Brian J' Marquardt

1
FDA-PAT Spectroscopy TrainingRaman Spectroscopy

• Brian J. Marquardt
• Center for Process Analytical Chemistry
• University of Washington, Box 351700
• Seattle, WA 98195

2
Raman Scattering

• Two types of scatter of electromagnetic
  radiation occur
• Elastic (Rayleigh scatter, very intense)
• Inelastic (Raman scatter, weak phenomenon)

3
The Raman Scattering Effect

• Incident Radiation is absorbed by the molecule
  in the ground state and excites it to a virtual
  state
• The molecule is polarized by the incident
  radiation and emits radiation shifted in
  frequency from the incident
• The radiation can be either higher (Anti-stokes)
  or lower energy (Stokes) compared with the
  incident Stokes shifted Raman is the most
  common due to higher probability

4
Mechanisms of Infrared and Raman
- for a vibrational mode to be IR active, the
dipole moment of the molecule must change
- for a transition to be Raman active, there must
be a change in polarizability of the molecule
5

• Due to the Boltzman distribution we see stokes
  lines with a much greater intensity than
  anti-stokes (i.e. the ground state is more
  populated at room temperature)

Rayleigh
Raman
Ehv
Virtual States
Ehv
stokes
Anti-stokes
3 2 1 0
Ground Electronic States
6
Raman and IR Spectra
7
Raman Scattering Efficiency
Raman Intensity is proportional to the following

• Number of Raman scatter centers (Concentration of
  Analyte)
• Intensity of Laser (Power)
• Exposure time of the detector (integration of
  photons)
• Raman Cross Section (physical constant)
• Frequency of Laser (n4) (Excitation Wavelength)

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Advantages of Raman Spectroscopy

• Little or no sample preparation is required
• Water is a weak scatterer - no special
  accessories are needed for measuring
• aqueous solutions
• Water and CO2 vapors are very weak scatterers -
  purging is unnecessary
• Inexpensive glass sample holders, non-invasive
  probes and immersion probes
• are ideal in most cases
• Fiber optics (up to 100's of meters in length)
  can be used for remote analyses
• Since fundamental modes are measured, Raman
  bands can be easily related to
• chemical structure (very good for
  fingerprinting)
• Raman spectra are "cleaner" than mid-IR spectra
  - Raman bands are narrower, and
• overtone and combination bands are generally
  weak
• The standard spectral range reaches well below
  400 cm-1, making the technique
• ideal for both organic and inorganic species
• Raman spectroscopy can be used to measure bands
  of symmetric
• linkages which are weak in an infrared
  spectrum (e.g. -S-S-, -C-S-, -CC-)

9
Disadvantages of Raman Spectroscopy

• Inherently not sensitive (need 1 million
  incident
• photons to generate 1 Raman scattered photon)
• Fluorescence is a common background issue
• Typical detection limits in the parts per
  thousand
• range
• Fluorescence Probability versus Probability of
• Raman Scatter ( 1 in 103-105 vs 1 in 107-1010)
• Requires expensive lasers, detectors and filters

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Example of Raman Spectra of Alcohols
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High Performance Raman System
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Typical Filtered Fiber-Optic Raman Probes
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Raman Sampling Applications
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CPAC Ball Probe Optical Benefits

• no moving parts
• sapphire spherical lens
• constant focal length and sample volume
• focus is at tangent of sphere
• probe is ALWAYS aligned when in
• contact with sample
• effective sampling of liquids, slurries,
• powders, pastes and solids
• high sampling precision allows it to
• be used effectively to monitor dynamic
• mixing systems (powders/slurries)
• particle size has minimal effect on
• optical performance (lt 1mm 5mm)

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Raman Immersion Ball Probe Design
Probe barrel when used with collimated
laser length is not an issue.
Probe Tip weld sealed to barrel
Ball sapphire ball lens (focal point is on face
of lens)
Ball Fastener pressure seals ball into the
probe tip
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Ball Probe Analysis of PDMS Coated Silica
Particles
1200
1000
PDMS ( SD 15.1)
800
Intensity
Silica ( SD 2.4)
600
400
200
400
600
800
Raman Shift (cm-1)

