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 BhuvaneshTexas A & M University

XDL

X-ray Diffraction LaboratoryTAMU/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/Foilsa,b,c

Mylar FoilKAPTON

aTeng, (1990) J.Appl.Cryst. 23, 387b 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

Sample Mounts : PET CapillariesX-ray Transparency* Copper Radiation

(2.0mm)OD wallPET 86% 0.05mm(0.3mm) 96% 0.02

Boron Glass 62% 0.01Quartz 61% 0.01Glass 54% 0.01----------------------------------*SAXS reference Glassy Carbon

µ(cm-1)* Cu MoPET 8 0.67Boron Glass 71 7Quartz 75 8Glass 111 12

0.3 mmPET tube0.02mm wall

PET = Poly(ethylene terephthalate)Thin walled Heat Shrink TubingSource : Advanced PolymersAdvantages •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

PET Tubing X-ray ScatterLi

n (C

ps)

0

1

2

3

4

5

6

7

8

9

2 -T h e ta - S c a le5 1 0 2 0 3 0 4 0 5 0 6

angl

e =

16.3

51 °

, d=

5.41

663

angl

e =

43.3

11 °

, d=

2.08

734

angl

e =

26.8

14 °

, d=

3.32

210

angl

e =

21.6

07 °

, d=

4.10

948

PETGLASS

QuartzB-Glass

C h i - S c a le-1 5 0 -1 0 0 0 1 0 0

PET

PETCu radiation2.0 mm OD0.05 mm wall PET0.01 mm wall rest

Loading Sample*

5 ng sample on a 0.1mm loop1. The sample is adhered to the loop by gently dragging the loop over the sample surface.

2. The sample is “shaped” into a sphere and attached to the loop with mineral oil.

60 µg in a 0.7mm loop

1. A 1mm PET tube is attached to a small brass pin.

2. The open end of the tube is “inserted” in the powder and quickly removed

200 µg on a Kapton loop

1 mg in a PET tube

Bhuvanesh & Reibenspies (2003) J. Appl. Cryst. 36, 1480-1481Bhuvanesh & Reibenspies (2006) J. Appl. Cryst. (in prep)

Data Collection on the GADDS

Raw Data Frame

Operations: X Offset -0.067 | Displacement 0.062 | Y Scale Add -60 | Y Scale Add 54 | Y Scale Add 20 | Smooth 0.092 | 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: 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_18002.001 - File: aa_mesh_18002.raw - Type: 2Th alone - Start: 3.000 ° - End: 74.300 ° - Step: 0.020 ° - Step time:

Lin

(Cou

nts)

510

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

2-Theta - Scale8 10 20 30 40

• 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.

CorundumStandard in loop

Powder Pattern Collection

1. No movement – Still2. ϕ scan only - Spin

Debye-Scherrer 3. ω scan + ϕ scan – Tumble

Gandolfi

ω scan

Gandolfi2θ ω ϕ χ scan width time-30 -30 0 54.7 ω -179 600

use ϕ spin option

ϕ scan

Gandolfi G. Miner. Petrogra. Acta (1967). 67-74.

“Cake” FRAME Integration

Conventional “Slice”

“Area” IntegrationFIT2D

GADDS NT Software Reference Manual pp 1-5 to 1-15

Foil/Capillary/Loop(unprepared aspirin sample)

d - S c a le31 0 4567892 0

predicted

loop

capillary

foilSample Displacement error

Sample hard to center

Sample Preparation and Scan Types

Still/foil Still/Foil Gandolfi/Loopno sample preparation sample prepared no sample preparation

2-Theta - Scale20 21 22 23 24

D8 Vario FWHM

GADDS 0.334o

Vario 0.087o

(Ultra)Micro-samples on the Bruker Vario Powder Diffractometer

18 mm beam width0.5mm slit I/Io ~ 0.03 D8

GADDS

Argonne: Advanced Photon Source*

MARS image plateCollimatorBeam stop

Line : 1-BM-C (XOR line)20.016 KeV λ = 0.61938ÅMARS 345 IP

*Lee, P et.al. (1999) Rev. Sci. Inst. 70, 4457-4462

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-Theta - Scale

.2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

3-amino-4-hydroxybenzoic Acid

*Hammersley, A. P. (1995) ESRF Internal Report, EXP/AH/95-01, FIT2D V5.18 Reference Manual V1.6

Applications

Pharmaceutical Sciences

Structure Determination from Powder Data : 3-Bromophenylboronic Acid*

Br B(OH)2

BOHOH

Br3

Br

BrBO

BOO

B

Br

-3H2O

2,4,6-Tris-(3-bromo-phenyl)-cyclotriboroxane

3-Bromophenylboronic acidλ (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.

