high resolution lc or lc-ms analysis of oligonucleotides ... · the world leader in serving science...
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The world leader in serving science
5/24/2016Julia Baek , Jim Thayer, Shanhua Lin, Yizhu Guo, Yoginder Singh, Jessica Wang, Xiaodong Liu, Ilze Birznieks
High Resolution LC or LC-MS Analysis of Oligonucleotides and Large DNA Fragments Using a New Polymer-Based Reversed Phase Column
Oligonucleotides as Tools for Molecular Biology Research
DNA Amplification (PCR)
DNA Sequencing
Fluorescent in situ hybridization(FISH)
Primers
2
Molecular Diagnostics
DNA Nanomaterials
Oligonucleotides as Therapeutic Agents
Antisense Oligonucleotide
siRNA(small interfering RNA)
Aptamer
Ribozyme
2
1
3
isRNA(immunostimulatory RNA)
Ribozyme
IVT mRNA(in vitro transcribed mRNA)
CRISPR/Cas9
Chemical Modifications of Oligonucleotides
4
• N-1, N-2, N+1
• PO impurity in PS sample
• Imperfect deprotection (DMT)
• Depurination
Oligonucleotide Impurities
5
HPLC Analysis of Nucleic Acids
Anion Exchange Chromatography
• Provides high resolution separation oligonucleotides
• Low or no organic solvent
Ion-Pair Reversed Phase Chromatography
• Provides high resolution separation oligonucleotides
• Can be directly coupled to
6
• Low or no organic solvent
• Cannot be directly coupled to Mass spectrometer
• Can be directly coupled to Mass spectrometer
Anion Exchange Chromatography
ResinCl-
Base
Base
O
O R
P
O
O–
O
O
O R
Na+
Na+
Cl-
Oligonucleotide
7
Na+
Na+
Cl-
Cl-
Oligonucleotide
Ion-Pair Reversed-Phase Chromatography
Base
Base
O
O R
P
O
O–
O
O
O RResin
Ion-pairing
reagent
Oligonucleotide
8
Oligonucleotide
Triethylammonium acetate (TEAA)
Triethylamine (TEA) + Hexafluoroisopropanol (HFIP)
LC/UV
LC/MS
FF F
FF
FOH
HPLC Analysis of Nucleic Acids
Anion Exchange Chromatography
DNAPac PA100DNAPac PA200
DNAPac PA200 RSDNASwift SAX-1S
Ion-Pair Reversed Phase Chromatography
DNAPac RP
NEW
9
DNASwift SAX-1S
• Reversed phase column designed for separation of oligonucleotides and large DNA/RNA fragments
• High resolution and high efficiency
• Excellent MS compatibility
• Wide operating pH range: 0-14
• High temperature stability: Up to 100 ºC
• High throughput
DNAPac RP Column
10
• High throughput
Substrate Poly(styrene-divinylbenzene) particles
Particle Size 4 µm
Pore Size 1,000~2,000 Å
Column Formats 3.0 × 100 mm3.0 × 50 mm2.1 × 100 mm2.1 × 50 mm
Applications
0
100
Rel
ativ
e A
bund
ance
Ion current
MS compatible
0
28
Abs
orba
nce
(mA
U)
12
40
5Retention Time (min)0
High resolution / Fast separation
pH 9.9
11
2 14
0
80
72
1353
1078
872
Retention Time (min)
Abs
orba
nce
(mA
U)
dsDNA & long oligonucleotides61 Retention Time (min)
1655
.96
[M-4H]4-
0
100
Rel
ativ
e A
bund
ance
Fast Sparation of 12-40mer Deoxythymidines (dTs)
