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UBC Theses and Dissertations

Studies on the crystallinity and phase transitions of calcium gluceptate Suryanarayanan, Rajagopalan 1985

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STUDIES ON THE CRYSTALLINITY AND PHASE TRANSITIONS OF CALCIUM GLUCEPTATE by R. SURYANARAYANAN A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES ( F a c u l t y of Pharmaceutical Sciences) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1985 © R. Suryanarayanan, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. requirements for an advanced degree at the University Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) i i A B S T R A C T Calcium g l u c e p t a t e (calcium a-glucoheptonate, CaC, t tH 2gO, 6) occurs as a c r y s t a l l i n e hydrate (I) c o n t a i n i n g 3 1/2 molecules of water of c r y s t a l l i z a t i o n per atom of c a l c i u m or as an amorphous a n h y d r a t e ( I I I ) . Calcium g l u c e p t a t e was s y n t h e s i s e d commercially as I I I u n t i l 1980 but s i n c e then only I has been commercially a v a i l a b l e . The maximum aqueous s o l u b i l i t y of I I I at room temperature was found to be > 2 molal while the e q u i l i b r i u m s o l u b i l i t y of I at 25.5°C was 0.06 molal (^3.3% w/v). A c r y s t a l l i n e anhydrate (II) wtiich had an apparent water s o l u b i l i t y of 1.3 molal was prepared from I by d e h y d r a t i o n . The U n i t e d S t a t e s Pharmacopeia (USP) i n j e c t i o n i s an aqueous s o l u t i o n c o n t a i n i n g 20.8 to 23.3% w/v CaC 1 i (H 2 60 1 6 which i s greater than the e q u i l i b r i u m s o l u b i l i t y . S o l u t i o n s prepared using II or I I I were s u p e r s a t u r a t e d with respect to I and c r y s t a l l i z e d on s t o r a g e . P h a r m a c e u t i c a l l y s t a b l e s o l u t i o n s c o u l d be prepared from II by a u t o c l a v i n g the s o l u t i o n s at 121°C f o r 20 minutes immediately a f t e r p r e p a r a t i o n which presumably destroyed seed c r y s t a l s of I. When s t o r e d at r e l a t i v e h u m i d i t i e s (RH) g r e a t e r than 66% at 25°C, II was converted i n t o I and the reverse process o c c u r r e d at 0% RH. Above 0% and below 66% RH n e i t h e r I nor II underwent a phase t r a n s i t i o n d u r i n g one year of s t o r a g e . The c o - e x i s t e n c e of I and II over a range of RH would be c o n t r a r y to the phase r u l e . I t i s suggested that at RH l e s s than 66%, the a d s o r p t i o n of a small amount of. atmospheric water vapor i n h i b i t s the II to I i i i t r a n s i t i o n probably due to the formation of a s u r f a c e l a y e r of I which l i m i t s f u r t h e r d i f f u s i o n of water. In a d d i t i o n to the phase t r a n s f o r m a t i o n s i n aqueous s o l u t i o n and the dehydration and r e h y d r a t i o n r e a c t i o n s , the e f f e c t s of f r e e z e d r y i n g and g r i n d i n g on the i n t e r c o n v e r t i b i l i t y of I, II and I I I were s t u d i e d . On g r i n d i n g II f o r i n c r e a s i n g times, there were marked i n c r e a s e s i n apparent water s o l u b i l i t y , decreases in the i n t e n s i t y of x-ray d i f f r a c t i o n peaks, and heats of s o l u t i o n changed from endothermic to exothermic. The r e s u l t s were a t t r i b u t e d to decreases in c r y s t a l l i n i t y , s i n c e surface area measurements showed that they c o u l d not be due to p a r t i c l e s i z e r e d u c t i o n . D e n s i t y i s independent of p a r t i c l e s i z e , and values obtained f o r II u s i n g a l i q u i d suspension method changed p r o g r e s s i v