ft-ir microspectroscopy: a powerful tool for spatially resolved studies on supports for solid phase...
TRANSCRIPT
FT-IR microspectroscopy: a powerful tool for spatially
resolved studies on supports for solid phase organic
synthesis
Lisa Vaccari
Outline
Source for Imaging and Spectroscopic Studies in the Infrared
Beamline layout MidIR experimental station FT-IR Microspectroscopy
Solid phase organic synthesis Introduction Reaction Kinetic Diffusion Process
Acknowledgements
M1 Plane mirrorM2 Ellipsoidal mirrorM3 Plane mirrorM4 Ellipsoidal mirror
Radiation is collected over a solid angle of 65 mrad (H) x 25 mrad (V)
M1 Plane mirrorM2 Ellipsoidal mirrorM3 Plane mirrorM4 Ellipsoidal mirror
a b
c d
e f
Layout of SISSI
a = 3.5 m d = 1.5 mb = 1.0 m e = 1.0 mc= 11.5 m f= 2.5 m
Source for Imaging and Spectroscopic Studies in the Infrared
Experimental stations
IFS66/v
VERTEX Hyperion3000
Hyperion2000
2nd branch
1st branch
1st Branch (CNR-INFM)Solid State PhysicsHigh PressuresTime-resolved spectroscopy
2nd Branch (Elettra)Biophysics/BiochemistrySpectroscopy and Imaging
SwitchingMirror (M5)
MidIR Microspetroscopy
Mercury-Cadmium-Telluride Detector
Active area of 250X250 mm2
Operation range: 600- 9000 cm-1
Focal Plane Array detector
64X64 pixels 2.5X2.5 mm2 active area
Operative range: 900-4000 cm-1
Visible
Microwaves
Chemical ImagingGenerate Image Contrast by Using Vibrational Spectral Properties
Chemical Sample Mapping Chemical Sample Imaging
Vibrational spectra of a sample point by point irradiating small sample areas
Vibrational spectra of many sample points Irradiating the full field of view
Single point MCT detector
64X64 pixels of FPA detector
Lateral Resolution
Objective NA Wavelength
15X 0.410 m (1000cm-1) 15 m
2.5 m (4000cm-1) 4 m
36 X 0.510 m (1000cm-1) 12m
2.5 m (4000cm-1) 3m
Diffraction Limited = 0.61 / NA
FPA Detector
Objective Pixel resolution
15X 2.6
36X 1.1
Acquisition Time vs Sensitivity
MCT Detector
Scan Velocity 20 KHz
Number of scans 32
Spectral resolution 4 cm-1
2.36 spectra per second
SNR 10-5 au
Easily usable with SR
FPA Detector
Scan Velocity 6 KHz
Number of scans 32
Spectral resolution 4 cm-1
3048 spectra per second
SNR 10-3 au
Special applications with SR
MCT detector128 scanRes:4 cm-1
MIR Performance of SISSI
FPA detector32 scanRes:8 cm-1
Development of Globar-FPA/Synchrotron-MCT combined approach
• Fast acquisition of sample images with FPA detector to check sample quality and to identify regions of interest
• Higher quality map collection exploiting the brightness advantage of SR and major sensitivity of MCT detector
Solid Phase Synthesis
Large compound libraries of peptides, oligonucleotides and small molecules (drugs)
Distribution of reaction products into the bead can gives information on pore wettability and accessibility, efficiency of the reactant diffusion process, load capacity of the bead and reaction kinetics
BEAD PERFORMACES
Inert resin support
I block II block Product
Optical Transparent Polymeric Resins
Non-Optical Transparent Polymeric Resins
ATR powderFlatten Single bead microscopy
Annie Y. Bosma, Rein V. Ulijn, Gail McConnell, John Girkin, Peter J. Halling and Sabine L. Flitsch
Using two photon microscopy to quantify enzymatic reaction rates on polymer beads
Chem. Commun., 2003, 2790 - 2791
ATR Microscopy
Synbeads
In collaboration with Pharmaceutical Science Department of Trieste University (Prof. L. Gardossi, A. Basso, S. Cantone, L. Sinigoi) and Resindion Mitsubishi Chem. Corp. (Milano)- www.resindion.com-
Rigid methacrylic polymeric beadsNon-swelling and rigid support – High mechanical stabilityVersatile - Controlled porosity and different chemical functionalitiesRecyclable
Synbeads typeAverage pore diameter (nm)
30 ÷ 40 80 ÷ 100 200 ÷ 250
Amino-Methacrylate
A110 A210 A310
Carboxyl-Methacrylate
X110 X210 X310
Hydroxymethyl-Methacrylate
H110 H210 H310
Chloromethyl-Methacrylate
C110 C210 C310
Test reaction - kinetic - 1 -
Amino-Methacrylate beads – A310 – average pore diameter of 200-250 nm
Reaction time:5,10,20,30,40,60 min; 1eq polymer- 3 eq nitropropionic acid; Bead loading: 0.85 mmol/ gr dry
Detector Source
Ge
Samplepd
z
eEE
0
221
21 sin2 nn
dp
Evanescent field propagation
Ge (n1=4), = 45˚, organic medium n2 = 1.5
dp (1550cm-1) = 428 nm
Test reaction - kinetic - 2 -
Time (min)
0 20 40 60
Rel
ativ
e a
bso
rba
nce
at
15
50
cm
-1
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
min8.4
s 101.2
2/1
-13
t
k
kt
ba
eB
B
kk
ABA
kk
1][
])[1k(dB/dt
reagents of excess large ,
[B]}-][K1/{])[1[B])(]([KkdB/dt
/K K[A]) K[A]/(1 θ
θ[B])1k(dB/dt
B A Bead
00
da
Test Reaction – Diffusion - 1 -Infrared Microscopy is a label free assay
5μm thin bead sections
Average diameter 150-170 μm
FPA Images
64 scans, 4cm-1
Reaction time: 10 min
Test Reaction – Diffusion - 2 -
Reaction time: 60 min
FPA Images, 64 scans, 4cm-1
Reaction time: 30 min
Test Reaction – Diffusion - 3 -
Test Reaction – Diffusion - 4 -
10 min 20 min 30 min
SR-FTIR Microspectroscopy. 5μm spatial resolution, 256 scans, 4cm-1
Conclusion and future developments We propose a new approach for spatially resolved studies of chemical distribution based on the combination of two FTIR microscopic techniques: Conventional Source-FPA/SR-MCT detector
The high spatial resolution and fast acquisition time of FPA detector are exploited for a rapid screening of the samples to identify the best ones to be measured
The major sensitivity of MCT detector and high brightness of SR source are exploited to highlight spectral features otherwise not easily detectable
The proposed approach is sensitive and fast enough to be employed for a systematic study of reaction kinetics and diffusion mechanism for solid phase chemistry and to be extended to others scientific problems
6 min
0 min
Thanks for your attention
Acknowledgements
Trieste University – Pharmaceutical Science Department Prof. Lucia Gradossi, Alessandra Basso, Sara Cantone and Loris Sinigoi
SISSI groupM. Kiskinova, D. Eichert, F.Morgera G. Birarda and D. Bedolla
S. Lupi, A. Perucchi, R. Sopracase,