accucore - thermo fisher...

Accucore Ultimate Core Performance LC Column Technology to Maximize Your Investment

Dave Jarzinski Thermo Fisher Scientific Account Manager – Greater Boston/Cambridge & North Phone: 978-408-1576 Email: [email protected]

October 2011

mailto:[email protected]�

2

Fast Chromatography - Understanding the Drivers

• Analytical Scientist want

FAST CHROMATOGRAPHY

Speed 62%

Resolution 23%

Sensitivity 7%

Other 8%

3

Accucore - Core Enhanced TechnologyTM

Solid Core Particles – 2.6µm diameter particles with a solid core generate high speed, high resolution separations without excessive backpressure

Advanced Bonding Technology – Optimised phase bonding creates a series of high coverage, robust phases

Tight Control of Particle Diameter – Enhanced selection process keeps particle size distribution to a minimum and produces high efficiency columns

Automated Packing Process - Enhanced automated procedures ensure that all columns are packed with the highest quality

4

Particle Evolution

Pellicular particles, 50μm

1970 2000 1990 1980 2010 1960

Higher Efficiency

5

Resolution Equation

+

−

=

'1'1

41 5.0

kkNRs α

α

Efficiency Particle size / packing

Selectivity (Chemistry)

Retention Factor

(Surface area)

6

The Theory … Van Deemter Equation

uCuCuBAHETP sm +++=

Eddy Diffusion

Longitudinal Diffusion

7

A Term - Eddy Diffusion or Multiple Paths

• The A term depends on; • Quality of the packing (better packing smaller A term) • Particle size (smaller the particle the smaller is the A term)

8

Core Enhanced Technology – Eddy Diffusion

Fully Porous Particles D90/10 ~ 1.5

Core Enhanced Technology D90/10 ~ 1.1

9

C Term - Resistance to Mass Transfer (s)

Fully Porous Particle Core Enhanced Technology Particle

• The C term depends on: • Differences in diffusion

path in the silica pores

10

Core Enhanced Technology – Packed Bed

Core Enhanced Technology

Porous Silica

11

0.0

5.0

10.0

15.0

20.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Hei

ght E

quiv

alen

t The

oret

ical

Pl

ate

Linear velocity of mobile phase (mm/s) Accucore 2.6µm 5µm 3µm <2µm

Core Enhanced Technology - Efficiency

Highest efficiency and lowest rate of efficiency loss with flow rate with Core Enhanced Technology

12

Core Enhanced Technology – Low Pressure

0

100

200

300

400

500

600

700

800

900

1000

0 200 400 600 800 1000

Pres

sure

(bar

)

Flow rate (µL/min)

Accucore RP-MS 2.6µm <2µm 3µm 5µm

Columns: 100 x 2.1 mm Mobile phase: H2O / ACN (1:1) Temperature: 30 °C

600 bar limit

HPLC pressure limit

Pressures below 600 bar over optimum flow rate range with Core Enhanced Technology

13

Kinetic Plots

• Allows for fairer comparisons of analytical systems • Van Deemter just compares pure separation ability

• Incorporates time of analysis

• Analysts want FASTER chromatography • Van Deemter plots do not specify the time of analysis

• Incorporates pressure limitations of systems

• Van Deemter does not account for a pressure limitation on system

14

0.0003

0.0030

0.0300

1,000.00 10,000.00 100,000.00 1,000,000.00

Accucore RP-MS 2.6um 5um 3um <2um N (/)

Nopt

t0/N (s)

Nopt Tim

e re

quire

d to

obt

ain

a se

t effi

cien

cy

Kinetic Plot – Efficiency & Speed

Efficiency increasing

Highest plate generation rate with Core Enhanced Technology

15

Impedance

• Devised by Knox and Bristow in 1977 • Defines the resistance a compound has to moving

down a column relative to the performance of that column

• Allows for pressure to be incorporated

• Often plotted with a reverse axis • Mimics van Deemter plot • Minimum value optimum conditions

• Often plotted as a dimensional form

• t/N2

• t0 or tr both used

η2N

PtE ∆=

16

Core Enhanced Technology – Low Impedance

Lowest impedance (best combination speed, efficiency and low pressure) of with Core Enhanced Technology

1,000

10,000

100,000

0.10 1.00 10.00

Accucore RP-MS 2.6µm 5µm 3µm <2µm

Impe

danc

e

Linear velocity of mobile phase (mm/s)

