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Novel Infrared Spectroscopic and Imaging Tools For Quality Control
Christian Huck
Institute of Analytical Chemistry and Radiochemistry, University Innsbruck
Austria
AOAC, October 11th 2011
Seite 2
Agenda / Summary slide
Motivation
NIR - Applications
Imaging Techniques
1
2
3
4
Guideline
Fundamentals
Motivation
Plant
Phytomics/Metabolomics
Production Patient
Extraction
Pretreatment
Product
Step 1 Step 2 Step 3 Step 4 Step 5
Approaches
MALDI-TOF-MS/MS
Matrixfree-MALDI
MELDI-TOF
MALDI-imaging/mapping
Near-infrared
Mid-Infrared
Imaging/mapping
Enrichment
Desalting
High-sample througput
LC, LC-MS/MS
µ-LC, µ-LC-MS/MS
CE, CE-MS
CEC
Innovations
Motivation
Non-invasive
Simultaneous determination of
several parameters
Physical and chemical
parameters
Increase in selecticity, speed
High-Throughput
Alternative method
COSTS
Nr of Samples
Fingerprint technique
off-, on- and even in-line
W. Herschel, "Investigation of the powers of the prismatic colours to heat and illuminate objects", Phil. Trans. (1800) 255.Herschel, W., "Experiments on the refrangibility of the invisible rays of the sun.", Phil Trans. (1800) 284.
Frederick William Herschel
(1738 - 1822)
Stretching vibration
Discovery of Infrared Vibration
Infrared spectroscopy
The Electromagnetic Spectrum
Micro- Far Mid NIRVisible
Ultra- Vacuum X-Ray
10 5000 2500 800 400 170 20 nm
10 200 4000 12500 25000 60000
6
5*10 5 cm -1
Overtones
ofMolecular
vibrations
Electronic transitions
rotation vibrations Inner shell
electrons
wave IR IR violet UV
stretch
bend
v
Valenceelectrons ionisation
Molecular bond
HIGH
PHOTON
ENERGY
LOW
PHOTON
ENERGY
Model of the Oscillator
2
1oscosc hE
2
2
1
2
1oscoscosc hhE
χ = anharmonicity constant
Mid-Infrared Near-Infrared
Instrumentation
MIR/NIR chemical
imaging
NIR spectroscopy
FTIR-ATR
spectroscopy
IR fingerprint1082
1170
1244
1301
1395
1451
1450
1656
2872
2957
3057
3304
2921
966
1068
1171
1239
1376
1467
1706
2853
3012
699
1090
1243
1395
1486
1601
1706
2957
1000 1500 2000 2500 3000 3500
Wavenumber [cm-1]
Absorb
an
ce
DNA
Lipids
Proteins
The NIR spectrum is a
fingerprint of the
investigated material.
It is characteristic of its
chemical and physical
properties.
Spectral characteristics
Wavenumber [cm-1] Assignation
~3300 amide A, νN-H of proteins
~3100 amide B, ν N-H with 1. overtone of the amide I band resonant (Fermi), proteins
~3010 ν C-H, lipids, cholesterols, esters
~2920
alternatively 2850
ν C-H (>CH2, Methyl) antisymmetric/symmetric, lipids, proteins, carbohydrates, esters
~2956
alternatively 2872
ν C-H (CH3, Methyl) antisymmetric/symmetric, lipids, proteins, carbohydrates, nucleic
acids
~1745-1735 ν C=O, esters and phospholipids
~1620-1695 amide I-band, proteins
~1550 amide II-band, proteins
~1400 ν C=O of COO--group, fatty acids and amino acids
~1360-1260 amide III-band absorptions (predominantly C-N stretching) with significant
contributions from ν CH2 of carbohydrate residues
~1350-1260 ν PO2-
~1310-1240 amide III-band, proteins
~1250-1220 ν P=O symmetric of the>PO2- groups, phospholipids, nucleic acids
~1225 ν PO2- asymmetric of nucleic acids and phospholipids
~1185-1120 C-O ring vibrations of nucleic acid “sugars”
~1084 ν P=O symmetric of the >PO2- groups nucleic acids, phospholipids
Overview of prominent bands of complex biological samples (4000 cm-1 to 750 cm-1)
RAMAN MID-INFRARED NEAR-INFRARED
M m
HOMONUCLEAR POLAR
e.g. C=C FUNCTIONALITIES e.g. C=O FUNCTIONALITIES
HIGH STRUCTURAL SELECTIVITYLOW STRUCTURAL
SELECTIVITY
0q
0q
0q
ANHARMONICITY
m<<M
CH / OH / NH
/
OFTEN CHEMOMETRICS
NECESSARY
polarizability dipole moment mechanical anharmonicity
Why can we see a signal ?