e l y with g r i n d i n g . The x-ray d i f f r a c t i o n , c a l o r i m e t r i c and d e n s i t y measurements were used both to q u a n t i f y the degree of c r y s t a l l i n i t y of II and to t e s t v a r i o u s models of c r y s t a l l i n i t y . A c c o r d i n g to the USP, s o l i d s are c r y s t a l l i n e , n o n - c r y s t a l l i n e (amorphous) or a mixture of the two. The degree of c r y s t a l l i n i t y depends on the f r a c t i o n of c r y s t a l l i n e m a t e r i a l i n the mixture (two-state model). An a l t e r n a t i v e concept i s that the degree of c r y s t a l l i n i t y has a value between 100% ( p e r f e c t c r y s t a l ) and 0% (amorphous) depending on the s t a t e of o r d e r / d i s o r d e r i n the l a t t i c e (one-state model). I t was concluded that g r i n d i n g decreases the c r y s t a l l i n i t y of II by i n c r e a s i n g l a t t i c e d i s o r d e r a c c o r d i n g to the one-state model. i v T A B L E O F C O N T E N T S Page A b s t r a c t i i L i s t of T a b l e s v i i i L i s t of F i g u r e s i x L i s t of Schemes x i Acknowledgements x i i INTRODUCTION 1 A. S o l i d s 1 B. Concepts of c r y s t a l l i n i t y 1 1. Two-state model of c r y s t a l l i n i t y 3 2. One-state model 4 C. Methods of q u a n t i t a t i n g c r y s t a l l i n i t y 8 1. Powder x-ray d i f f r a c t i o n 9 2. C a l o r i m e t r y 12 2.1 S o l u t i o n c a l o r i m e t r y 12 2.2 Fusion c a l o r i m e t r y 14 3. D e n s i t y 14 4. I n f r a r e d spectroscopy 17 5. Other methods 20 6. I d e n t i f i c a t i o n of models of c r y s t a l l i n i t y -l i m i t a t i o n s of the methods 22 7. Comparison of q u a n t i t a t i v e c r y s t a l l i n i t y v a l u e s o b t a i n e d by d i f f e r e n t methods 23 D. Methods of d e c r e a s i n g the degree of c r y s t a l l i n i t y of s o l i d s 23 V Page E. E f f e c t of changes i n degree of c r y s t a l l i n i t y on v a r i o u s p r o p e r t i e s 26 F. N o n - c r y s t a l l i n e s o l i d s 29 1. P r e p a r a t i o n 29 2. Pharmaceutical i m p l i c a t i o n s 31 3. S t r u c t u r e of n o n - c r y s t a l l i n e s o l i d s 33 4. "Polymorphism" i n n o n - c r y s t a l l i n e s o l i d s 37 G. I n t r o d u c t i o n to c a l c i u m g l u c e p t a t e 39 H. O b j e c t i v e s 45 EXPERIMENTAL 46 A. Apparatus 46 B. M a t e r i a l s 48 C. C h a r a c t e r i z a t i o n of c a l c i u m g l u c e p t a t e 50 1. Gas Chromatography (GC) 50 2. Powder x-ray d i f f r a c t i o n 52 3. Thermal methods 52 3.1 Thermogravimetric a n a l y s i s (TGA) 52 3.2 D i f f e r e n t i a l scanning c a l o r i m e t r y (DSC) 52 3.3 Thermomicroscopy 53 4. E q u i l i b r i u m s o l u b i l i t y 53 D. Treatment of c a l c i u m g l u c e p t a t e 55 1. G r i n d i n g 55 1.1 E f f e c t of g r i n d i n g on apparent s o l u b i l i t y 55 1.2 E f f e c t of g r i n d i n g on s u r f a c e area 56 1.3 E f f e c t of g r i n d i n g on powder x-ray d i f f r a c t i o n p a t t e r n 56 2. Freeze d r y i n g 57 3. Constant humidity s t u d i e s 57 v i Page E. P r e p a r a t i o n of s t a b l e c a l c i u m g l u c e p t a t e s o l u t i o n s 57 F. Determination of degree of c r y s t a l l i n i t y of anhydrous c a l c i u m g l u c e p t a t e 58 1. Powder x-ray d i f f r a c t i o n 59 2. S o l u t i o n c a l o r i m e t r y 64 3. Suspension d e n s i t y method 65 4. D i f f e r e n t i a l scanning c a l o r i m e t r y (DSC) 69 5. P o l a r i z e d l i g h t microscopy 70 RESULTS AND DISCUSSION 71 A. C h a r a c t e r i z a t i o n of c a l c i u m g l u c e p t a t e 71 1. Gas chromatographic a n a l y s i s 71 2. Powder