Micro Sample X-ray Diffraction (µXRD) :
Applications to Pharmaceutical Sciences
NATURA IN MINIMIS MAXIMA
(Nature is greatest in the smallest things)
Joseph H. Reibenspies* & Nattamai Bhuvanesh
Texas A & M University
XDL
X-ray Diffraction Laboratory
TAMU/Chemistry
www.chem.tamu.edu/xray
Micro-sample X-ray Powder Diffraction
• Why we use micro-samples at TAMU
– Economy of resources
– Simple/robust mounting technique
– Minimize preferred orientation, transparency/sample
displacement
– Green Chemistry
• When
– SDPD investigations
– Initial stages of Discovery
– Identification of by-products
– In situ experiments
– RMA (routine materials analysis)
Micro-sample Diffraction
•
•
•
Parameters to Optimize Micro-sample
Diffraction experiment
Maximize Intensity
Decrease the Background
Achieve acceptable particle statistics
– Grinding
– Mechanical Tumbling
Debye, P. & Scherrer, P. Phys Z. (1916) 17, 277-283.
Hull, A. W. Phys. Rev. (1917) 10, 661-696.
Micro-sample Diffractometer
•
Instrumentationa
– Bruker D8 GADDS diffractometer with fixed χ stage.
•
•
•
•
•
Copper radiation (40kv/40ma) with graphite
monochromator and 17cm pinhole collimator.
Transmission mode (capillary, loop, foil)b
Multi-wire (HI-STAR) detector.
– Single – crystal : 5cm sample to detector distance.
– Powder :
12 or 25cm sample to detector distance.
Oxford 600 Cryostream LN2 cold stream (110K).
“Short” beam stop.
aDepero
et.al. J. Appl. Cryst. (2001). 34, 663-665
Sample Mounts : Loops/Foils
a,b,c
KAPTON
aTeng,
Mylar Foil
(1990) J.Appl.Cryst. 23, 387
Thorne et.al. (2005) J. Appl. Cryst. 38, 333.
c Thorne et.al. (2003) J.Appl.Cryst. 36, 1455.
www.hampton.com www.mitegen.com
www.moleculardimensions.com
b
Sample Mounts : PET Capillaries
0.3 mm
PET tube
0.02mm wall
PET = Poly(ethylene terephthalate)
Thin walled Heat Shrink Tubing
Source : Advanced Polymers
Advantages
•Inexpensive $0.25 /cm
•Transparent to X-rays
•Smooth low X-ray Scatter Pattern
•Simple to Cut, Shape and Seal
•Replaces Glass Capillaries and Fibers
•Good at low temperatures
*www.charles-supper.com
X-ray Transparency* Copper Radiation
(2.0mm)OD
wall
PET
86%
0.05mm
(0.3mm)
96%
0.02
Boron Glass
62%
0.01
Quartz
61%
0.01
Glass
54%
0.01
---------------------------------*SAXS reference Glassy Carbon
µ(cm-1)*
PET
Boron Glass
Quartz
Glass
Cu
8
71
75
111
Mo
0.67
7
8
12
PET Tubing X-ray Scatter
Lin (Cps)
6
5
Quartz
4
angle = 16.351 ° , d=5.41663
7
-15 0
0
100
PET
B-Glass
3
GLASS
2
-1 0 0
C h i - S c a le
angle = 43.311 ° , d=2.08734
8
angle = 26.814 ° , d=3.32210
angle = 21.607 ° , d=4.10948
PET
9
PET
Cu radiation
2.0 mm OD
0.05 mm wall PET
0.01 mm wall rest
1
0
5
10
20
30
2 -T h e ta - S c a le
40
50
6
Loading
1.
2.
1.
2.
*
Sample
The sample is adhered to the
loop by gently dragging the loop
over the sample surface.
The sample is “shaped” into a
sphere and attached to the loop
with mineral oil.
A 1mm PET tube is attached to a
small brass pin.
The open end of the tube is
“inserted” in the powder and
quickly removed
5 ng sample on a 0.1mm loop
60 µg in a 0.7mm loop
200 µg on a Kapton loop
1 mg in a PET tube
Bhuvanesh & Reibenspies (2003) J. Appl. Cryst. 36, 1480-1481
Bhuvanesh & Reibenspies (2006) J. Appl. Cryst. (in prep)
Data Collection on the GADDS
•
•
•
The detector distance (12cm or
25cm) and beam center are
calibrated. (Corundum).
The sample is centered.
Thee frames (at three different
2θ angles) are collected.
The frames are un-warped and
an area integration routine (cake
integration) is employed to
reduce the data.
Corundum
Standard in
loop
Raw Data Frame
180
170
160
150
140
130
120
Lin (Counts)
•
110
100
90
80
70
60
50
40
30
20
10
5
8
10
20
30
40
2-Theta - Scale
?
