molecular spectroscopy in compliant environments: qc-qa to ... · handheld analyzers cary 630 ftir...

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Molecular Spectroscopy in Compliant Environments:

QC-QA to troubleshooting

Jean-Louis Cabral, Ph.D. MBA

Product Specialist/Application Scientist – Molecular Spectroscopy


S1 | Agilent Restricted |

Molecular Spectroscopy in Compliant

Environment

Examples of “Compliant” Environments

1. Pharma/Biopharma • Most stringent level of control

2. Food/Cosmetics/Nutraceutics • Changing regulations – traceability (incoming and released products)

3. Forensics/Legal • Trace of evidence

4. High Tech Industries • IP – high level of control on data



Analytical Challenges in Compliant

Environment

1. Performance validation and verification • System performance: IQ-OQ-PQ-PV

• Method development and validation

2. 21CFR11 - Data compliance • User access and control

• E-signature and data approval

• Data traceability and integrity – same applies when outsourcing (audits)

3. Requirement for high throughput • Fast response time and more samples being tested

– Incoming raw material ID and release of finished products

4. Always looking further • Smaller samples (e.g. defects, contaminations)

• Requirement for higher sensitivity



Analytical Challenges in Compliant

Environment

5. HR related challenges • Multitasking and complexity:

– More analytical techniques – less and less dedicated specialists

– More administrative tasks

• Rotation of personnel

– Training

– Requirement for simple integrated software platforms



Agilent Molecular Spectroscopy in Compliant

Environment - FTIR

Hand Held

Portable

Analyzers

Routine Research




Environment - FTIR

Handheld Analyzers Cary 630 FTIR Microscope

Raw Material ID – Ingredients & Materials (ex: packaging)

ID - Multilaminate

packaging

Quantitation – ingredients and finished good

ID – Finished product

ID of defects and

contaminations

(down to 1.1um)

Example of applications




Environment – UV-Vis/NIR

Model Spectral range

(nm) Design Samples

Cary 8454 190-1100 Photodiode array Liquids

Cary 60 190-1100 Dual beam, single mono, Si

detector

Liquids/Solids

Cary 100 190-900 Double beam, single mono,

PMT

Cary 300 190-900 Double beam, pre-mono, PMT

Cary 4000 175-900 Double beam, double mono,

PMT

Cary 5000 175-3300 Double beam, double mono,

PMT and PbS

Cary 6000i 175-1800 Double beam, double mono,

PMT and InGaAs





Example of applications

8454 Cary 60 Cary 100/300

ID - Raw Material and finished products

Quantitation

Fast Kinetics

Dissolution

Microvolume (down to 0.5uL)

Fiber optics (ex: process)

Thermal denaturation

(biomelting)

USP 671 (containers)



Most reliable, flexible and ruggest and FTIR…

630 Engine DialPath TumblIR

Di, Ge, ZnSe ATR Diff/Spec Reflectance Transmission

Introduction to Cary 630 FTIR



Anywhere at anytime…no need to recalibrate


Entrance to evacuation

chamber

• Toxic API (ex: anticancer drugs)

• Air sensitive compounds

• Explosive/Hazard risks



Most intuitive software…No need for training


Software guides the user

through the selected

method. Also recognizes

the correct sampling

interface to guide proper

sampling techniques and

cleaning.



Most intuitive software with visual results


Library results are shown with

the hit list and can be overlaid

or stacked

Quant results can be programmed

for critical action levels with color

coded queues (red, green, yellow)



Dialpath – No need for liquid cells


Pathlength (μm) Typical Conc. Range

30 Neat - 0.1%

50 50% - 500 ppm

100 20% - 100 ppm

200 10 % - 50 ppm

1000 > 10 ppm

TRADITIONAL APPROACH

250mL Fill Volume

3mL Fill Volume



Dialpath – Raw Material ID


Spectra collected with 30um DialPath setting

glycerol

propylene glycol

dipropylene glycol

triacetin

Spectra, are then searched against reference spectra in a library to produce matches

scaled from 0 to 100 (with 100 being a perfect match)



Dialpath – Determination of impurity


Spectra collected with 75um DialPath setting

ethylene glycol in glycerol diethylene glycol in glycerol

R2=0.9895 R2=0.9745

Limit of detection – 0.04% Limit of detection – 0.02%

This fits well with FDA limits of <0.1% impurities in glycerol



Application - Simethicone

Cary 630 with Dialpath

Pathlength: 500 µm

Spectral Range: 1330 to 1180 cm-1

No of scan: 64

Resolution: 4 cm-1

Software used: Microlab

Blank: Use toluene as blank.




