q1PTC
TA12S05-1046PTC
TA12S05-1010PTC

TA12S05-15Q1PTC
TA12S05-1546PTC
TA12S05-1510PTC

YMC Accura Triart
Bio C18

2.1
4.6
10

TA30S05-05Q1PTC
TA30S05-0546PTC–

TA30S05-10Q1PTC
TA30S05-1046PTC
TA30S05-1010PTC

TA30S05-15Q1PTC
TA30S05-1546PTC
TA30S05-1510PTC

YMC Accura Triart
C8

2.1
4.6
10

TO12S05-05Q1PTC
TO12S05-0546PTC–

TO12S05-10Q1PTC
TO12S05-1046PTC
TO12S05-1010PTC

TO12S05-15Q1PTC
TO12S05-1546PTC
T012S05-1510PTC

YMC Accura Triart
Bio C4

2.1
4.6
10

TB30S05-05Q1PTC
TB30S05-0546PTC–

TB30S05-10Q1PTC
TB30S05-1046PTC
TB30S05-1010PTC

TB30S05-15Q1PTC
TB30S05-1546PTC
TB30S05-1510PTC

5 µm HPLC columns (max. pressure 20–25 MPa)
Phase

ColumnID[mm]

Column length
[mm]
30/33

Hydrosphere
C18

2.1
3.0
4.6

50

75

Guard cartridges*
with 10 mm
length

100

150

250

HS12S05-03Q1WT HS12S05-05Q1WT HS12S05-L5Q1WT HS12S05-10Q1WT HS12S05-15Q1WT HS12S05-25Q1WT
HS12S05-0303WT HS12S05-0503WT HS12S05-L503WT HS12S05-1003WT HS12S05-1503WT HS12S05-2503WT
HS12S05-0346WT HS12S05-0546WT HS12S05-L546WT HS12S05-1046WT HS12S05-1546WT HS12S05-2546WT(pack of 5)
HS12S05-01Q1GC
HS12S05-0103GC
HS12S05-0104GC*Guard cartridge holder required, part no. XPGCH-Q1 (for EMEA)/XPGCHP1 (outside EMEA)

5 µm YMC-Actus high-throughput (semi)preparative columns (max. pressure 20–30 MPa)
Phase

YMC-Triart
C18
YMC-Triart
Bio C18
YMC-Triart
C8
YMC-Triart
Bio C4
Hydrosphere
C18

Column
ID
[mm]
20
30
50
20
30
50
20
30
50
20
30
50
20
30

Column length[mm]

Guard cartridges*
with 10 mm
length

50

75

100

150

250

(pack of 2)

TA12S05-0520WXTA12S05-0530WXTA12S05-0553DX

TA12S05-L520WXTA12S05-L530WX–TA12S05-1020WX
TA12S05-1030WX
TA12S05-1053DX

TA12S05-1520WXTA12S05-1530WXTA12S05-1553DX

TA12S05-2520WXTA12S05-2530WXTA12S05-2553DX

TA12S05-0120CCN
TA12S05-0130CCN
TA12S05-0553DXG**

TA30S05-0520WXTA30S05-0530WXTA30S05-0553DX

TA30S05-L520WXTA30S05-L530WX–TA30S05-1020WX
TA30S05-1030WX
TA30S05-1053DX

TA30S05-1520WXTA30S05-1530WXTA30S05-1553DX

TA30S05-2520WX TA30S05-0120CCN
TA30S05-2530WX TA30S05-0130CCN
TA30S05-2553DX TA30S05-0553DXG**

TO12S05-0520WXTO12S05-0530WX
TO12S05-0553DX

TO12S05-L520WXTO12S05-L530WX–

TO12S05-1020WX
TO12S05-1030WX
TO12S05-1053DX

TO12S05-1520WXTO12S05-1530WX
TO12S05-1553DX

TO12S05-2520WX TO12S05-0120CCN
TO12S05-2530WX TO12S05-0130CCN
TO12S05-2553DX TO12S05-0553DXG**TB30S05-0520WXTB30S05-0530WXTB30S05-0553DX

TB30S05-L520WXTB30S05-L530WX–TB30S05-1020WX
TB30S05-1030WX
TB30S05-1053DX

TB30S05-1520WXTB30S05-1530WXTB30S05-1553DX

TB30S05-2520WX TB30S05-0120CCN
TB30S05-2530WX TB30S05-0130CCN
TB30S05-2553DX TB30S05-0553DXG**

HS12S05-0520WXHS12S05-0530WX

HS12S05-L520WXHS12S05-L530WX

HS12S05-1020WX
HS12S05-1030WX

HS12S05-1520WXHS12S05-1530WX

HS12S05-2520WXHS12S05-2530WX

HS12S05-0120CCN
HS12S05-0130CCN*Guard cartridge holder required, part no. XPGHF2P20ID (20 mm ID)
XPGHF2P30ID (30 mm ID)
no holder required for 50 mm

Further dimensions and guard cartridges available in regular stainless-steel hardware.

