Miller Indices

William Hallowes Miller (1801 1880)

Miller indices
A Miller index is a series of coprime integers that are inversely proportional to the intercepts of the crystal face or crystallographic planes with the edges of the unit cell. It describes the orientation of a plane in the 3-D lattice with respect to the axes. The general form of the Miller index is (h, k, l) where h, k, and l are integers related to the unit cell along the a, b, c crystal axes.

Miller indices
1. Miller indices of directions [hkl]
2. Miller indices of planes (hkl) 3. Miller indices of a family of directions < hkl > 4. Miller indices of a family of planes {hkl}

Miller Indices of 1. Choose a lattice point on the Directions direction as the origin
z 2. Choose a crystal coordinate system with axes parallel to the unit cell edges y 3. Find the coordinates, in terms of the respective lattice parameters a, b and c, of another lattice point on the [110] direction.

1a+1b+0c

4. Reduce the coordinates to smallest integers. 5. Put in square brackets []

Miller Indices of Directions (contd.)

z Q OA=1/2 a + 1/2 b + 1 c

1/2, 1/2, 1
[1 1 2] y

y P x

PQ = -1 a -1 b + 1 c
-1, -1, 1 __ [111]

Miller Indices for planes

1. Select an origin not on the plane

z
(0,0,1)

2. select a crystallographic coordinate system 3. Find intercepts along axes 2 3 1 y 4. Take reciprocal 1/2 1/3 1

(0,3,0)

(2,0,0)

5. Convert to smallest integers in the same ratio: 3 2 6 6. Enclose in parenthesis : (326)

Family of Symmetry Related Planes

(110) (101) (011) _ (1 1 0) _ (101) _ (011)

{110}
{110}= Plane ( 1 1 0 ) and all other planes related by symmetry to ( 1 1 0 )

Planes in Lattices and Miller Indices

A summary of notation that you will see in regard to planes:

(hkl) denotes a set of planes [hkl] designates a vector (the direction of the planes) {hkl} set of faces made equivalent by the symmetry of the system, thus: {100} for point group 1 this refers only to the (100) face {100} for point group -1 this refers to (100) and (-100) faces for mmm {111} implies (111),(11-1),(1-11),(11-1),(-1-11),(-11-1),(1-1-1),(-1-1-1)

Conclusion about Miller Indices

We can imagine planes within a crystal. Each set of planes is uniquely identified by its Miller index (h k l) We can calculate the separation, d, for each set of planes (h k l) Each diffraction spot can be regarded as a X-ray beam reflected from a lattice plane, and therefore has a unique Miller index.

What is a polymorph?
Occur when a specific chemical compound exists in nature in more than one structural or atomic arrangement. Arise when molecules of a compound stack in the solid state in different ways Same chemical composition but different properties Vary in stability, bioavailability, solubility, and many other properties. Important in drug production Pseudopolymorphs are hydrate or solvate forms.

Acetaminophen polymorphs

Form 1

Form 2

Two polymorphic forms of aspirin

Polymorph I Melting point: 143 144 C E of dissolution: 0.862 Polymorph II Melting point: 123 125 C E of dissolution: 1.350

Efavirenz (a HIV protease inhibitor)

Efavirenz (100 mg) was dissolved in a boiling mixture of hexane (5 ml) and ethyl acetate (10 ml), under continuous stirring. The hot solution was filtered in a beaker and allowed to evaporate slowly at room temperature. Needles and a few fragile plates were obtained on complete evaporation

XRPDs and crystal structures of Efavirenz

Needles

Plates

Ravikumar & Sridhar, Mol. Cryst.Liq.Cryst. 515, 190-198, 2009

Efavirenz (a HIV protease inhibitor)

a() b() c() V(3) Crystal System Space group Z/Z

Form I 5.2111(7) 15.754(2) 17.182(2) 1410.5(3) Orthorhombic P212121 4/1

Form II 8.1403(4) 13.586(1) 24.962(2) 2760.6(3) Orthorhombic P212121 8/2

Form III 16.781(1) 27.258(1) 9.698(2) 4436(1) Orthorhombic P21212 12/3

Ravikumar & Sridhar, Mol. Cryst.Liq.Cryst. 515, 190-198, 2009 Cuffini et al., Acta Cryst. E65, o3170, 2009 Mahapatra et al., Cryst. Growth & Design., 10, 3191, 2010

Efavirenz (a HIV protease inhibitor)

Form-I Form-II

Form-III

Polymorphism screening

Methanol

Methanol : Water

Isopropyl alcohol

Ethanol

Ethanol: water

Acetonitrile

Safinamide
Safinamide, a derivative of the chemical class of aminoamides, has been developed by Newron Pharmaceuticals, Bresso, Italy, and was initially reported by Pharmacia & Upjohn as a potent anticonvulsant

Due to its excellent therapeutic properties and safety margin, safinamide has been developed as an anti-Parkinsonian and anticonvulsant agent.