Measurement Principle
detector
diffuse
reflection
detector
transmission
monochromator
sample
physical properties
particle size, porosity,…
chemical properties
Surface modification,…
sp
ec
tra
chemometrics /
multivariate data analysis
qualitative analysis
quantitative analysis
INTERACTANCE
Fibre optic probe
T R I
I0 It
I0
TRANSMISSION REFLECTANCE
or transflectance
0-45 optics or
integrating sphere
Ir+
-
I0
∞
It=0
NIR – Sample presentation modes
MID-INFRARED NEAR-INFRARED
NO SAMPLE PREPARATION
ONLY VIA ATR
NO SAMPLE
PREPARATION
• NON-INVASIVE FOR HIGH SAMPLE THROUGHPUT
Sample preparation
Source Monochromator Detector
Globar
Tungsten-
Halogen Lamp
Grating
Interferometer
AOTF
MCT
InGaAs, InSb
PbS, Si, Ge
Diode-Array
Sample Sample
or
Instrumentation
Source
Mirrors
Detector
Sample
Beam Splitter
Lightfiber-Bundle
Probe Head with Sapphire Window
Optical Fiber Probe
M: Mirrors S: Sample
: Angle of Incidence
M
4
M
1
M
2
M
3
S
S
Attenuated Total Reflection
Multiple Sample Interaction
Chemometrics
PCA
PLS
NIR - Chemometrics
Univariate / Multivariate Analysis
NIR Strategy of Analysis
Sinupret
Optimization of thereference method
Optimization of theNIR-parameters
NIRHPLCContent of the
leading compound3´,4´,5´-trimethoxyflavone
Content of Flos Primulae veris
HPLC Content of the otherleading compounds
Content of other
plants in Sinupret
Optimization of thereference method
®
®
CH3
O
O
O
O
O
CH3
H
HH
H
H
H
H
CH3
Choice of Sample Set
Frequent Distribution of
Calibration Samples
CONCENTRATION
FR
EQ
UE
NC
Y
Preferential Distribution of
Calibration Samples
CONCENTRATION
FR
EQ
UE
NC
Y
NIR Strategy of Analysis
Charge Area Primula Conc. LC NIR-MW Water EtOH(µV*sec) (g/100g) (ng/µl) (ng/µl) (%) (%)
91102064 28.10.91 70490 0,493 0,219 0,219 80,38 15,7
91112182 05.11.91 71740 0,500 0,222 0,210 80,83 15,2
91112191 08.11.91 61346 0,442 0,197 0,230 80,25 15,7
91112302 22.11.91 66799 0,472 0,210 0,186 81,33 14,5
91112311 28.11.91 81551 0,555 0,245 0,193 79,69 15,7
O
O
O
O
O
CH3
H
HH
H
H
H
H
CH3
CH3
3´,4´,5´- TrimethoxyflavoneLeading Compound 0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
0.30
0.16 0.18 0.20 0.22 0.24 0.26 0.28 0.30
True value (HPLC)P
red
icte
d v
alu
e (N
IRS)
R2 0,9594SEE 0,0057SEP 0,0099BIAS –0,0389
1st derivativeNormalisation
10 14 18 22 26 30
10
14
18
22
26
30
vor a
usgesagt e
Wer t
e
True values
EtOH
Reference method: GC
vo
rau
sg
esagte
Wert
e69 73 77 81 85 89
True values
69
73
77
81
85
89
Water
Reference method: Karl-Fischer
NIRS-Analysis of Water- and Ethanol Content
NIRS of Naphthodianthrones and
Phloroglucines in St. John´s Wort
a
b
t ime [ min]
t ime [ min] t ime [ min]
t ime [ min]
AU
AU AU
AU
Determination of (a) naphthodianthrones and (b) phloroglucines.Conditions: Hypersil BDS-C18 (5 µm, 130 Å, 250 × 4 mm); mobilephase, A: 888.0 g buffer (880.0 g bidest, 2 ml 85% H3PO4, TEA (pH2.80)), 80.0 g ACN, B: 49.64 g buffer (50.0 g Bidest, 1 ml 85% H3PO4,TEA (pH 6.10)), 85.04 g methanol, 275.28 g ACN; linearer gradient;sample volume, 20 µl; peak assignment, PrPH, protopseudohypericin;PH, pseudohypericin; CPH, cyclopseudohypericin; PrH, protohypericin;H, hypericin; Hf, hyperforin; Ahf, adhyperforin.