35

YMC Oligonucleotide Columns

AEX – Ordering information
3 µm bioinert coated HPLC columns (max. pressure 15–20 MPa)
Phase

YMC Accura
BioPro IEX QF

Column
ID
[mm]

2.1
4.6

Column length[mm]50 (15 MPa)

100 (15 MPa)

150 (20 MPa)

QF00S03-05Q1PTC
QF00S03-0546PTC

QF00S03-10Q1PTC
QF00S03-1046PTC

QF00S03-15Q1PTC
QF00S03-1546PTC

5 µm bioinert coated HPLC columns (max. pressure 10–30 MPa)
Phase

YMC Accura
BioPro IEX QF

Column
ID
[mm]

2.1
4.6

Column length[mm]50 (10 MPa)

100 (12 MPa)

150 (18 MPa)

250 (30 MPa)

QF00S05-05Q1PTC
QF00S05-0546PTC

QF00S05-10Q1PTC
QF00S05-1046PTC

QF00S05-15Q1PTC
QF00S05-1546PTC–
QF00S05-2546PTC

6 μm semiprep. columns, stainless-steel hardware (max. pressure 3–9 MPa)
Phase

ColumnID[mm]

Column length
[mm]
100

BioPro IEX QF

36

10
20
30

QF00S06-1010WTQF00S06-1020WTQF00S06-1030WT

SEC – Ordering information
2 µm UHPLC columns (max. pressure 45 MPa)
Phase

Column ID[mm]

Column length [mm]Guard cartridges*
with 10 mm
length150

300(pack of 5)

YMC-Pack
Diol-200

4.6

DL20S02-1546PTHDL20S02-3046PTHDL20S02-0104GC

YMC-Pack
Diol-300

4.6

DL30S02-1546PTH

DL30S02-3046PTH

DL30S02-0104GC*Guard cartridge holder required, part no. XPGCH-Q1 (for EMEA)/XPGCHP1 (outside EMEA)

3 µm HPLC columns (max. pressure 14–20 MPa)
Phase

Column ID[mm]

Column length [mm]Guard cartridges*
with 10/30 mm
length

150

250

300

(pack of 5)

YMC-Pack
Diol-120

4.6
6.0
8.0

DL12S03-1546WT–DL12S03-1508WTDL12S03-2546WT––DL12S03-3046WTDL12S03-3006WTDL12S03-3008WTDL12S03-0104GC–
DL12S03-0308WTG**

YMC-Pack
Diol-200

4.6
6.0
8.0

DL20S03-1546WT–DL20S03-1508WTDL20S03-2546WT––DL20S03-3046WTDL20S03-3006WTDL20S03-3008WTDL20S03-0104GC–
DL20S03-0308WTG**

YMC-Pack
Diol-300

4.6
6.0
8.0

DL30S03-1546WT–DL30S03-1508WTDL30S03-2546WT––DL30S03-3046WT
DL30S03-3006WT
DL30S03-3008WT

DL30S03-0104GC–
DL30S03-0308WTG***Guard cartridge holder required, part no. XPGCH-Q1 (for EMEA)/XPGCHP1 (outside EMEA)**no holder required for 30 x 8 mm ID guard columns (1 piece)
recommended column coupler part no. XRCP1602

5 µm HPLC columns (max. pressure 20 MPa)
Phase

Column ID[mm]

Column length [mm]Guard cartridges*
with 10/30 mm
length

250

300

500

(pack of 5)

YMC-Pack
Diol-120

4.6
6.0
8.0
10.0

DL12S05-2546WTDL12S05-2506WT–DL12S05-2510WTDL12S05-3046WTDL12S05-3006WTDL12S05-3008WTDL12S05-3010WT–
DL12S05-5006WTDL12S05-5008WTDL12S05-5010WT

DL12S05-0104GC–
DL12S05-0308WTG**
DL12S05-0310WTG**

YMC-Pack
Diol-200

4.6
6.0
8.0
10.0

DL20S05-2546WTDL20S05-2506WT–DL20S05-2510WTDL20S05-3046WTDL20S05-3006WTDL20S05-3008WTDL20S05-3010WT–
DL20S05-5006WTDL20S05-5008WTDL20S05-5010WT