17

Core Enhanced Technology – Features & Benefits

• Features • More uniform particle sizing • Better packing of particles • Reduced pore depth • Reduced mass transfer effects in mobile phase

• Benefits

• More Efficient Chromatography • Allows the use of low pressure systems

• Competitive Edge

• Bar for bar gives better separations than porous materials

18

Faster than Fully Porous 5 and 3µm

Gradient and flow rate: • Fully porous 5 μm 150 x 4.6 mm

35 – 60 %B in 10.0 min 1000 µL/min solvent used 17 mL

•Fully porous 5 μm, 100 x 2.1 mm 35 – 60 %B in 6.7 min 210 µL/min solvent used 2.4 mL

• Fully porous 3 μm, 100 x 2.1 mm 35 – 60%B in 4.0 min 350 µL/min solvent used 2.4 mL

• Accucore RP-MS 2.6 μm, 100 x 2.1 mm 35 – 60 %B in 3.5 min 400 µL/min solvent used 2.4 mL

Method Transfer Calculator: www.thermoscientific.com/crc

Minutes 0 1 2 3 4 5 6 7 8 9 10

-100

Fully porous 3µm, 100 x 2.1 mm


ACCUCORE 2.6µm, 100 x 2.1 mm


Rs = 2.64

Rs = 1.64

Rs =1.96

Rs = 2.50

5µL injection

1µL injection

1µL injection

1µL injection

ΔP = 59 bar

ΔP = 23 bar

ΔP = 218 bar

ΔP = 97 bar

Reduced analysis time and solvent costs

19

More Peak Capacity than Fully Porous 5 or 3µm

Minutes 0.0 0.5 1.0 1.5 2.0 2.5 3.0

220

240

3µm, 100 x 2.1 mm

ACCUCORE 2.6µm, 100 x 2.1 mm

5µm,100 x 2.1 mm Gradient: 65 – 95%B in 2.1 min, 95% B for 0.4 min Flow rate: 400 μL/min

0

20

40

60

80

100

120

140

160

180

Accucore 2.6µm 3µm 5µm

Nor

mal

ised

pea

k ca

paci

ty

Higher peak capacity – more peaks can be separated per injection

20

More Sensitive than Fully Porous 5 and 3µm Gradient and flow rate:

• 5μm, 100 x 2.1mm 35 – 60 %B in 6.7 min 210 µL/min

• 3μm, 100 x 2.1mm 35 – 60 %B in 4.0 min 350 µL/min

•Accucore RP-MS 2.6μm, 100 x 2.1mm 35 – 60 %B in 3.5 min 400 µL/min

Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

mAU

S/N = 169

S/N = 368

S/N = 399

3µm, 100 x 2.1 mm

ACCUCORE 2.6µm, 100x2.1mm

5µm, 100 x 2.1 mm

Higher S/N ratios – detection and quantitation of low level impurities

21

Lower Backpressure than Fully Porous sub-2µm

• Flow rate: 500 μL/min • Mobile phase: A - Water; B – Acetonitrile

Minutes 0.0 0.5 1.0 1.5 2.0 2.5

Sub 2µm, 100 x 2.1 mm

Accucore RP-MS 2.6µm, 100 x 2.1 mm

Accucore RP-MS 2.6 µm, 100 x 2.1 mm

Sub 2 µm, 100x2.1mm

Maximum pressure (bar)

171 338

Equivalent performance, lower pressure (50% lower)

22

UHPLC Systems Not Required • Column: Accucore RP-MS 2.6µm, 100 x 2.1 mm

• Gradient: 65 – 95 % B in 2.1 min

95 % B for 0.4 min

• Flow rate: 400 µL/min

Accela 1250

Surveyor

min 0 0.5 1 1.5 2 2.5 3 3.5

Minutes 0.00 1.00 2.00 3.00 4.00

Agilent 1100

Accela 1250

Surveyor Agilent 1100

Run time (min)

2.5 3.0 3.5

Average PW (1/2 Height)

0.02 0.02 0.04

Dwell volume: 100 µL



Accucore can deliver performance on a number of different systems

23

Equivalent Loading Capacity to Fully Porous sub-2µm

Columns:

• Accucore RP-MS 2.6µm,100 x 2.1mm

• <2µm,100 x 2.1mm

0

0.2

0.4

0.6

0.8

1

1.2

Nor

mal

ised

Val

ue

Load on Column (µg)