28
Abs
orba
nce
(mA
U)
20
25
30
15
Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water / Acetonitrile
(75:25 v/v)Gradient:
Time (min) %A %B-2.0 76 240.0 76 244.0 59 414.1 10 905.0 10 90
Gradient curve: 3
12
0
Abs
orba
nce
(
12
4035
1 2 3 4 5Retention Time (min)
0
Gradient curve: 3Flow rate: 0.80 mL/minInj. volume: 4 µL Temperature: 80 ºCDetection: UV (260 nm)Sample: poly dT12-40 (1 A/mL)Peak label: Length of DNA
High Resolution Separation of 12-60mer dTs
Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water / Acetonitrile
(75:25 v/v)Gradient:
Time (min) %A %B-2.0 76 240.0 76 247.0 57 437.1 10 905.0 10 90
Gradient curve: 3
Abs
orba
nce
(mA
U)
15
30
20
13
Gradient curve: 3Flow rate: 0.80 mL/minInj. volume: 10 µL Temperature: 80 ºCDetection: UV (260 nm)Sample: poly dT12-60 (0.5 A/mL)Peak label: Length of DNA
0
Abs
orba
nce
(
Retention Time (min)2 40 6 8
60
50
4012
LC/UV Analysis of Mixed-Base DNA
70Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water / Acetonitrile
(75:25 v/v)Gradient:
Time (min) %A %B-2.0 92 80.0 92 83.0 68 323.1 10 905.0 10 90
Gradient curve: 3
Abs
orba
nce
(mA
U)
12
16
20
24 28
32
36
40
14
1 2 3 4
Gradient curve: 3Flow rate: 0.80 mL/minInj. volume: 2 µL Temperature: 80 ºCDetection: UV (260 nm)Sample: 8-Combo DNA (5 µM)*Peak label: Length of DNA
Abs
orba
nce
(
Retention Time (min)
LC/MS Analysis of n and n-1 Sequences
0
100
Rel
ativ
e A
bund
ance
n
n-1
Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 15 mM TEA, 400 mM HFIP, pH 7.9Mobile phase B: 15 mM TEA, 400 mM HFIP in
Water / Methanol (50:50 v/v)
Gradient:Time (min) %A %B0.0 70 303.0 48 533.1 10 905.0 10 905.1 70 30
11.0 70 30
Ion current
15
1 2 3 4 5Time (min)
0
24
Abs
orba
nce
(mA
U)
n
n-1
11.0 70 30
Temperature: 60 ºCFlow rate: 0.25 mL/minInj. volume: 4 µL Detection: UV (260 nm)
MS (Negative-ion mode)Mass Spec: Q Exactive PlusSample: 21mer DNA
GATTGTAGGTTCTCTAACGCT
UV
LC/MS Analysis of n and n-1 Sequences: Mass Spectrum of n Sequence
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
802.2548z=8
1605.5155z=4
917.0068z=7
1604 1606 1608 1610 1612 16140
1001605.0160
1610.5123
[M+Na-5H]4-
[M-4H]4-
16
1100 1200 1300 1400 1500 1600 1700
m/z
0
10
20
30
40
50
Rel
ativ
e A
bund
ance
1070.0088z=6
1284.2119z=5
1299.5910z=5
1000900800
1604 1606 1608 1610 1612 1614
LC/MS Analysis of n and n-1 Sequences: Mass Spectrum of n-1 Sequence
60
70
80
90
100
Rel
ativ
e A
bund
ance
1529.5098z=4
1527.2569z=4
Mi=1526.7534
Mi=1529.0094
Mi=1532.7539
1533.2577z=4 1538.9978
z=4
-T
-A
-C
17
1520 1522 1524 1526 1528 1530 1532 1534 1536 1538 1540 1542 1544 1546 1548 1550
m/z
0
10
20
30
40
50
Rel
ativ
e A
bund
ance
z=4
z=4
1523.26z=4
Mi=1522.76021535.0061
1542.7451z=4
1536.7438
1548.4852z=4
1544.4927z=4 1546.4824
z=4