DL20S05-0104GC–
DL20S05-0308WTG**
DL20S05-0310WTG**

YMC-Pack
Diol-300

4.6
6.0
8.0
10.0

DL30S05-2546WTDL30S05-2506WT–DL30S05-2510WTDL30S05-3046WT
DL30S05-3006WT
DL30S05-3008WT
DL30S05-3010WT–
DL30S05-5006WT
DL30S05-5008WT
DL30S05-5010WT

DL30S05-0104GC–
DL30S05-0308WTG**
DL30S05-0310WTG***Guard cartridge holder required, part no. XPGCH-Q1 (for EMEA)/XPGCHP1 (outside EMEA)**no holder required for 30 x 8 mm ID guard columns (1 piece)
recommended column coupler part no. XRCP1602 (for 8 mm ID) and XRCP1605 (for 10 mm ID)

mns
Bioinert SEC colu
quest.
re
n
o
le
b
a
il
a
v
a
are
37

YMC Oligonucleotide Columns

HILIC – Ordering Information
1.9 µm bioinert coated UHPLC columns
Phase

Column ID [mm]

YMC Accura Triart
Diol-HILIC

2.1

Column length [mm]
50

100

150

TDH12SP9-05Q1PTC

TDH12SP9-10Q1PTC

TDH12SP9-15Q1PTC

3 µm bioinert coated HPLC columns
Phase

Column ID [mm]

YMC Accura Triart
Diol-HILIC

2.1
4.6

Column length [mm]
50

100

150

TDH12S03-05Q1PTC
TDH12S03-0546PTC

TDH12S03-10Q1PTC
TDH12S03-1046PTC

TDH12S03-15Q1PTC
TDH12S03-1546PTC

5 µm bioinert coated HPLC columns
Phase

Column ID [mm]

YMC Accura Triart
Diol-HILIC

2.1
4.6

Column length [mm]
50

100

150

TDH12S05-05Q1PTC
TDH12S05-0546PTC

TDH12S05-10Q1PTC
TDH12S05-1046PTC

TDH12S05-15Q1PTC
TDH12S05-1546PTC

Further dimensions and guard cartridges available in regular stainless-steel hardware.

The following brands, trademarks, or service marks are the property of the listed company and/or its subsidiaries.
Every effort has been taken to ensure this list is accurate at the time of printing this brochure.
ACQUITY, BEH, XBridge are trademarks of Waters Corp.
AdvanceBio is a trademark of Agilent Technologies Inc.
DNAPac is a trademark of Thermo Fisher Scientific Inc.
Every effort has been taken to ensure this list is accurate at the time of creating this brochure.

38

Download the following documents
BROCHURESYMC Biochromatography
Columns

YMC-Triart

YMC
BioPro IEX Resins

WHITE PAPER

YMC-Triart Prep

APPLICATION NOTEAPPLICATION NOTE

RP analysis of RNA markers using
YMC Accura Triart Bio C4
Tstandards are necessary for various analytical methods.
One of these standards are RNA markers which are
RNA fragments of a certain size which are used ingel electrophoresis to estimate the size of other RNA
fragments.

oday, oligonucleotides have become more and more
important in genetic testing, research, and forensics.
To date a variety of oligonucleotide-based approaches
have been developed for different pharmaceuticalapplications. Therefore, clean and reliable oligonucleotide

Figure 1: Gel electrophoresis.

surfaces. This effect is enhanced when working at low
to neutral pH as metals become more electropositive.
To overcome these challenges bioinert systems andcolumns such as the recently introduced YMC Accura
Triart columns are beneficial. YMC Accura Triart columns
have a bioinert coating on all surfaces, including the frits,
to prevent any unwanted ionic interactions.

The electron-rich backbone of oligonucleotides can cause
irreversible adsorption on metal parts. This is a major
issue for reversed phase liquid chromatography analyses
as standard equipment and hardware such as tubing, frits
or the standard column-body is made from stainless steel.
The oligonucleotides bind through ionic interactions withthe positively charged metal oxide layer of the metallic

YMC Europe GmbH · Schöttmannshof 19 · 46539 Dinslaken · Phone +49 2064 427-0 · Fax +49 2064 427-222 · Email info@ymc.eu · www.ymc.eu
Page 1/3

Analysis and Purificationof Oligonucleotides
by AEX and IP RP

RP analysis of
RNA markers using
YMC Accura Triart Bio C4TECHNICAL NOTESTechnical Note

Technical Note

Technical Note

HILIC analysis of oligonucleotides using different
bioinert columns

How to influence oligonucleotide analysis by IP-RP

Oligonucleotides have become more important for medical
applications, currently they are used to treat several diseases.
Therefore, robust and highly sensitive analysing methods
are required. Meanwhile ion pair reversed phase liquid
chromatography (IP-RP) and anion exchange chromatography(AEX) seem to be the gold standards for the characterisationof oligonucleotides and their by-products. In addition,hydrophilic interaction liquid chromatography (HILIC) can
provide an alternative approach due to the highly polar natureof oligonucleotides.