Effect of Loading - Accucore

As

N

Tr

As

N Tr

R² = 0.9998

R² = 0.9993

R² = 0.9721

0

500,000

1,000,000

1,500,000

2,000,000

2,500,000

0 0.5 1 1.5 2 2.5

Peak

are

a

Load on column (µg) <2µm Accucore 2.6μm Competitor

No loss in performance with 2µg loaded on a 2.1mm ID Accucore column

Accucore Evaluation for Metabolite Profiles

Courtesy of: Anila Desai - Scientist

25

ESI+ LC-MS Profile of a Drug / Hypersil Gold, 50 x 2.1, 1.9 micron

TIC (RT 5.22)

M1 – Oxidation (RT 4.55)

M2 – Hydrolysis of X (RT 4.36)

M3 – Glucuronidation (RT 4.61)

M4 – O+Glucuronidation (RT 4.52)

M5 – O+SO3H (RT 4.39)

ESI+ LC-MS Profile of a Drug / Hypersil Gold, 50 x 2.1, 1.9 micron ESI+ LC-MS Profile of a Drug

Hypersil Gold, 50 x 2.1, 1.9 micron RT: 0.06 - 6.97

1 2 3 4 5 6Time (min)

0

50

1000

50

1000

50

1000

50

100

Relat

ive A

bund

ance

0

50

1000

50

1005.22

2.582.402.15 2.95 3.401.77 4.13 4.884.58 6.936.075.541.030.255.322.40 4.55

2.58 2.92

4.663.506.244.13 4.882.29 6.613.32 5.542.131.420.880.64

4.36

3.033.81 5.254.773.322.92 6.582.451.51 5.932.101.030.44

4.61

4.133.892.58 3.74 5.735.222.66 5.902.45 6.440.595.40

4.52

2.92 3.715.22

3.58 3.922.61 4.952.53 6.022.01 6.906.411.770.44 0.644.39

2.212.77

3.683.423.76 6.613.292.55 5.274.63 5.40 5.991.791.57

26

ESI+ LC-MS Profile of a Drug Accucore – RP-MS, 50 x 2.1, 2.6 micron

TIC (RT 4.64)





Decrease in retention time while keeping same resolution

M5 – O+SO3H (RT 4.05)

RT: 0.02 - 7.33

1 2 3 4 5 6 7Time (min)

0

50

1000

50

1000

50

1000

50

100

Relat

ive A

bund

ance

0

50

1000

50

1004.64

2.272.08 2.63 2.93 3.56 7.074.414.13 4.97 6.931.59 5.880.10 0.494.97

4.022.27

4.132.63 3.562.08 5.862.98 7.026.931.87 5.484.411.370.790.25

3.69

2.707.18

6.773.13 4.232.19 6.655.13 5.534.821.891.510.98 5.970.054.15

6.774.33 4.713.97 5.212.16 3.64 6.633.082.29 5.291.89 5.94 7.120.830.25

4.954.18

2.70 3.23 4.313.95 5.08 5.48 6.795.832.452.061.471.234.05

2.47

1.65 3.842.86 3.59 4.67 7.072.27 5.944.71 6.085.510.10 0.88

27

ESI+ LC-MS Profile of a Drug Accucore – C18, 50 x 2.1, 2.6 micron

TIC (RT 4.44)





Decrease in retention time while keeping same resolution

M5 – O+SO3H (RT 3.95)

RT: 0.04 - 6.93

1 2 3 4 5 6Time (min)

0

50

1000

50

1000

50

1000

50

100

Relat

ive A

bund

ance

0

50

1000

50

1004.44

2.472.212.00 2.68 3.06 4.28 4.661.59 3.82 4.82 6.335.38 5.970.05 0.494.82

3.84

2.213.95

2.55 5.702.912.00 4.053.41 6.876.081.70 5.111.370.34 0.833.48

2.686.76

6.646.314.46 5.921.781.59 2.47 3.11 5.434.410.05 0.69

4.02

6.763.61 5.144.53 6.622.11 3.132.42 5.431.97 5.76

4.844.05

3.16

2.68 4.483.23 3.892.47 5.002.26 6.766.005.54 6.421.810.44 0.793.95

2.42

4.442.21 3.06 3.331.54 5.97 6.284.53 5.894.950.980.15

28

Retention Time Comparison – Metabolite Profiles

3

3.5

4

4.5

5

5.5

HypersilGOLD 50x2.1; 1.9um Accucore RP-MS 50x2.1; 2.6um Accucore C18 50x2.1; 2.6um