-G
LC/MS Analysis of Phosphorothioate Modified siRNA
Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 35 mM TEA, 40 mM HFIP, pH 9.9Mobile phase B: 35 mM TEA, 40 mM HFIP in Water /
Methanol (75:25 v/v)
Gradient:Time (min) %A %B0.0 93 75.0 52 485.1 10 907.0 10 907.1 93 7
13.0 93 7 0
100
Rel
ativ
e A
bund
ance
1
2
Ion current
18
Temperature: 30 ºCFlow rate: 0.25 mL/minInj. volume: 3 µL Detection: UV (260 nm)
MS (Negative-ion mode)Mass Spec: Q Exactive PlusSample: 21mer siRNA
AGCUGACCCUGAAGSUUCAUdCdT
1 2 3 4 5 6 70
Abs
orba
nce
(mA
U)
40 12
Time (min)
UV
Rp Sp
Base
Base
O
O
O
PS–
OO
O R
R
LC/MS Analysis of Phosphorothioate Modified siRNA
[M-4H]4-
Peak 1: RT 4.36 min
0
20
40
60
80
100
1656.7181z=4
1656.9645z=4
1656.4702z=4
1656.2194z=4 1657.2207
z=4
1657.4720z=41655.9644
z=4 1657.7151z=4R
elat
ive
Abu
ndan
ce
19
Peak 2: RT 4.93 min [M-4H]4-
1653 1654 1655 1656 1657 1658 1659 1660 1661
m/z
1656.4683z=4
1656.9705z=41656.2192
z=41657.2174
z=4
1657.4664z=41655.9706
z=4 1657.7197z=4R
elat
ive
Abu
ndan
ce
0
20
40
60
80
100
LC/MS Analysis of Phosphorothioate Modified siRNA
0
100 Ion current Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 35 mM TEA, 40 mM HFIP, pH 9.9Mobile phase B: 35 mM TEA, 40 mM HFIP in Water /
Methanol (75:25 v/v)
Gradient:Time (min) %A %B0.0 93 75.0 52 485.1 10 907.0 10 907.1 93 7
13.0 93 7
Rel
ativ
e A
bund
ance
1
3
2
4
1
20
2 3 4 5 6 7 8
0
56 UV13.0 93 7
Temperature: 30 ºCFlow rate: 0.25 mL/minInj. volume: 3 µL Detection: UV (260 nm)
MS (Negative-ion mode)Mass Spec: Q Exactive PlusSample: 21mer siRNA
AGCUGACCCUGAAGUUCAUSdSdT
Abs
orba
nce
(mA
U)
1
3
2
4
LC/MS Analysis of Phosphorothioate Modified siRNA
[M-4H]4-Peak 4:RT 4.96 min
Peak 1: RT 5.57 min [M-4H]4-
0
100
0
100
1656.7231z=4
1662.2181z=4
1661.7166z=4
1656.9744z=4
1662.7181z=41655.9773
z=4
1660.4620z=4
1660.2134z=4
1660.9648z=4
1659.9624z=4
1661.4653z=4
1660.9640
Rel
ativ
e A
bund
ance
PS/PO
21
Peak 2: RT 5.76 min [M-4H]4-
Peak 3: RT 5.93 min[M-4H]4-
1655 1656 1657 1658 1659 1660 1661 1662 1663m/z
0
100
0
1001660.9640z=41660.4652
z=4 1661.2151z=4
1660.2202z=4 1661.7079
z=41659.9653z=4
1660.7145z=41660.2139
z=4
1661.4708z=4
1659.9581z=4
Rel
ativ
e A
bund
ance
PS/PS
Analysis of CpG methylation
2Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 15 mM TEA, 400 mM HFIP, pH 7.6Mobile phase B: 15 mM TEA, 400 mM HFIP in
Water / Methanol (50:50 v/v)
Gradient:Time (min) %A %B0.0 75 254.0 56 444.1 10 906.0 10 906.1 75 25
11.0 75 250
100
Rel
ativ
e A
bund
ance
1
2
Ion current
22
11.0 75 25
Temperature: 60 ºCFlow rate: 0.25Inj. volume: 3 µL Detection: UV (260 nm)Sample: 1) CGGCATCCTTATTGG
2) /iMe-dC/GGCATCCTTATTGG
1 2 3 4 5 6 7Time (min)
0
040
Abs
orba
nce
(mA
U)
12UV
Analysis of CpG methylation: Mass spectra
0
20
40
60
80
100
1518.5880z=3