T

he natural structure of siRNA with an electron-richanionic phosphate backbone hampers its cellular uptake and is responsible for a poor pharmacokinetic profile.
The additional resistance towards nuclease-mediateddegradation led to the development of chemical modifications of the nucleotides structure. A common option is theintroduction of synthetic artificial linkages such as phosphorothioate linkages (PS) instead of the natural phosphodiester linkages (PO). The exchange of the oxygen
atom with sulphur in PS linkages decreases the polarityof the nucleotides and sensitivity to enzymatic degradation. As a result, PS oligonucleotides are more commonly
synthesized and used for in vivo and in vitro applications.
The chemical synthesis of oligonucleotides is a multistep
process, where the nucleotide chain is extended succes-

sively. During synthesis, impurities with only minor structural differences such as shortmers (n-x) and longmers(n+x) can accumulate. Therefore, the purity and qualityof oligonucleotides needs to be controlled closely since
impurities can affect their efficacy. The analysis of oligonucleotides is highly challenging due to their negatively
charged phosphate groups in the backbone. Ion pair reversed phase liquid chromatography (IP-RP) is considered
the gold standard for oligonucleotide analysis. The selection of several parameters such as pH, gradient and column temperature highly influences the performance. Also
the choice of ion pair reagent, pore size and hardware
plays an important role. This technical note demonstrates
the most important factors that need to be consideredregarding the analysis of oligonucleotides by IP-RP.

How to choose the optimum conditions for analysis
of denatured or non-denatured siRNA duplexes
Small interfering RNAs (siRNAs) usually occur as
oligonucleotide duplexes. They can be analysed under nondenaturing conditions so that duplex formation is assured
and excessive single-strands are detected. Alternatively,
analysis of siRNAs can be conducted under denaturingconditions so that both sense and antisense strands aremonitored. Analysis of siRNA under denaturing and nondenaturing conditions can be achieved by ion pair reversed
phase high performance liquid chromatography (IP-RP
HPLC). Several parameters influence siRNA separation:

1. Column temperature – key factor for the denaturation of siRNA
2. Ion pair reagents and acids – affect double-strand stability and separation efficiency

Expert tip
The phosphate-rich backbone of siRNA molecules enables the siRNA to adhere to metal surfaces, includingcolumn hardware. To prevent metal surface interaction, all experiments were conducted with a bioinert
YMC-Accura Triart Bio C18 column. The bioinert surface coating of column body and frit considerablyincreases the sensitivity and sample recovery.
This technical note introduces all relevant aspects of denaturing and non-denaturing IP-RP HPLC. As a sample thesiRNA duplex targeting the firefly luciferase gene GL2 was used (table 1). The siRNA consists of 19 nucleotides.

O

O

O

O

B1

Table 1: Oligonucleotide sequence

B1

siRNA

O
-

O

X

O

The conventionally used materials for tubing and column
hardware pose a special challenge for the analysis ofoligonucleotides. Stainless steel provides mechanicalresilience and compatibility with most solvents althoughmany eluents such as methanol or acetonitrile can cause
corrosion. The resulting positively charged surface can leadto metal leaching as well as undesired ionic interactions withthe analytes. Electron rich analytes such as oligonucleotides
can be irreversibly adsorbed. This non-specific adsorption

has a negative influence on recovery and peak shape.
This effect is even more critical when working at low toneutral pH, as metals are more electropositive under these
conditions.
To overcome this problem HPLC systems and columns
can be passivated with strong acids or pre-conditioned
with a similar sample. However, these procedures are time
consuming and a recurring task. Furthermore, a change ofsample can lead to non-specific adsorption again.

O
-

P O

S

O

O

HO

X

HO

X

B2
X

Phosphorothioate Linkage(All PS)

X = -H (DNA), -OH (RNA)
Figure 1: RNAs with phosphodiester linkage (All PO) and with sulphur modified phosphorothioate linkage (All PS).

