Holistic HPLC Analytical Strategies for Pharmaceutical Labsapps.thermoscientific.com/media/SID/LSMS/PDF/LSMSUsersMtg/Bosto… · Holistic HPLC Analytical Strategies for Pharmaceutical

Holistic HPLC Analytical Strategies for Pharmaceutical Labs by Rainer Bauder, Solutions Manager Pharmaceutical Markets

2 Proprietary & Confidential

Overview

1. Column Efficiency and Selectivity 2. Your Workflow and Our Automation Tools 3. Boosting Nebulizer Based Detectors

5 Proprietary & Confidential

0 10 0

100

H [µ

m]

Efficiency

Efficiency -Using Improved Diffusion Properties And Plates-

5 µm particles 3 µm particles 2 µm particles

Pressure

2 4 6 8 10 u [mm/s] 0

200

400

600

800

1000

1200

0

Col

umn

pres

sure

[bar

]

2 µm particles 3 µm particles 5 µm particles

0.7 1.4 2.1 2.8 3.5 F [mL/min] 0.7 1.4 2.1 2.8 3.5 F [mL/min] 2 4 6 8

u [mm/s]

*Linear velocity translated to flow rate for a 3 mm i.d. column

6 Proprietary & Confidential

Selectivity -Using Increased Chemical Interaction-

Rs – Resolution N – Theoretical plates α – Selectivity k – Retention factor

Example: To separate two analytes on a 5-µm,150 mm C18 column Assuming N = 10,000 plates/column, k >> 1, and α = 1.04 ⇒ Rs = 1 Objective: increase Rs to 2 Approach 1 – increase N needs 400% increase or N = 40,000 plates Approach 2 – increase α requires only 4% increase or α = 1.08

12 Proprietary & Confidential

Speed-Up Strategy

15 Proprietary & Confidential

Speeding-up Gradient Separation of Parabenes

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

min

150 x 4.6 mm, 3 µm

F = 2.5 mL/min

tG = 4.3 min

Speed 2.8x

No loss in resolution

100 x 4.6 mm, 2 µm

F = 3.75 mL/min

tG = 1.9 min

Speed 6.2x

No loss in resolution

10 µm particles 5 µm particles 3 µm particles 2 µm particles

0 10 5 Linear Velocity u [mm/s]

0

100

H [µ

m]

uopt, 5 µm uopt, 2 µm

uopt, 3 µm

2062

2 udu

dH pp

⋅++⋅=

90 bar

270 bar

575 bar

L/dp = 50

L/dp = 50

L/dp = 50

250 x 4.6 mm, 5 µm

F = 1.5 mL/min

tG = 12 min

L/dp must be kept constant

16 Proprietary & Confidential

4.6 x 250 mm Column, 5 µm 1.5 mL/min

0 10 15 22 0

100

Minutes

%B

Original First Gradient Step 0–10 min tG = 10 min F = 1.5 mL/min VC = 4.15 mL

Col. Volume (VC) = 4.15 mL

2.1 x 150 mm Column, 3 µm 0.52 mL/min (Increased u)

Col. Volume (VC) = 0.52 mL

New First Gradient Step 0–? min

= 3.6 min

0

100

%B

Minutes 0 3.6 5.4 7.9

15.452.0

52.05.1102 ⋅⋅=Gt1

2

2

112

C

CGG V

VFFtt ⋅⋅=

How do you deal with these complex considerations?

How to Adapt the Gradient Program?

17 Proprietary & Confidential

Gradient table recalculation performed automatically

Dionex RSLC Method Speed-Up Calculator

18 Proprietary & Confidential

Harnessing Efficiency Improvement?

19 Proprietary & Confidential

21 Proprietary & Confidential

22 Proprietary & Confidential

Potency RS Method Before and After

23 Proprietary & Confidential

Harnessing Selectivity Improvement

24 Proprietary & Confidential

Acclaim Trinity Nanopolymer Silica Hybrid (NSH) technology

silica bead

nano-polymer particles

anion-exchange/reversed-phase

cation-exchange

26 Proprietary & Confidential

Column: Acclaim Trinity P1, 3 µm Dimensions: 3.0 x 50 mm Mobile Phase: A - CH3CN, B - D.I. H2O, C – 0.2 M NH4OAc, pH4 Gradient: Time (min) -10 0 2 7 15 A 60 60 60 10 10 B 35 35 35 0 0 C 5 5 5 90 90 Temperature: 30 °C Flow Rate: 0.5 mL/min Inj. Volume: 5 µL Detection: ELS detector (Charged Aersosol as Preferred Alternative) Peaks: (80 to 150 ppm)