Ret

entio

n Ti

me

29

Long Column Lifetime

• Stability at extremes of pH

• Stability at elevated temperature

• Packed bed ruggedness

30

Reproducible Chromatography - Batch-to-Batch

Accucore C18 Test 1 - Hydrophobic Interactions HS SS HBC

EV419 1.76 1.41 0.18 EV422 1.77 1.41 0.19 EV435 1.75 1.40 0.18 EV442 1.77 1.40 0.18 EV436 1.77 1.41 0.19 EV446 1.76 1.40 0.18

Average 1.76 1.41 0.18 %RSD 0.32 0.36 2.98

Fully characterised primary and secondary interactions

31

Reproducible Chromatography – Run-to-Run

~2,400 injections on Accucore column with TLX system – no change in retention

3.5000

3.6000

3.7000

3.8000

3.9000

4.0000

4.1000

4.2000

4.3000

4.4000

0 500 1000 1500 2000 2500

Minutes

Injection

Rosuvastatin Retention

32

Accucore Column Stability – Low pH

0

5

10

15

20

25

30

35

40

0 5000 10000 15000 20000 25000 30000 35000

Ret

entio

n Fa

ctor

Column Volumes

Column Stability at pH < 2

Acetaminophen p-HBA o-HBA Amitriptyline Nortriptyline DIPP DNPP

Accucore columns are stable at low pH

pH ≅ 1.8 (0.1% TFA)

30,000 column volumes (5.5 days)

33

Accucore Column Stability – High pH

pH ≅ 10.5 (0.1% ammonia)

30,000 column volumes (5.5 days) R

eten

tion

Fact

or

Accucore columns are stable at high pH

34

Accucore Column Stability – Elevated Temperature

0

1

2

3

4

5

6

7

8

9

0 2000 4000 6000 8000 10000

Ret

entio

n Fa

ctor

Column Volumes

Column Stability at 70°C

Phenol

Butylbenzene

o-Terphenyl

Pentylbenzene

Mobile phase: MeOH/H2O (65:35) Flow rate: 0.4 mL/min

Column temperature: 70 °C Run time: 5 min

9,000 column volumes

(400 injections)

Accucore columns are stable at high temperature

35

Accucore Column Stability – Ruggedness

Efficiency (o-Xylene)

Asymmetry (o-Xylene)

Accucore RP-MS 2.6µm 100x 2.1mm ID Mobile Phase: 60/40 ACN/H2O Flow Rate: 400 µL/min Injection Volume: 1 µL Column Temp: 30°C

Accucore RP-MS 2.6µm 100x 2.1mm ID Mobile Phase A: Water (0.05% TFA) Mobile Phase B: Acetonitrile (0.05% TFA) Wash: H2O (0.05% TFA) Injection Volume: 1 µL Column Temperature: 30°C

4,000 + isocratic test injections with no decrease in performance

6,000 + fast gradient injections with no change in retention

36

Accucore HPLC Columns Proof of Concept

Ultimate Core Performance Maximize Your Investment

Fast Separations Increase sample throughput and decrease solvent costs

High Peak Capacity Separate and identify more peaks

Increased Sensitivity Detect trace amounts

Lower Pressure Use existing HPLC instruments or prolong the life of UHPLC instruments

Loading Capacity Analyze a wide range of concentrations

Wide Selectivity Use the right column for your analysis

Reproducible Chromatography Have confidence in your separations

Long Lifetime Use columns for longer

37

• Ultimate Core Performance to Maximize Your Investment

• Rugged and reproducible solid core particles • Fast separations with superb resolution • Low backpressures

Theory & Practice Conclusion

Core Enhanced Technology

Accucore Evaluation

Chris Singleton Biogen Idec Email: [email protected]

14 Oct 2011

mailto:[email protected]�

http://www.biogenidec.com/Default.aspx?ID=5402�

39

Outline

• Isocratic and gradient evaluation on C18

• Multiple analyte evaluation on aQ

• Temperature effect on aQ separations

• Selectivity comparison between columns


40

TIC of +MRM (3 pairs): from Sample 1 (isocratic_accucore_01) of AccuCore eval FINAL.wiff (Turbo Spray) Max. 7.3e5 cps.