• each spectrum was taken after sample was
  agitated
• Std Dev of silica and PDMS on non-agitated
  sample (5 reps)
• were 0.76 and 0.72 respectively

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Raman Analysis of Polymorphic Steroid Standards
80000
70 x103
60
50
60000
40
Intensity
30
20
40000
10
1580
1600
1620
1640
1660
1680
20000
0
1000
1100
1200
1300
1400
1500
1600
1700
Raman Shift (cm-1)
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Real-Time Reaction Monitoring of Polymorph
Formation _at_ 40º C

• two crystalline forms of a commercial active
  pharmaceutical
• data was collected during vigorous stirring in a
  slurried sample

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Polymorph Reaction Data Over 24 Hrs
Form 1
Intensity
Form 2
3.3
6.7
10
13.3
16.7
20
Time (hrs)
20
Dry Powder Mixing Analysis in a Fluidized Bed
with Raman Ball Probe
Transparent view of bed with probe inserted
Probe Seal and Air Bypass
Ball Probe
Fluidized Bed
2400
Sugar Citric Acid
2000
1600
Air flow
1200
200
400
600
800
1000
1200
1400
200
400
600
800
1000
1200
1400
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Dry Powder Mixing Analysis PLS Model

• The scores and factors plots show substantial
  mixing
• information as a function of fluidization time

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Online Analysis of a Shear Blender
Pharma Solid _at_ 1199.688
35
25
2 Spectra taken _at_ 5 min. after start
Arbitrary Y
active pharama.
15
lactose
5
80
0
2
4
6
8
Time (min)
Arbitrary Y
40
Lactose _at_ 1092.006
0
70
Arbitrary Y
1100
1200
1300
50
Raman Shift (cm-1)
30
0
2
4
6
8
Time (min)

• each spectrum is the result of 10 sec.
  acquisition

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Raman Analysis of Pharmaceutical Blending
Mixture Pharmaceutical(10) lactose(90)
500
Mixture
400
300
Intensity (arbitrary)
lactose(90)
200
100
Pharmaceutical(10)
0
400
600
800
1000
1200
1400
1600
Raman Shift (cm-1)
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Further Applications of RamanBall Probe for
Mixing Analysis
Proposed use of a ball probe(s) in a V-blender
fiber to probe(s)
optical interface

• use of fibers has the potential of
• coupling multiple probes to one blender

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Monitoring an Industrial Slurry Stream with
Percent Level Contaminants

• Raman ball probe inserted directly
• into flowing stream
• Analysis performed 20 meters from
• instrument
• Analysis performed under moderate
• process conditions (temp. and flow)

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PLS Model of Constituent A

• Range 2.82 3.65 A remainder of which is
  water
• Model was PLS, 3 factors, SNVT pretreatment,
  300-1500 cm-1

Slope 0.972 /- 0.188 Bias 0.00022 /-
0.029 Intercept 0.093 SEP 0.097 R2 0.844
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Analysis of a Parallel Batch Chemical Reaction
Using Raman Spectroscopy
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Esterification Reaction Monitored by Raman
Spectroscopy
H
Acetic Acid Methanol Methyl
Acetate H2O
Heat
ROI
Acetic Acid Methanol Methyl Acetate
20000
15000
Intensity
10000
5000
0
400
600
800
1000
1200
1400
1600
1800
Raman Shift (cm-1)
15 second exposure, 3 accumulations, 785 nm,
Inphotonics probe
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Acetic Acid to Methyl Acetate Conversion over 1.5
hours _at_ 25 C
Region of Interest 3D
Region of Interest 2D
5000
Acetic Acid
4000
3000
Intensity (counts)
Intensity (counts)
Acetate
2000
1000
0
Spectrum Number
800
820
840
860
880
900
920
940
Raman Shift (cm-1)
Raman Shift (cm-1)
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Reaction Profiles for the Formation of Methyl
Acetate at Different Temperatures
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MW Determination of a Polymer