Frame: f:\loop_test\aa_mesh_Frame: f:\loop_test\aa_mesh_18002.001 - File: aa_mesh_18002.raw - Type: 2Th alone - Start: 3.000 ° - End: 74.300 ° - Step: 0.020 ° - Step time:
Operations: Smooth 0.141 | Y Scale Add 5 | Background 1.000,0.000 | X Offset -0.082 | X Offset -0.164 | Range Op. Merge | Import
?
Frame: f:\loop_test\aa_mesh_Frame: f:\loop_test\aa_mesh_18001.001 - File: aa_mesh_18001.raw - Type: 2Th alone - Start: 2.965 ° - End: 39.467 ° - Step: 0.020 ° - Step time:
Operations: X Offset -0.067 | Displacement 0.062 | Y Scale Add -60 | Y Scale Add 54 | Y Scale Add 20 | Smooth 0.092 | Import
Powder Pattern Collection
ω scan
1. No movement – Still
2. ϕ scan only - Spin
Debye-Scherrer
3. ω scan + ϕ scan – Tumble
Gandolfi
ϕ scan
Gandolfi
2θ ω ϕ χ scan width time
-30 -30 0 54.7 ω
-179
600
use ϕ spin option
Gandolfi G. Miner. Petrogra. Acta (1967). 67-74.
“Cake” FRAME Integration
FIT2D
“Area” Integration
Conventional “Slice”
GADDS NT Software Reference
Manual pp 1-5 to 1-15
Foil/Capillary/Loop
(unprepared aspirin sample)
Sample Displacement error
Sample hard to center
foil
capillary
loop
predicted
20
10
9
8
7
6
5
4
d - S c a le
3
Sample Preparation and Scan Types
Still/foil
Still/Foil
no sample preparation sample prepared
Gandolfi/Loop
no sample preparation
(Ultra)Micro-samples on the Bruker
Vario Powder Diffractometer
18 mm beam width
0.5mm slit I/Io ~ 0.03
D8
Vario
FWHM
GADDS 0.334o
Vario
0.087o
D8
GADDS
20
21
22
23
2-Theta - Scale
24
Argonne: Advanced Photon Source*
Line : 1-BM-C
(XOR line)
20.016 KeV
λ = 0.61938Å
MARS 345 IP
*Lee, P et.al. (1999) Rev. Sci. Inst. 70, 4457-4462
Beam stop
MARS image plate
Collimator
Data Collection at the APS
•
•
•
•
•
•
The detector distance and beam
center are calibrated.
Background frames are
collected (loop less the sample).
The sample is centered.
Three 1-60sec exposure frames
are collected.
The background frame (s) are
subtracted from the data frame.
An area integration (FIT2D*)
routine is employed to reduce
the data.
.2
4
5
3-amino-4-hydroxybenzoic Acid
6
7
8
9
10
11
12
*Hammersley, A. P. (1995) ESRF Internal Report, EXP/AH/95-01,
FIT2D V5.18 Reference Manual V1.6
13
14
15
2-Theta - Scale
16
17
18
19
20
21
22
23
24
25
Applications
Pharmaceutical Sciences
Structure Determination from Powder Data :
3-Bromophenylboronic Acid*
Br
HO
B
OH
B
-3H2O
O
Br
O
B
B
Br
O
Br
B(OH)2
3
Br
2,4,6-Tris-(3-bromo-phenyl)-cyclotr
iboroxane
3-Bromophenylboronic acid
λ (Å)
0.6194
Spacegroup
P21/c
a (Å)
15.778(1)
b (Å)
5.3083(6)
c (Å)
9.375(1)
β(°)
93.3(1)
3 rotation, 3 translation, and 1 torsion
* β-lactamase inhibitor :
Martin, R. et.al. (1994) Bioorganic & Med. Chem. Let. 4, 1299-1234.
Ab Initio Structure Solution / Refinementa
• Solution : FOXb
• Refinement : TOPASc
• Structure Parameters
–
–
–
–
–
–
–
–
2θ range
Wavelength (Å)
Chebychev bck
number parameters
Rp(%)
Rwp(%)
Rexp(%)
RBrag (%)
aBhuvanesh
1.35 – 20.0
0.619383
10
30
16.060
10.543
19.818
4.25
et.al. J.Appl.Cryst. (2005) 38 632.
bFOX : Favre-Nicolin et.al. J. Appl. Cryst. 35 (2002), 734.
cTOPAS : Bruker-AXS Karlsruhe, Germany
OH
Treholase Hydration/Dehydration at
Room Temperaturea
OH
O
HO
OH
O
OH
O
OH
OH
OH
2H2O
C12H22O11 ↔ C12H22O11 • 2H2O
Form II & Form III
(Tα)
(Tβ)
Vacuum 1mbar (RT)
slow
Form II
aFuruki
Form I
(Th)
Form I
1atm / RT fast
150oC
Heat 80oC 10mins
1atm / RT
very slow
amorphous
Form III
T., Kishi, A. & Sakurai M. Carbohydrate Research (2005) 340 429.