• Solution : FOXb

• Refinement : TOPASc

• Structure Parameters– 2θ range 1.35 – 20.0– Wavelength (Å) 0.619383– Chebychev bck 10– number parameters 30– Rp(%) 16.060 – Rwp(%) 10.543– Rexp(%) 19.818– RBrag (%) 4.25

Ab Initio Structure Solution / Refinementa

aBhuvanesh et.al. J.Appl.Cryst. (2005) 38 632.bFOX : Favre-Nicolin et.al. J. Appl. Cryst. 35 (2002), 734.cTOPAS : Bruker-AXS Karlsruhe, Germany

Treholase Hydration/Dehydration at Room Temperaturea

O

OOH

OHOHOH

OOH

OH

OH

OH

C12H22O11 ↔ C12H22O11 • 2H2O2H2O

Form I(Th)

Form II & Form III(Tα) (Tβ)

Form I

Form II

Vacuum 1mbar (RT)slow

Heat 80oC 10mins

amorphous150oC

1atm / RT fast 1atm / RT very slow

Form III

aFuruki T., Kishi, A. & Sakurai M. Carbohydrate Research (2005) 340 429.

2-Theta - Scale7.6 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

C12H22O11 ↔ C12H22O11 • 2H2O2H2O

Trehalose Hydration

Vacuum Chamber1. 1mbar Vacuum for 24 hr2. Sample moved to cold stream3. Cold stream stopped4. Data collected

Cold Stream

Dynamic

120sec/pattern53% R.H.~ 10 µg

Calibrated Humidity ControlLN2 cold stream

Vacuum ChamberSalt Humidity %LiCl 15KNO2 45NaBr 58NH4Cl 79.5K2HPO4 92---------------------------At 20oC CRC 59 (1978) E-46.

K2HPO4 NH4Cl LiCl

2-Theta - Scale5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

LN2 cold stream 110KVacuum Chamber

79.5%R.H

6min

3min

9min

0min

Flash Frozen

Static

20min/scanT = 110KOXFORD LT

High Throughput Screening

7 10 20 30 40

Polymorph II

Mannitol

2-Theta - Scale7 10 20 30 40

Polymorph IIIPolymorph I2-Theta - Scale

6 10 20 30 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.

Evaporation from a binary mixture of solvents*Spot plate evaporation methodA saturated solution of D-Mannitol in ethanol and water is prepared

well EtOH/H2O well EtOH/H2O1 100/0 5 8/22 50/1 6 7/33 20/1 7 6/44 9/1 8 5/5

100 98 95 90%

Evaporation

mount

80 70 60 50%

*Guillory, J.K. in Polymorphism in Pharmaceutical Solids (2000) pp 184-227.

50%

60

70

80

90

95

98

100Polymorph III

Polymorph II

Polymorph I

%Ethanol

Mannitol : Results*

*Reibenspies & Bhuvanesh J. Pharm. Biomed. Analysis 37 (2005) 611-614

X-Rays

In-Loop Evaporation

DRY

Cleaning

The Molecular Structure of Samples that are Liquid at RT by Micro-Sample X-ray

Powder Diffraction

• 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– 12cm (sample-detector)– Gandolfi scans– 2theta set 20,40,60 deg– 1200,2400,3600 sec

• Area Integration/Reduction

Tetradecane C14H30

Space Group P -1_cell_length_a 4.27596 _cell_length_b 4.81530 _cell_length_c 18.66640 _cell_angle_alpha 87.225 _cell_angle_beta 79.020 _cell_angle_gamma 73.057 _structure_solution FOX

Norman et.al. Acta Chem. Scand. 26 (1972) 3913.Friedrich W. Physik. Z. 14 (1913) 317.

0 10 20 30 40 50

2-theta

ObservedCalculated

Bruker GADDS Diffractometer

The Future?

Source Detect. Optic. FWHM--------------------------------------Tube MWPC graph. 0.36o

RAG IP osmic 0.35o

Tube MWPC CCGmir. 0.14o

Sync. IP good 0.04o

3-bromophenyboronic acid

Area Detector/ Source/ Optics Resolution

SAXS

Long collimator

Focus at crystal

17 cm collimator

2-Theta - Scale24 30 40 50 60 70 80 90 100

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

*Kurt Erlacher, BRUKER-AXS

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

Acknowledgement

• Texas A & M University• Welch• NSF/MRI

Chemistry

Funding