Safinamide

Crystallization
Plate-shaped single crystals of polymorph (I) were obtained by slow evaporation of the Safinamide from benzene (10 ml) and hexane (2 ml) solutions stirred and warmed slightly over a steam bath for 10 min.

Needle-shaped crystals of polymorph (II) were obtained by slow evaporation from ethyl acetate (8 ml) and hexane (2 ml) solutions stirred and warmed slightly over a steam bath for 10 min

Safinamide
Plates

Form I Both forms are orthorhombic and regarded as conformational polymorphs due to the differences in the orientation of the 3-fluorobenzyloxy and propanamide groups.

Needles

Form II

XRDPs of safinamide forms

Plates

Needles

Safinamide

A superposition of the molecular conformations of safinamide molecules. Extracted structure of safinamide from safinamidehuman monoamine oxidase complex, (III)

The significant conformational differences between polymorphs (I) and (II) are in the orientations of the fluorobenzyloxy and alaninamide groups in relation to the central aromatic ring. It is interesting to note that the alaninamide chain is in an extended conformation in the extracted ligand structure and perhaps it may be speculated that a third polymorph of the title compound is possible with such features.

Safinamide

It can be seen that the two polymorphic structures, which differ significantly in the orientation of the two substituents at C8 and C11, give rise to two different modifications of the crystal packing.

Ravikumar & Sridhar, Acta Cryst. , C66, o317o320, 2010

Palonosetron Hydrochloride
Form I crystallized in orthorhombic form , while form II in monoclinic form Form I a() b() c() () V(3) Crystal System Space group 7.4972(7) 9.0228(8) 25.045(2) 90 1695.3 (3) Orthorhombic P212121 Form II 8.996(2) 7.555(1) 12.624(2) 98.080(1) 849.5(3) Monoclinic P21

Palonosetron Hydrochloride
XRPDs of Palonosetron Hydrochloride forms from patent US 2010/0105724 A1

Form I

Form II

Palonosetron hydrochloride

Form I (Orthorhombic)

Form II (Monoclinic)

Form I - Ravikumar and Sridhar, Acta Cryst., E63, o1404o1406, 2007 Form II Clark et al., J. Med. Chem. 36, 26452657, 1993

DONEPEZIL HCl (Quantification)

80

70

60

Lin (Counts)

50

40

10%

30

5%
20

2%
10

1% AL/04/2358

0 5. 5 5.6 5.7 5.8 5. 9 6.0 6.1 6. 2 6.3 6.4 6.5 6. 6 6.7 6.8 6.9 7.0

2-Theta - Scale
File: XUSV-14-118M.RAW - AL/04/3288 1% - Start: 5.500 - End: 7.000 - Step: 0.005 - Ste File: XUSV-14-118A.RAW - AL/04/2358 PURE SAMPLE (Form-VI) - Start: 5.500 - End: 7.000 Operations: Smooth 0.150 | Background 0.000,0.000 | Import Operations: Smooth 0.150 | Background 0.000,0.000 | Import File: XUSV-14-118E.RAW - AL/04/3292 5% - Start: 5.500 - End: 7.000 - Step: 0.005 - Step Operations: Smooth 0.150 | Background 0.000,0.000 | Import File: XUSV-14-118H.RAW - AL/04/3289 2% - Start: 5.500 - End: 7.000 - Step: 0.005 - Step Operations: Smooth 0.144 | Background 0.000,0.000 | Import File: XUSV-14-118B.RAW - AL/04/3290 10% - Start: 5.500 - End: 7.000 - Step: 0.005 - Ste Operations: Smooth 0.150 | Background 0.000,0.000 | Import

Quantification Of Form-IV in Form-VI

20 19 18 17 16 15 14 13 12

10%

Lin (Counts)

11 10 9 8 7 6 5

5%

2% 1%

AL/04/2465
4 3 2 1 0 5.6 5.7 5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8