Trehalose Hydration
Cold Stream
2H2O
C12H22O11 ↔ C12H22O11 • 2H2O
Vacuum Chamber
1.
2.
3.
4.
1mbar Vacuum for 24 hr
Sample moved to cold stream
Cold stream stopped
Data collected
120sec/pattern
53% R.H.
~ 10 µg
Dynamic
7.6
8
9
10
11
12
13
14
15
16
17
2-Theta - Scale
18
19
20
21
22
23
24
Calibrated Humidity Control
LN2 cold stream
Vacuum Chamber
Salt
Humidity %
LiCl
15
KNO2
45
NaBr
58
NH4Cl
79.5
K2HPO4
92
--------------------------At 20oC CRC 59 (1978) E-46.
K2HPO4 NH4Cl
LiCl
Flash Frozen
Vacuum Chamber
LN2 cold stream 110K
79.5%R.H
Static
20min/scan
T = 110K
OXFORD LT
9min
6min
3min
0min
5
6
7
8
9
10
11
12
13
14
15
16
2-Theta - Scale
17
18
19
20
21
22
23
24
25
High Throughput Screening
Mannitol
7
6
10
20
30
Polymorph I
2-Theta - Scale
40
7
10
20
30
Polymorph II
40
Fronczek, F. et. al. Acta Cryst. (2003) C59 o567-o570.
Bruget, J-O., et.al. Pharm. Sci. (2000) 89 457-468.
Roberts, S. et.al J. Pharm. Sci (2002) 28 1149-1159.
10
20
30
2-Theta - Scale
40
Polymorph III
Evaporation from a binary mixture of solvents*
Spot plate evaporation method
A saturated solution of D-Mannitol in ethanol and water is prepared
well
1
2
3
4
EtOH/H2O
100/0
50/1
20/1
9/1
100
98
95
well
5
6
7
8
EtOH/H2O
8/2
7/3
6/4
5/5
90%
Evaporation
mount
80
70
*Guillory,
60
50%
J.K. in Polymorphism in Pharmaceutical Solids (2000) pp 184-227.
Mannitol : Results*
50%
%Ethanol
60
70
80
Polymorph I
90
95
98
Polymorph II
100Polymorph III
*Reibenspies & Bhuvanesh J. Pharm. Biomed. Analysis 37 (2005) 611-614
In-Loop Evaporation
DRY
Cleaning
X-Rays
The Molecular Structure of Samples that
are Liquid at RT by Micro-Sample X-ray
Powder Diffraction
Bruker GADDS Diffractometer
•
•
•
•
•
0.7mm nylon loop dipped in
neat Tetradecane
Loop transferred to cold
stream maintained at 274K
Cold stream temperature
slowly lowered to 272K
Tetradecane was allowed to
crystallized (~2 mins)
Powder pattern collected
–
–
–
–
•
Tetradecane C14H30
Space Group
_cell_length_a
_cell_length_b
_cell_length_c
_cell_angle_alpha
_cell_angle_beta
_cell_angle_gamma
_structure_solution
Observed
Calculated
12cm (sample-detector)
Gandolfi scans
2theta set 20,40,60 deg
1200,2400,3600 sec
Area Integration/Reduction
0
10
20
Norman et.al. Acta Chem. Scand. 26 (1972) 3913.
Friedrich W. Physik. Z. 14 (1913) 317.
30
2-theta
40
P -1
4.27596
4.81530
18.66640
87.225
79.020
73.057
FOX
50
The Future?
Area Detector/ Source/ Optics Resolution
3-bromophenyboronic acid
Source Detect. Optic. FWHM
-------------------------------------Tube MWPC graph. 0.36o
17 cm collimator
Focus at crystal
RAG
SAXS
Tube
Long collimator
Sync.
IP
osmic
0.35o
MWPC CCGmir. 0.14o
IP
good
0.04o
Single Crystal/Powder Diffractometer
D8 Discover with GADDS*
TXS source with Cu anode operated
at 45kV / 100mA
Cross-coupled Goebel Mirror
0.5mm pinhole collimator
VÅNTEC-2000 detector
36cm sample to detector distance
0.3mm glass capillary
600 sec scan
24
30
40
50
*Kurt Erlacher, BRUKER-AXS
60
2-Theta - Scale
70
80
90
100
The Future Micro-Sample
Diffractometer
• Parallel optics
– mirrors double/single bounce
– Pin-hole collimation / Helium sealed/vacuum
• Full positioning goniometer
– Kappa or three-circle
• High Resolution Low Background Detector
– Long Exposures
• Movable Detector Base
– Close for small molecule single-crystal
– Distance for powders
• Low Temperature
Chemistry
Acknowledgement
• Texas A & M University
• Welch
• NSF/MRI
Funding