x-ray d i f f r a c t i o n 73 3. Thermogravimetric a n a l y s i s 73 4. D i f f e r e n t i a l scanning c a l o r i m e t r y and thermomicroscopy 73 5. E q u i l i b r i u m s o l u b i l i t y 80 6. C h a r a c t e r i z a t i o n of d i f f e r e n t samples of c a l c i u m g l u c e p t a t e 84 B. Treatment of c a l c i u m g l u c e p t a t e 86 1. G r i n d i n g 86 1.1 E f f e c t on apparent s o l u b i l i t y 87 1.2 E f f e c t on s u r f a c e area 87 1.3 E f f e c t on x-ray d i f f r a c t i o n 87 2. Freeze d r y i n g 91 3. Constant humidity s t u d i e s 91 v i i Page C. S t a b i l i z a t i o n of c a l c i u m g l u c e p t a t e s o l u t i o n s 102 D. Determination of degree of c r y s t a l l i n i t y of c a l c i u m g l u c e p t a t e 103 1. S e l e c t i o n of c r y s t a l l i n e and amorphous r e f e r e n c e standards 104 2. Comparison of c r y s t a l l i n i t y v a l u e s obtained by d i f f e r e n t methods 105 SUMMARY 114 REFERENCES 116 Appendix I 130 Appendix II 132 Appendix I I I 134 v i i i LIST OF TABLES Table Page I Heats of s o l u t i o n of some compounds i n both c r y s t a l l i n e and amorphous s t a t e s . 13 II Comparison of degrees of c r y s t a l l i n i t y o b t ained by d i f f e r e n t methods. 24 III E f f e c t of changes i n c r y s t a l l i n i t y on v a r i o u s p r o p e r t i e s . 27 IV P r e p a r a t i o n of n o n - c r y s t a l l i n e compounds by d i f f e r e n t methods. 30 V R e l a t i o n s h i p between the p r o p o r t i o n s of a and /3 epimers and the s t a b i l i t y of aqueous s o l u t i o n s of c a l c i u m g l u c e p t a t e (27% w/v) s t o r e d at room temperature (Suryanarayanan and M i t c h e l l , 1984). 42 VI Powder x-ray d i f f r a c t i o n data l i s t i n g the e i g h t most inte n s e l i n e s of I, the corresponding l i n e s of II and the e i g h t most inten s e l i n e s of the p r e c i p i t a t e from a s o l u t i o n of I I . '72 VII E q u i l i b r i u m s o l u b i l i t y of I at d i f f e r e n t temperatures. 81 VIII Some s o l i d - s t a t e p r o p e r t i e s of v a r i o u s samples of c a l c i u m g l u c e p t a t e . 85 IX E f f e c t of g r i n d i n g on the d i s s o l u t i o n behavior of some drugs. 89 X A c t i v a t i o n e n e r g i e s f o r some dehydration r e a c t i o n s . 99 XI E f f e c t of g r i n d i n g on some p r o p e r t i e s of I I . 106 ix LIST OF FIGURES F i g u r e Page 1 The r e l a t i o n s h i p between c r y s t a l l i n e and n o n - c r y s t a l l i n e s o l i d phases. The r e l a t i o n s h i p of l i q u i d s to g l a s s e s i s a l s o shown . 2 2 Schematic r e p r e s e n t a t i o n of the two models of c r y s t a l l i n i t y . 5 3 Schematic r e p r e s e n t a t i o n i n two dimensions of l a t t i c e of a compound of composition A 2 X 3 : (a) g l a s s and (b) c r y s t a l . 35 4 S t r u c t u r e s of (a) c a l c i u m a-glucoheptonate and (b) c a l c i u m 0-glucoheptonate. 40 5 Standard curve r e l a t i n g the c o n c e n t r a t i o n of aqueous s o l u t i o n s of I with t h e i r d e n s i t i e s . 54 6 Powder x-ray d i f f r a c t i o n p a t t e r n s of (a) II and (b) II ground f o r 4 h. 60 7 Standard curve r e l a t i n g the degree of c r y s t a l l i n i t y of II and the i n t e n s i t y r a t i o of the x-ray d i f f r a c t i o n peak of II at 20.1° 26 to that of l i t h i u m f l u o r i d e at 45° 26. 61 8 Powder x-ray d i f f r a c t i o n p a t t e r n of 20% c r y s t a l l i n e II c o n t a i n i n g 11% w/w l i t h i u m f l u o r i d e as the i n t e r n a l standard. 62 9 Standard curve r e l a t i n g the degree of < c r y s t a l l i n i t y and the heat of s o l u t i o n of II i n water at room temperature. 