1. Effect of ion pair reagents and pH on oligonucleotide separation

Bioinert column options
A much more robust and simple solution is to use a fullybioinert system and bioinert column hardware. The column
body represents more than 70% of the surface that theanalytes get in touch with [1]. Consequently, a bioinert
column body and frits will provide a distinct improvement in
performance. Therefore, YMC offer two bioinert hardware
options: YMC-Accura Triart (U)HPLC columns with a bioinert
coating of the column body and frits and YMC-Triart metalfree (U)HPLC columns with a PEEK-lining and PEEK frits.

Their main difference is their hydrophobicity: YMC-Accura
Triart columns are less hydrophobic compared to YMC-Triart
metal-free columns. Further, YMC-Accura Triart columns offer
a more robust end fitting without any preferences regarding
connection – in contrast to the PEEK-lined columns which
require special connecting options such as MarvelXACT
connectors. These are also available with a bioinert PEEK
layer or a PEEKsil capillary.

YMC Europe GmbH · Schöttmannshof 19 · 46539 Dinslaken · Phone +49 2064 427-0 · Fax +49 2064 427-222 · Email: info@ymc.de · www.ymc.eu

Chromatographic separation of charged biomolecules by
RP is challenging because they are not retained sufficiently on the column. Since oligonucleotides possess an ionic
structure, ion pairing reagents with an opposite charge
need to be added to the mobile phase. Their hydrophobic part of the agent bonds to the hydrophobic stationary phase while the charged part pairs with the analytes
from the mobile phase. This results in the oligonucleotides
being retained on the stationary phase and separated
by length, type and presence of modifications. Triethyl-sense strand
antisense strand

1. Choosing the optimum column temperatureO

B2

Phosphodiester Linkage(All PO)

5’-CGU ACG CGG AAU ACU UCG AdTdT-3’
3’-dTdTGCA UGC GCC UUA UGA AGC U-5’

P O

amine (TEA) is frequently used as the ion pair agent and isoften combined with the acidic counterion 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and methanol as organic modifier. The advantage of this mobile phase composition is its
compatibility with electrospray ionisation mass spectrometry (ESI-MS). This is based on the higher volatility of
HFIP, which results in faster evaporation of HFIP compared to TEA during ESI. The pH in the droplets increases and the TEA-paired oligonucleotide dissociates in thesource so that the detectability is increased.

YMC Europe GmbH · Schöttmannshof 19 · 46539 Dinslaken · Phone +49 2064 427-0 · Fax +49 2064 427-222 · Email info@ymc.de · www.ymc.eu

At high temperatures, double-stranded oligonucleotides
dissociate. The denaturation temperature of siRNAs depends on their length and composition. On-column denaturation can be observed by a peak shift since oligonucleotide duplexes adhere stronger to the stationary phase sothat single-strands elute earlier. Therefore, it is useful todetermine the denaturation temperature of the RNA sample.
Of course, LC can help to monitor the transition.

In figure 1, the different states of the siRNA at different temperatures are visible. At an elevated temperature of 65 °C,
the double-stranded oligonucleotide is separated into twosingle-strands. The retention corresponds to the two single-strands analysed separately. At 45 °C the siRNA is partially degraded and the retention time increases. At 25 °C
non-denatured siRNA is detected, which elutes significantly
later from the stationary phase.

Table 2: Overall chromatographic conditions unless otherwise stated.

Column:Part No.:
Eluent:Gradient:
Flow rate:
Temperature:
Detection:
Injection:
Sample:YMC-Accura Triart Bio C18 (1.9 µm, 30 nm), 50 x 2.1 mm ID
TA30SP9-05Q1PTC
A) 8 mM alkylamine - 100 mM HFIP* (pH 8)B) methanol
1%B/min (initial = 3%B)
0.42 mL/min
65 °C, 45 °C or 25 °C
UV at 260 nm
1 µl (5 nmol/mL)
siRNA duplex, sense strand, antisense strand*1,1,1,3,3,3-hexafluoro-2-propanol

YMC Europe GmbH · Schöttmannshof 19 · 46539 Dinslaken · Phone +49 2064 427-0 · Fax +49 2064 427-222 · Email info@ymc.eu · www.ymc.eu

Page 1/5

Page 1/10

Page 1/5

HILIC analysis ofoligonucleotides using
different bioinert columns

How to influenceoligonucleotide analysis
by IP-RP

How to choose the optimum
conditions for analysis ofdenatured or non-denatured
siRNA duplexes

39

YMC Oligonucleotide Columns

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www.ymc.co.jp

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YMC Oligonucleotide Columns 07/2024_int_V1.040