C.W. Huck, G. Abel, M. Popp, G.K. Bonn, „Analysis of naphthodianthrone and phloroglucin
derivatives in St. John´s Wort extracts by near infrared spectroscopy (NIRS) compared to
high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE)”, Anal.
Chim. Acta (2006) doi:10.1016/j.aca.2006.07.062
NIRS of Naphthodianthrones and
Phloroglucines in St. John´s Wort
NIR spectra(a) Original spectra, (b) pretreated spectra Predicted (NIRS) vs. True values (LC)
(a) Hypericin, (b) Hyperforin(n=80). (a) R2=0.99; SEP=0.68; (b) R2=0.99; SEP=0.72
a
b
Quality Control of Achillea Species
Loadings-plot (multiple compound model, MCM)of the four species of Achillea genus. A. millefolium(mil), A. clypeolata (cly), A. collina (col), A. nobilis(nob).
PLS regression lines of the SCM
model for determining the n-
decanoic acid content in the A.
millefolium.
Cluster Analysis Quantitative Analysis
Cooperation with Prof. Guo Lanping, CACMS, Beijing
and Prof. B. Kopp, University Vienna
Antioxidative Potential
Reference: DPPH
Total Phenolic Content
Reference: Folin-Ciocalteau
Total Flavonoid Content
Achillea millefolium, Achillea absinthium,
Artemisa annua, Fagopyrum esculentum,
Genista tinctoria, Gynostemma
pentaphyllum, Leonurus sibiricus,
Mentha arvensis, Origanum dictamnus,
Reseda luteola, Scutellaria baicalensis,
Spilanthes acmella, Tanacetum
parthenium
NIRS Method for the Non-Invasive Determination
of Total Flavonoid, Total Phenol Content and
Antioxidative Potential in Herba
- F -
- S -
1 65432 87 9
Preparative TLC of Flos Primulae veris and RadixPrimulae veris, stationary phase: SiO2 60 F254
20x20, thin layer thickness: 1 mm; mobile phase:chloroform/acetone = 55 / 10; volume: 100 µl;detection: UV 365 nm 1 - 3 = Flos Primulae(butanol), 4 = DC-isolated substance, 5 - 7 = RadexPrimulae, 8 - 9 = purified extract of Flos Primulae.
IR – TLC Mapping of Flavonoids and Leading
Compounds in Phytopharmaceutical Preparations
Quality control of Scutellaria via NIRS
Scatter plot of predicted (NIRS) vs. true
property (HPLC) for determination of baicalein
(B) in radix scutellariae. B shows anti-
inflammatory, antiviral (HIV) , anti-tumor,
antioxidant, free radical scavenging and anti-
SARS coronavirus effects
Principal components score image of the wild and
cultivated radix scutellariae samples.
Dr. Li Hua, China Academy of Traditional Chinese Medicine, Beijing, China
Surface enhanced infrared – Spectroscopy
(SEIRS)
Bsp.