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

7.0e5

7.3e5

Intensity, cps

0.66

0.54

0.48

Accucore C18, 2.1mm*50mm A: 20mM ammonium formate +0.1% formic acid B: Acetonitrile +0.1% formic acid

Mebendazole

Dibucaine Protryptyline

45 seconds

0.65ml/min 1µL injection Shimadzu HPLC MS/MS detection


41

TIC of +MRM (5 pairs): from Sample 2 (gradient_accucore_21) of AccuCore eval FINAL.wiff (Turbo Spray) Max. 2.9e5 cps.

0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5 2.9e5

Intensity, cps

1.18

1.00

1.28

1.37

Accucore C18, 2.1mm*50mm

1 min 27 sec

0.65ml/min

1µL injection

Shimadzu HPLC

MS/MS detect

30°C

Time %B 0.1 15 1.1 95 1.8 95 1.85 15 2.4 15

Chlorpheniramine Mebendazole Amitryptyline Loperamide

DPCPX

Same mobile phases as previous slide


42

Outline






43

TIC of +MRM (6 pairs): from Sample 3 (gradient_accucore_AQ_30C_01) of AccuCore eval FINAL.wiff (Turbo Spray) Max. 1.1e6 cps.

0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e6 1.09e6

Intensity, cps

1.21

1.42

1.12

1.27

0.98

1.36

Accucore aQ, 2.1mm*50mm

0.65ml/min

1µL injection

Shimadzu HPLC

MS/MS detect

30°C

Time %B 0.1 20 1.20 90 1.8 90 1.85 20 2.4 20

Same mobile phases as previous slide

Amitryptyline Protryptyline

DPCPX

Loperamide

Mebendazole

Chlorpheni-ramine


44

Outline






45

0.65ml/min 1µL injection Shimadzu HPLC MS/MS detect 30°C

Time %B 0.1 20 1.20 90 1.8 90 1.85 20 2.4 20

Accucore aQ, 2.1mm*50mm XIC of +MRM (6 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 3 (gradient_accucore_AQ_30C_01) of AccuCore eval FINAL.wiff (... Max. 8.1e5 cps.

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e6 1.09e6

Intensity, cps

1.27


46

0.65ml/min 1µL injection Shimadzu HPLC MS/MS detect 35 °C

Time %B 0.1 20 1.20 90 1.8 90 1.85 20 2.4 20


0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e6

1.10e6

Intensity, cps

1.25


47


Time %B 0.1 20 1.20 90 1.8 90 1.85 20 2.4 20


0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e6

1.10e6

1.15e6

1.20e6

Intensity, cps

1.24


48


Time %B 0.1 20 1.20 90 1.8 90 1.85 20 2.4 20


0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 Time, min

0.00 5.00e4 1.00e5 1.50e5 2.00e5 2.50e5 3.00e5 3.50e5 4.00e5 4.50e5 5.00e5 5.50e5 6.00e5 6.50e5 7.00e5 7.50e5 8.00e5 8.50e5 9.00e5 9.50e5 1.00e6 1.05e6 1.10e6 1.15e6 1.20e6 1.25e6 1.28e6

Intensity, cps

1.24


49


Time %B 0.1 20 1.20 90 1.8 90 1.85 20 2.4 20


0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 Time, min

0.00 5.00e4 1.00e5 1.50e5 2.00e5 2.50e5 3.00e5 3.50e5 4.00e5 4.50e5 5.00e5 5.50e5 6.00e5 6.50e5 7.00e5 7.50e5 8.00e5 8.50e5 9.00e5 9.50e5 1.00e6 1.05e6 1.10e6 1.15e6 1.20e6 1.25e6 1.28e6

Intensity, cps

1.23


50


Time %B 0.1 20 1.20 90 1.8 90 1.85 20 2.4 20


0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

Intensity, cps

1.22


51


Time %B 0.1 20 1.20 90 1.8 90 1.85 20 2.4 20


0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

Intensity, cps

1.22


52

1ml/min 1µL injection Shimadzu HPLC MS/MS detect 60 °C

Time %B 0.1 20 1.20 90 1.8 90 1.85 20 2.4 20

Accucore aQ, 2.1mm*50mm XIC of +MRM (6 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 11 (gradient_accucore_AQ_60C_1mL-min_01) of AccuCore eval F... Max. 8.6e5 cps.