1525.9135z=3 1531.2359
z=31532.9063
z=31538.5626
z=3
Rel
ativ
e A
bund
ance 1517.9194
z=3
Peak 1[M-3H]3-
23
1522.5926
1510 1515 1520 1525 1530 1535 1540 1545 1550m/z
0
20
40
60
80
100
01523.2603
z=3
1530.5860z=3 1535.9079
z=3
1524.2618z=3
1537.5784z=3
1543.2351z=3
1529.2527z=3
z=3
Rel
ativ
e A
bund
ance
Peak 2[M-3H]3-
Separation of Fluorescein-Labeled DNA
0
70
Abs
orba
nce
(mA
U)
Column: DNAPac RP, 4 µmFormat: 2.1 ×100 mmMobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water /
Acetonitrile (80:20 v/v)
Gradient:Time (min) %A %B0.0 75 25
10.0 0 10012.0 0 10012.1 75 2516.0 75 25
aUnmodified DNA
24
0 2 4 6 8 10
0
50
Abs
orba
nce
(mA
U)
Retention Time (min)
Flow rate: 0.60 mL/minInj. volume: 2.5 µL Temperature: 60 ºCDetection: UV (260 nm)Sample: a. 20mer DNA (60 µg/mL)
b. Fluorescein labeled 20mer DNA (60 µg/mL)
bFluoresceinlabeled DNA
Unmodified DNA
Separation of Restriction Enzyme Digests of pDNAs: Gel
pDNA
Restriction enzyme
25
Separation of Restriction Enzyme Digests of pDNAs: HPLC
80
0
45
51 57
64 80 89 104 12
3
184
192 21
3 234 26
7
434
458 50
254
058
7
124
1353
Abs
orba
nce
(mA
U)
a pBR322-BsuRI digest
b ΦΧ174-BsuRI digest
26
2 3 4 5 6 7 8 9 10 11 12 13 14
0
8072
1353
1078
872
603
310
281
271
234
194
118
Retention Time (min)
Abs
orba
nce
(mA
U)
b ΦΧ174-BsuRI digest
Separation of Thermo Scientific™ FastRuler™ DNA ladders
850
0
28
50
200
400
1.5k
85028
100
400
2k
5k
Abs
orba
nce
(mA
U)
a Low Range
b Middle Range
27
0
2 3 4 5 6 7 8 9 10 11 12 13 14
0
28
1k
2k4k
10k50
0
Abs
orba
nce
(
Retention Time (min)
c High Range
Purification of a PCR product
Column: DNAPac RP, 4 µmFormat: 2.1 ×100 mmMobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water /
Acetonitrile (75:25 v/v)
Gradient:Time (min) %A %B0.0 56 44
20.0 32 6820.1 10 9022.0 10 9022.1 56 44 32.0 56 44
200
300
400
Abs
orba
nce
(mA
U) PCR product
dNTP impurities
5000
2000
850
400
100
28
32.0 56 44
Flow rate: 0.25Inj. volume: 50 µL Temperature: 50 ºCDetection: UV (260 nm)Sample: PCR product
0 5 10 15 20 25 32
0
100
Abs
orba
nce
(
Retention Time (min)
100
41mer RNA: Comparison with Competitors
180
0
900
140
Brand B, 3 µm, 100Å
Brand D, 1.7 µm, 100Å
DNAPac RP 4 µm, 1,500ÅFormat: 2.1 ×50 mmMobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water /
Acetonitrile (90:10 v/v)
Gradient: Gradient slope: 1.73% B/minGradient time: 7.5 min
Temperature: 45 ºCFlow rate: 0.80 mL/minInj. volume: 1 µL Detection: UV (260 nm)Sample: 41mer RNA
Abs
orba
nce
(mA
U)
29
2 3 4 5 6 7
0
340
0
1400
Brand A, C18 2.5 µm, 130Å
Brand A, 1.7 µm, 130Å
Retention Time (min)
Abs
orba
nce
(
Method Development
Pre-Column Heater
Gradient Curve
Temperature
30
Temperature
Flow Rate
Ion-Pair Reagent
Adjustment of Gradient Curve
Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water / Acetonitrile