Sample Preparation

A certain sample of suspension equivalent to 50 mg of SMT was accurately weighed

and transferred to a capped conical flask. Then, 50 mL of HCl 6 N was added to the

conical flask and swirled until SMT dissolved completely. Next, 50 ml of toluene was

added for the extraction and the conical flask was mechanically shaken at a frequency

3000 rpm for 10 minutes (suspension). After allowing the layers to separate, toluene

layer was pipetted sufficiently and transferred to screw-capped tube. Any residual

water in the organic layer was then removed using anhydrous sodium sulfate.

Standards were prepared by the same extraction procedure using the known

quantities of SMT. Toluene layer was then analyzed by FTIR spectrophotometer and

the height of the band (absorbance) at ~1260, was used to determine SMT content in

sample preparations.




Method validation – Specificity

To check specificity Simeticone standard and blank analyzed in FTIR and found no

peak present in blank around ~1260.68 cm-1.

Fig-1: Comparison between 50 mg/mL standard vs blank solution.




Method validation – Linearity

Performed linearity with concentrations range of 0.5, 1, 2.5, 5 and 50 mg/mL.





8454 Key Features…

• Photodiode array detector for complete spectrum capture

• Fast full spectrum kinetics

• Multi-component analysis

• Small footprint – open sample compartment

Cary 60 Key Features…

• Fiber optics for improved workflow

• Analysis of small volumes

• Xenon flash lamp for low ongoing costs of ownership

• Photometric range (4.0 Abs with cuvette)

for turbid solutions



Cary 60 with fiber optics - flexibility

Fiber Optics

QA/QC on raw materials and finished

product in manufacturing

Chemical identification or study of

chemical processes

Wavelength Scans – What is it?

Kinetics – what processes are occurring?

Wavelength Scans – What is it?

Concentration – How much?



Cary 60 with fiber optics - flexibility

Replaceable Tips

Various selection of pathlengths

Cleanable and reusable.

Microprobes

Diameter: < 4 mm

10mm (2-5mm option) pathlength



Cary 60 with microprobe – small volume at

low temperature

Fiber Optics for improved workflow

- Take the instrument to the sample and improve workflow

- Measure samples in situ: hot, cold, radioactive, noxious

- Full UV and Vis spectrum – no expensive quartz cuvettes needed

- No compromise in data quality:

Ab

s

DNA concentration (ng/µl)

y = 0.0198x R² = 0.9996

0.0

0.5

1.0

1.5

2.0

0 20 40 60 80 100



Cary 60 for nano/microvolumes

Accurate spectro measurements

Small volume measurements - Traycell

- Save precious sample – measure 0.5 to 10 µL

- Accurate and reproducible results

- Excellent linear dynamic range

- Flexibility – no additional dedicated system

required

mg/mL

1

Abs

Abs



Cary 60 for surface - color

• Remote (<1.5m from instrument) diffuse

reflection measurements!

• Fiber optic accessory with movable detector

• 1.5 mm light patch – inbuilt CCD allows

visualization of position

• Needs fiber optic coupler (G6865A – for Cary

60)

• Understand the sample surface porosity ie

small beam

Accurate assessment of packaging and finished

products



Cary 60 for surface - color

Accurate assessment of packaging and finished

products



What about (very small) unknowns?

http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCJSP_J61gskCFchoPgodKfML6w&url=http://www.crainsdetroit.com/article/20150404/NEWS/304059992/great-lakes-researchers-target-microplastics-pollution&bvm=bv.106923889,d.cGc&psig=AFQjCNEtwEltIrAhKdOQj7xSfiB3oWQt4Q&ust=1447126661648639



What do you do when you need microscopic info?