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 Time, min

0.00 5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e6

1.10e6

1.15e6

1.20e6 1.25e6

Intensity, cps

0.96


53

XIC of +MRM (6 pairs): 305.0/221.1 Da ID: CPDPX from Sample 11 (gradient_accucore_AQ_60C_1mL-min_01) of AccuCore eval FINAL... Max. 4.2e5 cps.

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

Intensity, cps

1.06 1ml/min

1µL injection

Shimadzu HPLC

MS/MS detect 60 °C

XIC 305.0/221.1

Time %B 0.1 20 1.20 90 1.8 90 1.85 20 2.4 20

Accucore aQ, 2.1mm*50mm

1.8 second peak width at baseline


54

Outline






55

Typical bonded phases for Reversed-Phase LC

• Alkyl chain: C18, C8, C4, C1 • same selectivity, decreasing degree of

hydrophobicity/retention • Dispersive interactions

• Aromatic groups

• Phenyl, Phenyl-Hexyl, perfluorinated • Dipole-dipole, π-π, hydrogen-bonding

interactions

• Other • Cyanos, aminos, polar embedded • Dipole-dipole, hydrogen-bonding

interactions

Perfluorinated

56

Phase Characteristics

Hydrophobic Interactions

Secondary Interactions

Acidic Interactions

Hydrophobic retention

Base activity Acid interaction

HR BA AI Hydrophobic selectivity

Chelation

Ion exchange capacity pH 2.7

HS C IEX (2.7) Steric selectivity Ion exchange

capacity (pH 7.6)

SS IEX (7.6) Hydrogen bonding capacity

HBC

HR K’ Pentylbenzene

HS α Butylbenzene / Pentylbenzene

SS α Triphenylene / o-Terphenyl

HBC α Caffeine / Phenol

BA tf Amitripyline

C tf Quinizarin

IEX(7.6) α Benzylamine / Phenol

AI tf 4-Chlorocinnamic acid

IEX(2.7) α Benzylamine / Phenol

57

Accucore HPLC Columns – Phase Details

RP-MS Optimized for MS detection, excellent combination of speed and quality of separation

Phenyl-Hexyl Unique selectivity for aromatic and moderately polar

analytes

C18 Optimum retention for non-polar analytes

PFP Alternative

selectivity to C18, particularly for

halogenated analytes

aQ Compatible with 100% aqueous mobile phases, special selectivity for polar analytes

HILIC Enhanced

Retention of polar and hydrophilic

analytes

L11

Guard Columns Available !

58

XIC of +MRM (7 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 2 (Accucore_phenyl-hexyl_001) of Accurcore_selectivity.wiff (Turb... Max. 2.7e5 cps.

1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

1.6e6

1.7e6

Intensity, cps

1.15


Time %B 0.15 20 1.20 90 2.0 90 2.05 20 2.70 20

Accucore Phenyl-Hexyl, 2.1mm*50mm

Mebendazole

Protryptyline

Amitryptyline

DPCPX

Loperamide


59

XIC of +MRM (7 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 4 (Accucore_PFP_001) of Accurcore_selectivity.wiff (Turbo Spray... Max. 200.0 cps.

1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

1.6e6

1.7e6 1.7e6

Intensity, cps

1.33


Time %B 0.15 20 1.20 90 2.0 90 2.05 20 2.70 20

Accucore PFP, 2.1mm*50mm Mebendazole

DPCPX

Protryptyline, amitryptyline and loperamide are highly retained and elute in subsequent injections


60

XIC of +MRM (7 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 6 (Accucore_C18_001) of Accurcore_selectivity.wiff (Turbo Spray) Max. 2.7e5 cps.

1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

1.6e6

1.7e6

1.8e6

1.9e6

Intensity, cps

1.24

1.45


Time %B 0.15 20 1.20 90 2.0 90 2.05 20 2.70 20

Accucore C18, 2.1mm*50mm

Mebendazole

Protryptyline

Amitryptyline

DPCPX

Loperamide


61

XIC of +MRM (7 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 8 (Accucore_aQ_001) of Accurcore_selectivity.wiff (Turbo Spray) Max. 4.3e5 cps.

1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

1.6e6

1.7e6

1.8e6

1.9e6

Intensity, cps

1.53


Time %B 0.15 20 1.20 90 2.0 90 2.05 20 2.70 20

Accucore aQ, 2.1mm*50mm Mebendazole

Protryptyline Amitryptyline

DPCPX

Loperamide is a late eluter and elutes well after the gradient


62

Quest i ons

accucore - thermo fisher...

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