(25:75 v/v)Gradient:
Time (min) %A %B-3.0 88 120.0 88 124.0 62 384.1 10 906.0 10 90
Gradient curve: a) 5 (linear)
0
70
0
70
Abs
orba
nce
(mA
U)
a
b
31
Gradient curve: a) 5 (linear)b) 4c) 3d) 2
Temperature: 60 ºCFlow rate: 0.60 mL/minInj. volume: 2 µL Detection: UV (260 nm)Sample: 8-Combo DNA (5 µM)*
Abs
orba
nce
(
0
70
1 2 4 50
70
Retention Time (min)
c
d
3
Effect of Gradient Curve on Resolution
6
7
8
9
Res
olut
ion
Effect of Gradient Curve on Resolution
5
4
32
2
3
4
5
12-16 16-20 20-24 24-28 28-32 32-36 36-40
Res
olut
ion
Length of DNA
4
3
2
Separation of Oligonucleotides at Different Temperatures
500
Abs
orba
nce
(mA
U)
Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water / Acetonitrile
(25:75 v/v)Gradient:
Time (min) %A %B-3.0 94 60.0 94 68.0 54 468.1 10 90
10.0 10 90
Temperature: a) 30 ºC
a
b
c
33
4 5 6 7 8 9
0
Abs
orba
nce
(
Retention Time (min)
Temperature: a) 30 ºCb) 40 ºCc) 50 ºCd) 60 ºCe) 70 ºCf) 80 ºC
Flow rate: 0.40 mL/minInj. volume: 2 µL Detection: UV (260 nm)Sample: 8-Combo DNA (5 µM)*
d
e
f
Effect of Temperature on Resolution
7
8
9
10
11
12
Res
olut
ion
Effect of Temperature on Resolution
30°C
40°C
50°C
g
34
2
3
4
5
6
7
12-16 16-20 20-24 24-28 28-32 32-36 36-40
Res
olut
ion
Length of DNA
50°C
60°C
70°C
80°C
Separation of Oligonucleotides at Different Flow Rates
0
70
0
60
60
Abs
orba
nce
(mA
U)
Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water /
Acetonitrile (75:25 v/v)Gradient: a) 12 to 36%B in 12 min.
b) 12 to 36%B in 6 min.c) 12 to 36%B in 4 min.d) 12 to 36%B in 3 min.
Gradient curve: 3
Temperature: 60 ºCFlow rate: a) 0.20
a
b
c
35
0
60
1 2 3 4 5 6 7 8 9 10 11 12 13 14
0
50
Retention Time (min)
Abs
orba
nce
(
Flow rate: a) 0.20b) 0.40c) 0.60d) 0.80
Inj. volume: 2 µL Detection: UV (260 nm)Sample: 8-Combo DNA (5 µM)*
c
d
Effect of Flow Rate on Resolution
7
8
9
10
11
Res
olut
ion
Effect of Flow Rate on Resolution
0.2
0.4
e
36
2
3
4
5
6
12-16 16-20 20-24 24-28 28-32 32-36 36-40
Res
olut
ion
Length of DNA
0.4
0.6
0.8
Comparison of TEA and HA as Ion-Pairing Reagents
0
70
Abs
orba
nce
(mA
U)
Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mm
Mobile phasesa)Mobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water / Acetonitrile
(75:25 v/v)b)Mobile phase A: 0.1 M HA, pH 7.4Mobile phase B: 0.1 M HA in Water / Acetonitrile
(50:50 v/v)
Gradient:a) Time (min) %A %B
-2.0 92 80.0 92 8
a
37
1 2 3 4 5
0
70
Abs
orba
nce
(mA
U)
Retention Time (min)
0.0 92 83.0 68 323.1 10 905.0 10 90
b) Time (min) %A %B-2.0 78 220.0 78 223.0 36 643.1 10 905.0 10 90
Gradient curve: 3Temperature: 80 ºCFlow rate: 0.80 mL/minInj. volume: 2 µL Detection: UV (260 nm)Sample: 8-Combo DNA (5 µM)*
b
Other Ion-Pairing Reagents
38Rapid Commun. Mass Spectrom. 2014, 28, 339–350.