An FTIR imaging microscope has essentially two main purposes:

1. To allow users to visually see small (micron) sized samples

2. Collect accurate FTIR spectra/images from small samples (spatial resolution of a few microns, with

FOVs of up to cm2)

FTIR imaging can be performed in essentially three modes:

1. Single point mapping

2. Linear array mapping

3. 2-D Focal Plane Array (FPA) imaging

FTIR Imaging gives spatial (WHERE) and spectral (WHAT) information, and FTIR Chemical

Imaging with a IR Focal Plane Array (FPA), gives this simultaneously



FTIR Microscopy/imaging measurement modes:

1: Single Point Mapping Automated acquisition of spectra (one

by one) defined by a grid. A hundred

points can take several hours.

2: Linear array Mapping Acquisition of spectra by a row (1x16)

of detectors. Faster than single point

mapping, but still much slower than

FPA imaging



3: FPA Imaging

With an FPA detector (128x128), up

to 16384 spectra can be recorded

simultaneously in a single

measurement

700 micron (15x)

2.6 mm (4x)

Each pixel = 3.3um/0.66um (25x obj.)

5.5um/1.1um (15x obj.)

19um (4x obj.)




200 µm 200 µm

25x Objective in “high mag” mode in Transmission

Mixed Polymer Beads

3 micron polystyrene beads on CaF2 substrate are clearly

chemically distinguished.

Even at relatively long wavelengths (low

wavenumbers), 3 micron beads are clearly resolved

Pixel size: 0.66 micron

Obj mag: 25x, 0.81 NA

High mag: ON

FOV: 85x85 um

Total system mag: 61x

Working Distance: 12 mm

85mm

85 m

m

40x vis image Polystyrene, 3024cm-1 (3.3 um)

PMMA, 2950 cm-1 (3.4 um) Polystyrene, 1430cm-1 (7.0 um)

PMMA, 1492cm-1 (6.7 um)



Packaging – 25x Transmission High mag Chemical

Images

- Even in transmission mode, the ultra high NA and

very small pixel size has allowed for the

resolution of the 3 micron PA layer, hence

rivalling the spatial resolution of Ge micro ATR

- Most books and papers still talk of ~10um spatial

resolution for transmission imaging!

- The 25x, 0.81NA is a revolution in objective

design

40x obj. vis image (polarized)

EVOH, layer 3

PA, layer 2

PE, layer 1

3500 3000 2500 2000 1500 1000

Wavenumber

Abso

rbance

Pixel size: 0.66 micron

Obj mag: 25x, 0.81NA

High mag: ON

Total system mag: 61x

1

2 3

140 u

m

180 um



Questions? Comments?

For more information on Agilent’s Products or Applications

Visit Web Page at

www.chem.agilent.com

or contact your local Agilent rep

http://www.chem.agilent.com/




Pixel Size (obj mag, NA, mode) Achieved

Spatial

Resolution

3750 cm-1

Achieved

Spatial

Resolution

2500 cm-1

Single FPA tile

FOV (with

128x128FPA)

3.3 um (25x, 0.81NA, std mag) 4.3 um 5.0 um 420x420 um

0.66 um (25x, 0.81NA, high mag) 1.4 um 1.7 um 85x85 um

5.5 um (15x, 0.62NA, std mag) 6.9 um 7.6 um 700x700 um

1.1 um (15x, 0.62NA, high mag) 2.4 um 3.0 um 140x140 um

19 um (4x IR, 0.2NA, std mag) 20.4 um 20.0 um 2400x2400 um

Entire 2”x2” (50x50mm) USAF target imaged at

19 um pixel resolution with 4xIR objective in 90

minutes

(21x21 tile mosaic with128FPA)

USAF target (700x700um) imaged at 5.5 um pixel

resolution (normal mag. mode) with 15x objective in

2 minutes

Single 128FPA tile

1.4um

0.98um

25x obj, 0.81 NA

molecular spectroscopy in compliant environments: qc-qa to ... · handheld analyzers cary 630 ftir...

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