Enabling Generation III by Interfacing Measurement Science to the Generation 1 Platform

Brian Marquardt Ph.D. Charles Branham, Wes Thompson, Thomas Dearing, Michael Roberto, Lauren Hughs CPAC Applied Physics Laboratory University of Washington

Perfect Process Analyzer? Pressure, Temperature and Flow

Process IR

O2 Sensor

Process Raman

Mass Flow Control

Where Does NeSSI Fit in the Lab Instrument/Sensor Interfaces

Design standards make development simpler Reduced toolset to be mastered Reduced sample variability to account for

Calibration/validation built-in Consistent physical environment for measurement Stream switching and/or mixing allow generation of

standards to match analytical requirements

Reaction monitoring Microreactors and continuous flow reactors Batch reactors (with fast loop)

Sample Preparation Gas handling (mixing, generation, delivery) Liquid handling (mixing, dilution, conditioning, etc.)

NeSSI with an Array of Micro-Analytical Techniques will Impact Many Industries

• Process Control • Process Optimization • Product Development

Thermo/C2V Fast Micro-GC

http://www.c2v.nl/ as well as http://www.thermo.com

NeSSI™ Compatible GC/LC Diaphragm Valve

Inject valves

Applied Analytics Inc. Diode Array OMA-300 A Fiber-optics-diode-

array process analyzer For on-line concentration

monitoring

NeSSI Ballprobe - Raman/NIR/UV

Matrix Solutions: www.ballprobe.com

Ballprobe Specs. •Hastalloy c-276

Ti, SS, Monel •Sapphire optic •Std. temp range:

-40 – 350° C •Pressure:

0-350 Barr

Kaiser Airhead Raman Gas Probe

AirHeadTM Probe with compression mount

NeSSI interface

AirHeadTM Probe with

NPT- Threaded mount

NeSSI interface

NeSSI™ IR Gas Cell

micrOptix

NeSSI UV-Vis/NIR Sensor

Cactus, Inline Fluid Healthcare Monitor

Cactus is used to monitor the health of motor oils and hydraulic fluids. This transmitter combines physical/chemical, thermal & electrical measurements with high level computation algorithms to provide users with a single unit, named HEAL.

Output range & unit 0 to 100 HEAL

Measurement time 1 sec

Fluid operating range

Density 600 Kg.m-3 to 1100 Kg.m-3

Pressure Atm to 100 bar

Temperature -25 °C to 110 °C

Viscosity 0.3 cP to 200 cP

Water saturation Up to 100%

Performances Repeatability* 1 %

Accuracy* 3 %

H2Scan Adaptation to NeSSITM Platform • Hydrogen specific: 0.5% H2 to 100% H2 v/v

• Response time (T90) < 30 sec

• In-line, real-time measurements in process gas streams up to 100OC

• Unique models for CO, H2S, wet CL2

• 4-20mA, RS422 or RS232 serial connectivity

• Stable results

• On-site verification and calibration

• Approved for hazardous locations – Intrinsically safe design – ATEX certificate granted; UL pending

• Cost effective to buy / install / maintain

• Field verification and calibration kit available

http://www.h2scan.com/

Astute Sampling System

GAS TREATMENT

COLUMN

Sampling + Sensors & micro-analyzers

Astute System with C2V Micro GC and H2Scan hydrogen analyzer Process

www.eif-filters.com

Agilent NeSSI Dielectric Sensor Cable to Agilent

Network Analyzer

Swagelok 2-Port

Valve Base

Dielectric Probe

Inner Body

O-ring (inside)