Comparison of TEA and HA as Ion-Pairing Reagents
Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mm
Mobile phasesa)Mobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water / Acetonitrile
(75:25 v/v)b)Mobile phase A: 0.1 M HA, pH 7.4Mobile phase B: 0.1 M HA in Water / Acetonitrile
(50:50 v/v)Gradient: a) 8 to 32%B in 3 min.
b) 23 to 63.5%B in 3 min.
6.97
5.155.76
4.63 4.253.88
3.48
Abs
orba
nce
(mA
U)
0
80 a
39
b) 23 to 63.5%B in 3 min. Gradient curve: 3Flow rate: 0.80 mL/minInj. volume: 2 µL Temperature: 80 ºCDetection: UV (260 nm)Sample: 8-Combo DNA (5 µM)*Peak label: Resolution (ep)
8.42
6.327.01
5.444.73 4.22
3.62
1 2 3 4Retention Time (min)
Abs
orba
nce
(mA
U)
0
80 b
Effect of Oligonucleotide Diluent
0
70Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water /
Acetonitrile (75:25 v/v)
Gradient:Time (min) %A %B-2.0 92 80.0 92 83.0 68 323.1 10 905.0 10 90
Gradient curve: 3
a
Abs
orba
nce
(mA
U)
40
1 2 3 4 5
0
70
Retention Time (min)
Gradient curve: 3Flow rate: 0.60Inj. volume: 2 µL Temperature: 60 ºCDetection: UV (260 nm)Sample: 8-Combo DNA (5 µM)*Sample diluent: a) Water
b) Mobile phase A
b
Abs
orba
nce
(mA
U)
Separation of Diastereoisomers of siRNA at Different pHs
Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 15 mM TEA, 400 mM HFIP, pH 7.9Mobile phase B: 15 mM TEA, 400 mM HFIP in Water /
Methanol (75:25 v/v)
Gradient:Time (min) %A %B0.0 65 355.0 42 585.1 10 907.0 10 907.1 65 35
13.0 65 350
100
Abs
orba
nce
(mA
U)
pH 7.9
41
Flow rate: 0.25 mL/minInj. volume: 3 µL Temperature: 30 ºCDetection: UV (260 nm)
MS (Negative-ion mode)Mass Spec: Q Exactive PlusSample: 21mer siRNA
AGCUGACCCUGAAGSUUCAUdCdT
1 2 3 4 5 6 70
Abs
orba
nce
(mA
U)
40
Time (min)
pH 9.9
Rp Sp
Base
Base
O
O
O
PS–
OO
O R
R
Separation of Mobility Modifier DNA at Different pHs
Column: DNAPac RPFormat: 2.1 ×50 mm
a)Mobile phase A: 0.1 M TEAA, pH 7.0Mobile phase B: 0.1 M TEAA in Water / Acetonitrile
(90:10 v/v)
b)Mobile phase A: 0.05 M TEAA + 0.05% TEA, pH
10.0Mobile phase B: 0.05 M TEAA + 0.05% TEA in
Water / Acetonitrile (50:50 v/v)130
0
180
Abs
orba
nce
(mA
U)
pH 7.0
pH 10.0 Desired product
42
Gradient: a) 5-35% B in 7.5 minutesb) 5-40% B in 7.5 minutes
Temperature: 45 ºCFlow rate: 0.80 mL/minInj. volume: 2 µL Detection: UV (260 nm)Sample: Sequencing primer labeled with
fluorescein and contains at least 10 units of mobility modifier (ethylene oxide)
1 7
0
130
Abs
orba
nce
(
Retention Time (min)
pH 10.0
2 3 4 5 6
N-1 PEG
N-2 PEG
Desired product
DNAPac PA200 RS vs DNAPac RP
DNAPac PA200 RSAnion Exchange Chromatography
• Provides ultra-high resolution separation oligonucleotides
• Low or no organic solvent
DNAPac RPIon-Pair Reversed Phase
Chromatography
• Provides high resolution separation oligonucleotides
• Can be directly coupled to
43
• Low or no organic solvent
• Cannot be directly coupled to Mass spectrometer
• Can be directly coupled to Mass spectrometer
• Excellent separation of long oligonucleotides and large dsDNA fragments
ssDNA: 8-ComboA
bsor
banc
e (m
AU
)
15.83
11.488.94
7.185.94 5.02 4.23
0
60
140
DNAPac RP 3.0x100 mm
DNAPac PA200 RS 4.6x150 mm
44
4 5 6 7 8 9 10 11 12 13
Abs
orba
nce
(mA
U)
Retention Time (min)
25.76
17.9313.47
10.498.50
7.19 6.20
0
140 DNAPac PA200 RS 4.6x150 mm
dsDNA: ΦΧ174-BsuRI digest
0
45
Abs
orba
nce
(mA
U)
120
19.8
9
16.2
3
4.85 3.67 0.