2-Theta - Scale
File: XUSV-14-165B.RAW - AL/04/2465 PURE FORM-VI - Start: 5.600 - End: 6.800 - Step: Operations: Smooth 0.150 | Background 0.000,0.000 | Smooth 0.150 | Background 0.000,0.000 | File: XUSV-14-165F.RAW - AL/04/4076 (1%) - Start: 5.600 - End: 6.800 - Step: 0.005 - Step Operations: Smooth 0.150 | Background 0.000,0.000 | Smooth 0.150 | Background 0.000,0.000 | File: XUSV-14-165H.RAW - AL/04/4077 (2%) - Start: 5.600 - End: 6.800 - Step: 0.005 - Step Operations: Smooth 0.150 | Background 0.000,0.000 | Smooth 0.150 | Background 0.000,0.000 | File: XUSV-14-165K.RAW - AL/04/4078 (5%) - Start: 5.600 - End: 6.800 - Step: 0.005 - Step Operations: Smooth 0.150 | Background 0.000,0.000 | Smooth 0.150 | Background 0.000,0.000 | File: XUSV-14-165O.RAW - AL/04/4079 (10%) - Start: 5.600 - End: 6.800 - Step: 0.005 - Ste Operations: Smooth 0.150 | Background 0.000,0.000 | Smooth 0.150 | Background 0.000,0.000 |

Various types of solid forms

Crystal lattice & Diffraction lattice Real lattice & Reciprocal lattice Real space & Reciprocal space
The lattice of diffracted x-rays has an inverse or reciprocal relationship to the crystal lattice. For this reason the lattice of the diffraction pattern is called the reciprocal lattice, while the crystal is said to form the real or direct lattice.

Reciprocal lattice

Real lattice a b

a*
b*

It is important to remember that the crystal lattice, the reciprocal lattice, and Miller planes are not actual physical objects assuming you could see objects that small, you would not see an actual dot at the corner of a unit cell, etc. Nevertheless, these concepts are extremely helpful for crystal structure determination.

The Reciprocal Lattice

Because of the reciprocal nature of d spacings and q from Braggs Law, the pattern of the diffraction we observe can be related to the crystal lattice by a mathematical construct called the reciprocal lattice. In other words, the pattern of X-ray reflections makes a lattice that we can use to gain information about the crystal lattice.
The reciprocal lattice is constructed as follows: Choose a point to be the origin in the crystal lattice. Let the vector normal to a set of lattice planes in the real lattice radiate from that origin point such that the distance of the vector is the reciprocal of the d spacing for each family of planes. i.e. the vector for the plane (hkl) has a distance of 1/d(hkl) (or, more generally K/d(hkl)). Repeat for all real lattice planes.

You can see how this works at: http://www-sphys.unil.ch/x-ray/rlattice/

The Reciprocal Lattice

This procedure constructs a reciprocal lattice (RL) in which each lattice point corresponds to the reflection that is generated by a particular family of planes. This lattice can easily be indexed by assigning the proper (hkl) value to each lattice point.

Note that consequence of this reciprocal relationship include:

-Large d spacings correspond to small spacings in the RL this is an important feature that must be considered during data collection.

- Obtuse angles in the real lattice correspond to obtuse angles in the RL

The Reciprocal Lattice

For those of you who are comfortable with vectors, here is how the reciprocal lattice is built: Note that : a a* = 1 (etc. this is the reciprocal part) a b* = 0 (etc. the vectors are orthogonal in this geometry)
Thus the reciprocal lattice can be represented by vectors of the form: Rhkl = ha* + kb* + lc*, | Rhkl | = K / dhkl where h, k, and l are the indices of sets of planes in the crystal, and K can assume the value of 1, , or 2, depending on the user's convention (crystallography, solid-state physics, etc). In later discussions, K will be assumed to have a value of 1. K is shown in the relations below for completeness. Thus the individual lattice vectors have the following definitions: a* = K (b c) / (a b c) a = (b* c*) / K (a* b* c*) b* = K (c a) / (a b c) b = (c* a*) / K (a* b* c*) c* = K (a b) / (a b c) c = (a* b*) / K (a* b* c*) cos* = (cos cos - cos) /( sin sin) cos* = (cos cos - cos) /( sin sin) cos* = (cos cos - cos) /( sin sin) cos = (cos* cos* - cos*) /( sin* sin*) cos = (cos* cos* - cos*) /( sin* sin*) cos = (cos* cos* - cos*) /( sin* sin*)

V = a b c = 1/V* = abc (1 - cos2 - cos2 - cos2 + 2 cos cos cos) V* = a* b* c* = 1/V = a*b*c* (1 - cos2* - cos2* - cos2* + 2 cos* cos* cos*)

The Ewald Sphere

We have constructed the reciprocal lattice (RL) in terms of the reciprocal dspacings, 1/dhkl, another utility of this lattice in terms of crystallography is made apparent by the Ewald sphere, which tells us the angle at which each family of planes will diffract!
Consider a circle of radius r, with points X and Y lying on the circumference.