1. Procaine 2. Choline 3. Tromethamine 4. Sodium 5. Potassium 6. Meglumine 7. Mesylate 8. Maleate

9. Chloride 10. Bromide 11. Iodide 12. Phosphate 13. Malate 14. Tartrate 15. Citrate 16. Oxalate 0 6 12 9 3 15

Minutes

1

2

3

4

5 6

7 8

9 10

11 12 13 14 15

16

mV

0

1100

Gradient Separation of Pharmaceutical Counterions

27 Proprietary & Confidential

t0

0.5 mL/min

1.5 mL/min Column: Acclaim Trinity P1, 3 µm Dimensions: 3.0 x 50 mm Mobile Phase: 75/25 v/v CH3CN/30 mM (total) NH4OAc, pH5 Temperature: 30 °C Flow Rate: 0.5 and 1.5 mL/min Inj. Volume: 2.5 µL Detection: CAD Sample: Na, Naproxen (0.5 mg/mL in mobile phase) Peaks: 1. Na+

2. Naproxen

2

1

1

2

ONa

Minutes 0 2 4 3 1

mV

500

Hydrophobic Acidic API and Counterion – Na, Naproxen

t0

30 Proprietary & Confidential

3

4

Minutes

t0

0 2 4 3 1

mA

U

0 1

2

800

3

4

1 2

interference

pH 4.1

pH 5.2

Acclaim® Trinity Application: Advil ALLERGY and SINUS (pH (pH Effect)

Column: Acclaim Trinity P1, 3 µm Dimensions: 3.0 x 50 mm Mobile A: CH3CN Phase: B: D.I. H2O C: 0.2 M NH4OAc, pH 5.2 Gradient: Time (min) –4 0 0.1 1 4 A: 25 25 25 80 80 B: 65 65 65 0 0 C: 10 10 10 20 20 Temperature: 30 °C Flow Rate: 1.0 mL/min Inj. Volume: 2 µL Detection: UV, 254 nm

Sample 1. Ground one tablet (~0.49 g) Prep: into fine powder 2. Weigh 20 mg of above powder in a 20-mL sample vial 3. Add 10 mL of MeCN/H2O (v/v, 1/1) solution 4. Sonicate the vial containing sample suspension at 40 °C for 30 min 5. Filter the extract with 0.2-µm membrane filter Peaks: 1. Pseudo-ephedrine

2. Chlorpheniramine 3. Maleate 4. Ibuprofen * Background subtraction applied

34 Proprietary & Confidential

Overview

1. Column Efficiency and Selectivity 2. Your Workflow and Our Automation Tools 3. Boosting Nebulizer Based Detectors

43 Proprietary & Confidential

(Binary) RSLC (2008)

• Best flow-pressure footprint • Brought Dionex up to UHPLC • Full conventional LC capability

43

Bringing UHPLC to its Full Capabilities

x2 Dual RSLC

● Boost system use time ● Boost throughput ● Integrate sample prep ● Comprehensive LC

Quaternary RSLC

● Gradient Flexibility ● Cost-Effectiveness

Unique Choice of Pump Modules

44 Proprietary & Confidential

We Introduced Two Awesome Pump Modules

LPG-3400RS – A True Quaternary UHPLC Pump

• Low-pressure gradient proportioning of

up to four solvents • Integrated 4 channel degasser • Provides full application flexibility • Supports ultrafast automated method

scouting

DGP-3600RS – Unique Dual-Gradient UHPLC Pump

• Two ternary UHPLC pumps in one • Supports x2 Dual LC techniques

• Automated application switching • Parallel RSLC (2 systems in one) • Tandem RSLC • Multidimensional LC (max. resolution in

1st dimension and max. speed in 2nd dimension)

44

45 Proprietary & Confidential

The Powerful Two-stage SpinFlow™ Design

• Concept as introduced with HPG-3x00RS Initial small volume radial mixing Variable sized longitudinal mixing

• Outstanding mixing-per-volume performance • Patent-pending

46 Proprietary & Confidential

The Powerful Two-Stage SpinFlow™ Design

46 SpinFlow™ Volumes: 35uL, 100uL, 200uL, 400uL, 800uL, 1550uL

47 Proprietary & Confidential

Viper™ Finger Tight Fitting System

• Finger tight, correct fit every time!

• Universal (fits to Rheodyne, Valco, Swagelok...)

• No dead volume as soon as tightly connected

• Ease of use, no sleeve for small capillaries

• 1200 bar compatible

Ease of Use and Ergonomic

PEEK Seal

Removable knurl

Adapter (no ferrule!)