• Molecular Weight
• 550
• 1000
• 2300

Intensity (counts)
Raman Shift (cm-1)

• 5 measurements per sample (measured with
  ballprobe)
• 3 sec acquisition, 3 accumulations (avg), 785
  nm, 80 mW

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MW Calibration Data for a Polymer
MW Range 550-2300
Molecular weight (pred)
Molecular weight (actual)

• 5 measurements per sample (measured with
  ballprobe)
• 3 sec acquisition, 3 accumulations (avg), 785
  nm, 80 mW

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Analysis of a Continuous Chemical Reaction Using
Raman Spectroscopy
Raman Ball Probe on Outlet of CPC System
Real-time Reaction Monitoring
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Monitoring a Continuous Reaction Using CPC Reactor
Triethyl Amine 80º C, Dioxane
Acetic Anhydride Propyl Amine
Acetic Acid Propyl Amid
4
x 10
15
Raman Intensity
10
5
0
0
500
1000
1500
2000
2500
3000
Raman Shift (cm-1)

• 18 minute reaction time, 15 sec. per spectrum (5
  sec. Exposure, 3 accumulations)

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Raman C-H Stretch Spectral Region of Interest for
the Rxn.
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Reaction Profile Over 18 Minute Continuous
Reaction
Factor 1 Scores Plot
x 105
1.5
1
0.5
Score Values
0
-0.5
-1
0
5
10
15
20
25
30
35
40
45
50
Sample Number

• 18 minute reaction time, 15 sec. per spectrum (5
  sec. Exposure, 3 accumulations)

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Factor 1 Loading Plot for C-H Stretching Spectral
Region

• plot describes spectral variance in C-H region
  during rxn.

0.2
0.15
0.1
Products
0.05
Loading Values
0
Reactants
-0.05
-0.1
-0.15
2800
2850
2900
2950
3000
Wavenumber (cm-1)

• 18 minute reaction time, 15 sec. per spectrum (5
  sec. Exposure, 3 accumulations)

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Acknowledgments

• Center for Process Analytical Chemistry
• Dave Veltkamp UW/CPAC
• Larry Ricker UW/CPAC
• Jens Petter Wold - Matforsk
• JY-SPEX - Mike Carrabba
• Argonaut Technologies Terry Long
• Matrix Solutions LLC
• licensee of CPAC ballprobe technology
• www.ballprobe.com

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Spectroscopy Course Outline

• Raman Spectroscopy
• FTIR Spectroscopy
• Light Scattering
• Imaging/Microscopy
• NMR
• Ultrasonics
• NIR

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Excitation and Raman Scatter in an Optical
Waveguide

• Benefits of a Raman waveguide
• Enables the excitation of more molecules
• Because Raman scatter is isotropic it allows
  very
• efficient collection of the Raman scattered
  light

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Raman Spectra of 2-propanol
50x103

• comparison of 10 2-propanol in
• waveguide vs. vial
• 2 sec. Integration
• Sample flowing at 50 mL/min

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30
Intensity (counts)
20
10
0
3500
3000
2500
2000
1500
1000
500
0
Raman shift (cm-1)
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Waveguide Flow Cell Connected to an HPLC System
spectrograph/detector
Raman microscope
laser
beam splitter
CCD
filter
microscope objective
H20 purge
HPLC pump
waveguide
waste
injection valve
microscope lamp
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HPLC/Raman Analysis of Aromatic Compounds
500ppm on-column conc.
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Standard Raman Spectra of Aromatic Compounds
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Raman Waveguide Detection

• The Raman waveguide cell allows for the analysis
  of a variety of flowing systems with the benefit
  of signal enhancements of 2 to 3 orders of
  magnitude
• Raman provides precise structural information to
  greatly improve the selectivity of spectroscopic
  analysis
• The waveguide cell is both a sensitive detector
  and an efficient sampling interface for flowing
  systems
• An HPLC column allows separation of fluorescent
  analytes from a sample stream
• Second order instrumental advantages (molecular
  information/chromatographic separation)