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,