Trypsine
Absolute 1.5 µg
GOLD CARRIER
CaF2
Amid I+II Bande
Surface enhanced infrared – Spectroscopy
(SEIRS)
Tablet Production – On-line Analysis
PLS calibrations
Assay
Acetone
Water
Laboratory probe Process probe
R e f e r e n c e v a l u e s ( % )
N I R
P
r e
d i
c t
i o
n s
(
% )
Infrared Imaging Spectroscopy
Step The Stage To Generate Any Size
Image…
16 element array views sample in blocks of
100 x 6.25 or 400 x 25
Up to 5 steps per second and 80 spectra
per second at 16 cm-1
Hyperspectral cube
Spectrum: from 1 to k at pixel (ni,
mj)
IR image plane:
n m pixels at i
A typical
hyperspectral cube
has up to several
(ten)thousand
spectra.
1
k
ni, mj
i
P. Wilhelm et al.,
Spectroscopy Europe, pp14-19
(May 2004)
SEM
micrograph
Conservative versus imagingspectrosopy
Conservative
Spectroscopy
Single
Detector
Array
Detector
Imaging
Spectroscopy Irradiated
area
Sample
Sample
Irradiated
area
(A)
(B)
The spectrum is
representative
for total
irradiated sample
area
The spectrarecorded by the
individual detectors
represent an image of the
irradiated sample area
Infrared Imaging Spectroscopy
3.9 x 3.9 mm2
20 x ATR-Objective
(Microscope)
260 x 260 m2
50 x 50 m2
3.9 x 3.9 mm2
15 x Objective
(Microscope; Transmission)
1 : 1 Image
(Macro Chamber; Transmission, ATR)
61 m res
10 m res
4 m res
Detector
Workflow
TissueSectioning
Tissue mounting on IR-Window
IR Micro-spectrometry and Imaging
IR Spectra Spectral Diagnose
Image reassembling
Application – Urtica dioica root
1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800
1631
1426
1152
1020
1731
1592
15051456
13691317
1241 1159
1106
1051
1033
1731
1596
14211369
1316
1153
1101
1025
Wavenumber cm-1
Ab
sorb
ance
Rhizodermis
Plerom
Periblem
IR spectra from 800 to 1800 cm-1 of a Urtica sp. root
IR spectroscopic 3 D - imaging
(B)
(C) (D)
(A)
Rhizodermis
Periblem
Plerom
1662-1645 cm-1
1591-1528 cm-1 1138-988 cm-1
A) Optical microscopic image
B) amide I-band proteins
C) amide II-band proteins
D) carbohydrates, nucleic acids and phospholipids
univariate
Ester (1770-1708 cm-1)
Protein Amide II (1563-1480 cm-1)Cellulose OH (3350-3590 cm-1)
Total Scan
IR spectroscopic 2 D - imaging
univariate
Cluster analysis
(B)
(C) (D)
(A)
Rhizodermis
Periblem
Plerom
HCA-Clustering
FCM-Clustering KMC-Clustering
Optical Image
A) Optical microscopic
image
B) Hierarchical cluster
analysis
C) K-means clustering
D) Fuzzy C-means
clustering
multivariate
IR spectroscopic 3 D – hyper space imaging
IR spectroscopic 3 D – hyper space imaging
Prostate tissue
(A) HE stained diagnostic slide:1. cancer2. stroma3. benign glands
(B) nucleic acids, cholesterolphospholipids and ester
(C) proteins(D) lipids and carbohydrates
Principal component analysis (PCA)Each colored data point represents oneregion of interest (ROI). The data setswere chosen from tumor- (red) andstroma- (green) tissue region, which areco-registered with the scanned H&E
C. Pezzei, J. D. Pallua, G. Schaefer, C. Seifarth,V. Huck-Pezzei, L. K. Bittner, H. Klocker, G.Bartsch, G. K. Bonn, C. W. Huck*, Prostatecancer characterization by Fourier Transforminfrared microspectroscopy. Mol. Biosys, 2010,6, 2287
IR Imaging of Prostate Tissue
Imaging Active Ingredients in Tablets
Image Microscopic Picture
Acknowledgements
Prof. Yang Bin, China Academy of TCM, Beijing China
Prof. Dr. M. Popp, Prof. Dr. G. Abel
Prof. Dean Guo, Shanghai Institute of Materia Medica, China
Prof. Kopp, University Vienna, Austria
Prof. Bauer, University Graz, Austria
Prof. Stuppner, University Innsbruck, Austria
I will be happy to answer !
Questions?