Finger Tight Screw 1/32” capillary

48 Proprietary & Confidential

Viper™ Performance at 600bar/8700psi

ZERO Dead Volume

Standard finger tight fitting

VIPER

49 Proprietary & Confidential

Your Workflow and Our Automation Tools

• Eliminate Matrix

• 2 x UHPLC Systems in One

• Gradient Methods and Tandem Operation

• (Automated Method Scouting)

• Automated 2D-Offline Fractionation for Biotherapeutics

• System Startup and Shutdown

50 Proprietary & Confidential

Intelligent (UHP)LC Solution for On-line SPE-LC

• First dimension SPE (e.g., RAM) column separates analytes from matrix • After transfer to an analytical column, analytes are separated and detected • Unique Chromeleon on-line SPE-LC wizard helps develop methods Allows direct injection of complex samples, e.g., biological fluids, surface water Faster, more precise, cheaper and better automated than alternative techniques

56 Proprietary & Confidential

WPS-3000 FC Well-plate Sampler with Fractionation Capabilities

• Dual valve autosampler (bioinert fluidics)

• Optimized for automated workflows e.g.:

• (Off-line) 2D-LC • Protein purification combining different

modes of LC and Protein purification recovery studies.

• Sample fractionation and desalting prior to MS detection

• Sample derivatization, e.g. digestion,

neutralization, in-between LC separations

Key Technology for Advanced 2D Offline Workflows

57 Proprietary & Confidential

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 -500

0

1,000

2,000

3,000

3,500 mAU

min

1 - 0.414

2 - 3.320

RA3

- F1

(3-3

)

RA4

- F2

(4-4

)

Flow: 1.500 ml/min %B: 0.0 %

100

Automated Antibody Affinity//IEX: Antibody Purification and Isoform Analysis

Purified antibody

first dimension – protein A antibody affinity purification and collection

second dimension WCX ion-exchange analysis

0.0 20.0 40.0 60.0 80.0

0.0

10.0

20.0

30.0

mAU

min

Acidic Variants

Basic Variants

K

K K

58 Proprietary & Confidential

Overview

1. Column Efficiency and Selectivity 2. Your Workflow and Our Automation Tools 3. Boosting Nebulizer Based Detectors

59 Proprietary & Confidential

Boosting Nebulizer Detectors (MSx, CAD, ELSD)

• Stabilize Mobile Phase Viscosity

• Stabilize Nebulizer and Droplet Efficiency

• Stabilize Ionization and Detector Response

• Eliminate Baseline Drifts and Shifts

60 Proprietary & Confidential

Corona CAD – Inside The Engine

HPLC eluent

pneumatic nebulization

drying tube

liquid waste

dried particles Corona needle

mixing chamber

ion trap

collector / electrometer

data Station

61 Proprietary & Confidential

Universal Detector for Non-volatiles!

62 Proprietary & Confidential

1. Second liquid branch with given tubing volume and delayed gradient profile (to compensate for column)

2. Second liquid branch includes identical column

62

Implementation of Inverse Compensation Gradient

Pump 1

Pump 2

Injector Column

Column Thermostat

Detector

Pump 1

Pump 2

Injector Column 1

Column Thermostat

Detector

Column 2

63 Proprietary & Confidential

Improved Quantification with Inverse Gradient

Peaks 1. Citric Acid 2. Theophylline 3. Phenylalanine 4. Propranolol 5. Naproxen 6. Diclofenac 7. Progesterone

- 5.0

10.0

25.0 pA

1

- 1

- 2

- 3 - 4

- 5 - 6 - 7 - 8

0.0 1.0 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 15.0 16.0

- 1

- 2

- 3 - 4

- 5 - 6 - 7 - 8

0.0 1.0 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 15.0 16.0 17.0 18.0 19.0 20.0 - 5.0

10.0

20.0

35.0 pA

min

2

- 1

- 2

- 3

- 4

- 5 - 6

- 7 - 8

Inverse Gradient

Gradient only

64 Proprietary & Confidential

Broadening Your UHPLC Approach

Smart Method Development Using Column Efficiency And Column Selectivity

Use a Straight Forward and Easy to Eastablish Strategy to Migrate

LC Methods to Faster UHPLC Separations Utilize a Wide Detection Offering to Separate Chromophores and

Non-Chromophores Clever Hardware and Software Allows Us to Speed Up Our

Processes Outside the (UHP)LC (Workflow Integration)

65 Proprietary & Confidential

Good Luck and Have Fun!

Dionex is focused on making UHPLC more productive for

All Chromatographers and All LC Challenges