Outer Body

Close up of Coaxial

Probe Tip

Teledyne: NeSSI Oxygen Sensor Fuel Cell or Zirconium Oxide Sensors Offer an SP76 compliant oxygen

sensor Available concentration ranges

0-10 ppm, 0-1000 ppm, 0-3%, 0-25%, 0-100% oxygen

Response time T90% < 45 seconds, Fuel Cell T90% < 10 seconds, Zirconium Oxide

Operating Temperature: 0-50˚C Signal output

4-20 mA and 0-10 VDC

http://www.teledyne-ai.com/

Mechatest Sampling Solutions

NeSSI Liquid Sampler Compliant with ANSI/ISA 76.00.02 38.2 mm

(1.5 in.) footprint. Three Port configuration: Sample Inlet,

Bypass and Vent. Quick and safe operation, low dead volume

design with bypass. Manual or Automatic sampling In-line Sampling Closed Loop and Emission Free Sampling Easy in operation

http://www.mechatest.nl/

NeSSI based research and developments at APL

Initial NeSSI Gas Blending System

NeSSI Gas/Vapor System

Aspectrics EPIR w/ glass cell

N2 Waste

NeSSI Flow Cell

ASI microFAST GC

Analog NeSSI Gas Mixture System

Features of Circor System obtained for UM: 1. 4 gasses line, able to produce and maintain gas mixtures 2. Fully automated system, set and forget capability

Automated NeSSI System

4 x Gasses

Mixed Gases

Mass Flow Controllers

Calibrated Analog Gas Mixer

- 3 stream calibrated mixing with fiber optic oxygen/temperature/humidity sensing

Digital Calib. Gas Generation System Features of NeSSI System : • Fully digital

• RS 485 • 4 Stage dilution,

able to produce and maintain gas concentrations in 1-5 ppm range

• Fully automated system, set and forget capability

• Integrated C2V NeSSI compat. GC

• Calib, platform for gas sensor dev.

Digital Gas Calibration System

Digital Serial Gas Diluter

Gas Calib. System LabView Control Program • Perform automated DoE calibration runs • Input and log sensor, reference, temperature and pressure feeds • System designed for full digital (plug and play) performance as h/w matures

Modular NeSSI Oxygen Gas Sensor

Exploded View

1/16 in bifurcated fiber optic fiber

1/16 to 1/8 in Swagelok union

Agilent NeSSI Mount

Sensor Body

Close up of Outer Body Tip

Vapochromic Tip

Fiber optic ferrule with plastic housing

Sensor response to O2 Gas

Pre

dict

ed O

2 %

Calculated O2 %

R2 = 0.990, 3 PC RMSEC = 1.0744

20 replicates at each concentration Concentration range: 0 -100% Oxygen 16 ms reversible response full range

Inte

nsity

(cou

nts)

Wavelength (nm)

100 %

0%

120

100

80

60

40

20

0 100 90 80 70 60 50 40 30 20 10 0

0 10 20 30 40 50 600

10

20

30

40

50

60

Measured Oxygen Concentration(micromol/liter)

Pred

icte

d O

xyge

n C

once

ntra

tion(

mic

rom

ol/li

ter)

R2 = 0.9982 Latent VariablesRMSEC = 0.90163Bias = -7.1054e-015

Sensor Response for O2 in Solution

5 replicates at each concentration Concentration range: 1 μmol/L - 55 μmol/L 3.2 sec reversible response – full range

1 μmol/L = 32.5 ppb

500 550 600 650 700 7500

0.2

0.4

0.6

0.8

1

Wavelength(nm)

Rea

lativ

e In

tens

ity

55 μmol/L

1 μmol/L

Vapochromic Oxygen Sensor

Raman Filtration/Sampling Sys

Initial NeSSI Permeation System

NeSSI Gas/Vapor System

Permeation Tower

Aspectrics EPIR w/ glass cell

N2 Waste

NeSSI Flow Cell

ASI microFAST GC

Permeation Apparatus – ver. 2

- CPAC inspired and Parker built prototype permeation apparatus - Currently, used to generate calibrated organic vapor samples for sensor testing - Vapor calibration NeSSI system with Kaiser Airhead gas probe for analysis

Advanced NeSSI Perm. System Ver. 3

MFC 1

MFC

2

MFC 3

Perm

eatio

n Ap

para

tus

Vapor

Balance Gas

Sample to IR

Heater Set Temp. 65˚C Permeation App. Temp. 48˚C

Permeation System – Ver 4

Parker NeSSI Permeation Apparatus

Aspectric EP-IR

4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000-2

-1

0

1

2

3

4

5x 10-3

Wavenumber (cm-1)