892.
30 11.3
2
4.33
1.56
1.45
DNAPac RP 3.0x100 mm
DNAPac PA200 RS 4.6x150 mm
45
Retention Time (min)2 3 4 5 6 7 8 9 10 11 12 13
0
120
Abs
orba
nce
(mA
U)
28.5
9
15.4
5
7.07
4.50
1.38
7.27
DNAPac PA200 RS 4.6x150 mm
DNAPac RP vs Competitors: ssDNA
0
90
11.4
1
7.77 5.93 4.75 3.
98
3.37
2.89
0
90
12.2
5
8.19 6.09 4.
80 3.90
3.40
2.93
90
Abs
orba
nce
(mA
U)
12.7
8
8.48 6.
26 4.90 4.01 3.38
2.96
Brand B, 100 Å, 2.7 µm
DNAPac RP, 4 µm
Brand A 100 Å, 1.7 µm
46
0
Abs
orba
nce
(mA
U)
12.7
8
0
90
6.70 4.
52 3.39 2.68 2.14
1.79
1.53
3 4 5 6 7 8
0
90
Retention Time (min)
9.74 6.
50 4.66 3.65 2.99
2.52
2.14
Brand B 1000 Å, 5 µm
Brand C, 3 µm
DNAPac RP vs Competitors: dsDNA
0
50
0
80
35
Abs
orba
nce
(mA
U)
DNAPac RP, 4 µm
72
310
281
271
234
194
118
1353
1078
872
603
Brand B, 100 Å, 2.7 µm
DNAPac RP, 4 µm
Brand A 100 Å, 1.7 µm
47
0
Abs
orba
nce
(
0
12
3 4 5 6 7 8 9 10 11 12 13
0
35
Retention Time (min)
Brand C 1000 Å, 5 µm
Brand D, 3 µm
Lot-to-Lot Variability: ssDNA
0
90
Abs
orba
nce
(mA
U)
90
Abs
orba
nce
(mA
U)
Lot 01425078
Lot 01425079
48
0Abs
orba
nce
(mA
U)
1.00 2.00 3.00 4.00 5.00 6.00
0
90
Abs
orba
nce
(mA
U) Lot 01425080
Lot-to-Lot Variability: dsDNA
-5.0
20.0
45.0
Abs
orba
nce
(mA
U)
20.0
45.0
Abs
orba
nce
(mA
U)
Lot 01425078
Lot 01425079
49
-5.0
Abs
orba
nce
(mA
U)
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0-5.0
20.0
45.0
Abs
orba
nce
(mA
U)
Retention Time (min)
Lot 01425080
• DNAPac RP is a polymer based reversed phase column
• DNAPac RP provides high resolution separation of single-stranded oligonucleotides
• LC/MS analysis of oligonucleotides can be achieved by using DNAPacRP column coupled with Thermo Scientific™ Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer
• Wide-pore size of DNAPac RP column provides high resolution
Summary
50
• Wide-pore size of DNAPac RP column provides high resolution separation of large double-stranded DNA/RNA fragments
• DNAPac RP can be used with high pH mobile phase and at high temperature which often provides alternative selectivity and higher resolution for more challenging samples
• DNAPac RP has minimal lot-to-lot variability
Thank You!
51