If the angle XAY is defined as q, then the angle XOY will be 2q by geometry and sin(q) = XY/2r If this geometry is constructed in reciprocal space, then it has some important implications. The radius can be set to 1/l, where l is the wavelength of the X-ray beam. If Y is the 000 reciprocal lattice point, and X is a general point hkl, then the distance XY is 1/dhkl Thus: sin(q) = (1/dhkl)/(2/l) or, rearranged: l = 2 dhkl sin(q) , and Braggs law is satisfied!

The Ewald Sphere

When a reciprocal lattice point intersects the Ewald sphere, a reflection will occur and can be observed at the 2q angle of the inscribed triangle. To be able to collect as many different reflections as possible, it is thus necessary to be able to rotate the reciprocal lattice to a great extent

The Ewald Sphere

Our need to rotate the crystal in numerous ways to bring all of the families of planes into reflection (i.e. to make RL point intersect with the Ewald sphere) explains the design of the goniometer portion of the diffractometer. The 4circles of the goniometer allows the crystal to be rotated in virtually any direction while remaining in the X-ray beam. The labels for each of the goniometer axes (f, c, w, and q) are indicated on the diagram below. Note that there are different designs (such as the Kappa geometry) that accomplish the same task.

The Ewald Sphere

If one rotates the Ewald sphere completely about the (000) reciprocal lattice point in all three dimensions, the larger sphere (of radius 2/l) contains all of the reflections that it is possible to collect using that wavelength of X-rays. This construction is known as the Limiting sphere and it defines the complete data set. Any reciprocal lattice points outside of this sphere can not be observed. Note that the shorter the wavelength of the X-radiation, the larger the Ewald sphere and the more reflections may be seen (in theory).

The limiting sphere will hold roughly (4/3pr3/ V*) lattice points. Since r = 2/l, this equates to around (33.5/ V*l3) or (33.5 V/l3) reflections. For an orthorhombic cell with a volume of 16003, this means CuKa can give around 14,700 reflections while MoKa would give 152,000 reflections.

Crystallization Methods and Protein Crystal Properties

History

Crystallization An overview

Four major steps in crystallization

Obtain large amounts of pure protein samples Choose a protein buffer in which the protein is both soluble and stable Bring protein solution to supersaturation where spontaneous nucleation can take place Crystal growth now begins

As a rule, protein solubility will usually increase as you add salt to your aqueous solution, then begin to decrease when the salt concentration gets high enough to compete with the protein for hydration (interaction with water molecules).

Nucleation
A phenomenon whereby a nucleus, such as a dust particle, a tiny seed crystal, or commonly in protein crystallography, a small protein aggregate, starts a crystallization process. Nucleation poses a large energy barrier, which is easier to overcome at a higher level of supersaturation.

Common difficulties:
1. If supersaturation is too high, too many nuclei form, hence an overabundance of tiny crystals. 2. In supersaturated solutions that dont experience spontaneous nucleation, crystal growth often only occurs in the presence of added nuclei or seeds.

Oil Immersion Micro Batch

This method is rising rapidly in popularity- typical sample size 1-6 L
Figure 1- Paraffin oil allows for little to no diffusion of water through the oil. This is a true batch experiment because all the reagents are present at a specific and relatively unchanging concentration.

Figure 2- Als oil is a 1:1 mixture of silicon oil and paraffin oil which allows for evaporation through slow diffusion through the oil. This is an evaporation Method, and the concentration of the protein and reagents in the drop does increase over time.

Interpreting the Results of the Crystallization Experiment

View the Hampton Crystal Gallery to see in which labs each of these crystals originated and which biological molecule(s) they represent. http://www.hamptonresearch.com/stuff/gallery.html

Crystal Mounting
Capillary tubes (Glass or Quartz)

Cryo-loops (thin nylon)

Properties of protein crystals

Soft, easy to crush Contain large solvent channels
Relatively large organic and inorganic molecules can diffuse inside

Anisotropic physical properties

Birefrigence due to anisotropic refraction indices

Ability to diffract X-ray due to regular spaced lattices