Abs

orba

nce

(Arb

itrar

y U

nits

)

Temperature Permeation Results: IR Spectra

H2O

Ethanol CO2 As Temp Decreases the Signal Decreases 85˚C

65˚C

NeSSI Continuous Flow NMR System

Pump

Sampling System

Reactor

NMR

• Challenges with steel – polymer NeSSI system being manufactured to perform this measurement

PEEK NMR NeSSI Substrate

Goal: to improve reaction monitoring and optimization through the use of continuous glass flow reactors, NeSSI and analytics

Funded by the FDA to demonstrate the benefits of improved reactor design, effective sampling and online analytics to increase process understanding (QbD)

Demonstrating Quality by Design

CF Reactor and Raman Analyzer

4 channel, 785 nm Kaiser Optical Systems Rxn2 probes placed at different reactor zones

Raman Analysis of CF Reactor

3 4

1

2

• Monitor reaction with 4 channel 785 nm Raman system • NeSSI sampling systems (1-4) equipped with Raman ballprobes • Online GC also used as post quench online analyzer (4)

NeSSI™ Sampling System for Reactor

Raman Probe

monitor

bypass

clean

NeSSI™ and Raman Probe Images

Continuous Chemical Synthesis

- NeSSI flow management system (P, T, flow sensors, backpressure, filter) - NeSSI analytic sampling system (mulit probe Raman, IR, NIR, RI, Density, …)

Analytical Control System Design

Feedback & Control

Process Modeling

Data Fusion

Analytics and Data Handling

Hardware Control and Monitoring (Software)

Hardware – NeSSI, Raman, Other PAT, Reactor

Optimization

44

Interface with continuous flow reactor Mechanisms to ensure safe reactor

conditions Combined waste outlets from safety mechanisms

On-line analytics Temperature, flow, pressure

Product / Waste stream mechanism downstream of analytics

Open ports to allow for new analytics

Continuous Flow System Targets

HPLC Pump (Flow Rate)

Back Pressure Regulator Flow Meter Pressure

Gauge

•Reactant 1

Thermocouple Raman Other PAT

Flow Meter Pressure Gauge Thermocouple

•Product

Raman Infrared Needle Valve

Temperature Control

Control Analytics

Target System

HPLC Pump (Flow Rate)

Back Pressure Regulator Flow Meter Pressure

Gauge

•Reactant 2

Thermocouple Raman Other PAT

46

Preliminary Schematic

Reagent Sample Lines

Reactor Product Line

Current Reactor Iterative design

process, with multiple redesigns New analytics brought

to bear on system New safety and valve

mechanisms NeSSI allows for this

iterative process to proceed rapidly at practical costs

Esterification of benzoic acid

Straight-forward, pharmaceutically relevant

Strong dependence on temperature, flow rates

Chemical Reaction

Wiles, C., Watts, P. 2009

Temperature vs. Reaction Progress

PLS Raman Calibration Model

0 20 40 60 80 1000

10

20

30

40

50

60

70

80

90

100

Yield Measured by HPLC (%)

Yiel

d Pr

edic

ted

by R

aman

(%)

PLS Model - Raman Prediction vs. HPLCHigh Concentration Factorial Calibration

R^2 = 0.9992 Latent VariablesRMSEC = 0.91141Bias = 0

NeSSI for Fermentation Monitoring

Real-time Analysis of a Simulated Batch Fermentation Process

Simulated Fermentation liquor 900 mL of synthetic sugar water was

fermented with yeast for 8 hours The solution of sugar was held at 30˚C

and pH 6 for the duration of experiment. Glucose additions were added when

ethanol peak equilibrated. The final total sugar concentration in the reactor was 25 g/L glucose

NeSSI Fast-Loop Kaiser Optical Systems Raman

½” ballprobe with sapphire optic, 250mW power at probe tip

Average of six, 5 second exposures Mettler React-IR

MCT Detector Silver Halide Immersion Probe Diamond ATR

Fiber Optic O2 Sensor Cassini O2 Sensor

NeSSI Ferm. Fast-loop Design

• Custom designed NeSSI interfaces for all process analyzers in my lab • Plug and play analytics for any flowing system (liquid, slurry or gas)

