High Resolution LC or LC-MS Analysis of Oligonucleotides ... · High Resolution LC or LC-MS...

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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


• Can be directly coupled to

6


• Cannot be directly coupled to Mass spectrometer

• Can be directly coupled to Mass spectrometer


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


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



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


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

(


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)


13.0 93 7 0

100

Rel

ativ

e A

bund

ance

1

2

Ion current

18


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


[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


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 /



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



AGCUGACCCUGAAGUUCAUSdSdT

Abs

orba

nce

(mA

U)

1

3

2

4


[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)


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)


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


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

(


c High Range

Purification of a PCR product



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

(


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 /


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


Abs

orba

nce

(

Method Development

Pre-Column Heater

Gradient Curve

Temperature

30

Temperature

Flow Rate

Ion-Pair Reagent

Adjustment of Gradient Curve



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


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)



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

(


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)


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


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)


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 /


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


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 /



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)


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

(


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


DNAPac RPIon-Pair Reversed Phase

Chromatography


• Can be directly coupled to

43


• 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)


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


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 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


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


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)


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

High Resolution LC or LC-MS Analysis of Oligonucleotides ... · High Resolution LC or LC-MS...

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Transcript of High Resolution LC or LC-MS Analysis of Oligonucleotides ... · High Resolution LC or LC-MS...