Ethanol HPLC and Raman PC1

0 50 100 150 200 250 300 350 400 0

2

4

6

8

10

12

Time (Min)

Etha

nol (

mg/

mL)

-8

-6

-4

-2

0

2

4

6

8

Scor

es P

C1

HPLC Concentration Scores of Raman Data

After validating the loadings with the standard spectra, we can compare the scores plots (change in Raman spectra with time) with the reference method (change in concentration with time)

Glucose HPLC and Raman PC2

0 50 100 200 250 300 350 400 0

1

2

3

4

5

6

Glu

cose

(mg/

mL)

150 -3

-2

-1

0

1

2

3

4

Time (Min)

Scor

es P

C2

HPLC Concentration Scores of Raman Data

Sterilization of NeSSI Fast Loop? Concerns for sterilization

The flow path for a single NeSSI block has potential eddy points that can hold organisms

O-rings, seams and seals can allow holdup of biological material

Potential sterilization methods 10% bleach solution, initially

flowed through system, then held static to allow diffusion

Steam sterilization, heats entire block to 120°C

Autoclave

Substrate

Top Mount Component

Bleach Sterilization Method Rinsed 10% Bleach Solution for 1 hour Sterile Water Rinse

Filled Sterile Growth Media

Incubated 37°C for 72 hours

Swabbed

Culture plates

1. End piece (Start Flow) 2. Valve substrate 3. Top mount 3 4. Substrate 3 5. Top mount 4

6. Substrate 4 7. Top mount 5 8. Substrate 5 9. Valve substrate 10.End piece (End Flow)

1 2

3 5 7

9 4 6 8 10

contaminated

Conclusions and Challenges Demonstrated sterilization of NeSSI

components Possibly contaminated by residual cells

trapped in valve seat Pre autoclaving worked to remove

majority of possible contamination points O-rings and tubing

Next step – Sterile pumps and tubing Food grade diaphragm pump Stainless steel tubing Diaphragm valves to replace ball valves

More Dev. in Steam sterilization 120°C for 15 minutes (15 psi)

More work to be done!

Steam Sterilization Experiments

Steam Sterilization Method Sterilized Sterile Water Rinse ~ 500mL 120 °C for 15 min

Filled Sterile growth media is pumped by head pressure of compressed gas

Incubated 37°C for 72 hours

Swabbed Substrates sampled onto bacterial streak plates

Culture Plates

1. End piece (Start Flow) 2. Valve substrate 1 3. Substrate 3 4. Top mount 3 5. Substrate 4 6. Substrate 5 7. Valve substrate 6 8. End piece (End Flow)

1

2 3 5 7 4 6

8

contaminated

• Contamination only found in end connector components

• This has been a continuing challenge with all sterilization routines

• Difficult to keep end connectors sterile due to fill cycles for media to test long term sterility

Steam Sterilization Autoclave procedure, 120 °C for 15 minutes Temperature and pressure measured at end of block to

ensure the entire system is controlled New Valves

Pin valves were replaced with Hastelloy diaphragm valves to reduce possibility of hold up in valve seat

Sterile pumping mechanism Peristaltic pump replaced with compressed air pump Air is passed through a 0.2 µm filter into the headspace of

a sealed 2 L jar containing sterile media, forcing the media through the NeSSI substrate

All tubing is stainless steel

Differences from bleach sterilization

Demonstrated sterilization of NeSSI components Possibly contaminated by residual cells trapped in threads of end

caps Steam sterilization is a valid method of sterilization for NeSSI

components O-rings and tubing are compatible with autoclave conditions Ensures a sterile flow path without exposing external parts to hot and

humid conditions Next step – Top mount components

Sterilization of top mount components will be dependent on the materials and flow path of the components themselves

Next step – Full integration to NeSSI System Integrate rinse stream and boiler system to NeSSI fast loop sampling

system Create digital control system for automatic sterilization

Conclusions and Challenges

LinkedIn NeSSI Group www.linkedin.com - search in groups tab for NeSSI and join!!!

Questions?????

Thank You!!