66 10 Schematic diagram of apparatus f o r suspension d e n s i t y d e t e r m i n a t i o n s . 68 11 R e p r e s e n t a t i v e thermal curves of I (a) standard pan, (b) v o l a t i l e pan, (c) v o l a t i l e pan with a p i n h o l e and (d) v o l a t i l e pan with a p i n h o l e under reduc {c) v o l a t i l e pan witn a pinnoxe ana va; v o l a t i l e pan with a p i n h o l e under reduced p r e s s u r e . 75 X F i g u r e Page 12 Re p r e s e n t a t i v e thermal curves of II i n v o l a t i l e pans (a) f r e s h l y prepared sample and (b) sample exposed to ambient c o n d i t i o n s f o r 5 min. 79 13 The van't Hoff p l o t f o r I i n water. 82 14 E f f e c t of g r i n d i n g on the apparent s o l u b i l i t y of I and II i n water at room temperature. 88 15 Powder x-ray d i f f r a c t i o n p a t t e r n s of unground and ground 1. 90 16 The h y d r a t e d ) - anhydrate (11) t r a n s i t i o n s of c a l c i u m g l u c e p t a t e at 25°C." 95 17 K i s s i n g e r p l o t f o r c a l c u l a t i o n of the a c t i v a t i o n energy f o r d e h y d r a t i o n of I . 97 18 R e l a t i o n s h i p between the degree of c r y s t a l l i n i t y and apparent s o l u b i l i t y of II i n water at room temperature. 111 x i LIST OF SCHEMES Scheme Page I D e h y d r a t i o n r e a c t i o n s and t r a n s i t i o n s of I i n the d i f f e r e n t i a l s c a n n i n g c a l o r i m e t e r . 76 I I Some phases of c a l c i u m g l u c e p t a t e and t h e i r aqueous s o l u b i l i t i e s . 92 I I I Phase t r a n s i t i o n s of c a l c i u m g l u c e p t a t e . 93 IV Some thermodynamic v a l u e s of d e h y d r a t i o n of I t o I I . 99 ACKNOWLEDGEMENTS I wish to thank Dr. A.G. M i t c h e l l f o r h i s guidance and a l s o f o r p r o v i d i n g a s t i m u l a t i n g environment which made the study very e n j o y a b l e . I am t h a n k f u l t o : Dr. H.M. Burt f o r her he l p and a d v i c e . Mr. R.G. B u t t e r s , Dr. K.M. McErlane and Dr. J.H. M c N e i l l f o r t h e i r h e l p and guidance. Ms. C. Hannah f o r s k i l l f u l t e c h n i c a l a s s i s t a n c e . Dr. R.L. Armstrong, Mr. R. Burton, Dr. J.M. O r r , Ms. U. Oza, Dr. H. Ramaswamy and Ms. K. Watson f o r t h e i r h e l p . F i n a n c i a l support from the Science C o u n c i l of B r i t i s h Columbia, S t a n l e y Drug Products and the U n i v e r s i t y of B r i t i s h Columbia i s g r a t e f u l l y acknowledged. To Dr. T. Srinivasan . 1 INTRODUCTION A. SOLIDS S o l i d s as d e f i n e d by the a b i l i t y to t r a n s m i t shear waves, or by a minimum v i s c o s i t y of 1 0 1 3 Nm" 2 s ( 1 0 1 4 p o i s e ) , may be e i t h e r c r y s t a l l i n e or n o n - c r y s t a l l i n e (Roy, 1970). A s o l i d i s c o n s i d e r e d c r y s t a l l i n e , i f there i s long range p e r i o d i c i t y of the arrangement of the c o n s t i t u e n t atoms, ions or molecules to a g e n e r a l minimum of 3-5 nm(30-50 A). Another a r b i t r a r y d e s i g n a t i o n i s that c r y s t a l l i n e m a t e r i a l s are c h a r a c t e r i z e d by t h r e e - d i m e n s i o n a l p e r i o d i c i t y over d i s t a n c e s of s i x or more u n i t c e l l s (Klug and Alexander, 1974a). A l l remaining s o l i d s may be grouped as n o n - c r y s t a l l i n e s o l i d s . There i s no sharp boundary between c r y s t a l l i n e and n o n - c r y s t a l l i n e s o l i d s and the r e l a t i o n s h i p between the two i s given i n F i g . 1. N o n - c r y s t a l l i n e s o l i d s obtained from c r y s t a l l i n e s o l i d s may a l s o be c a l l e d "amorphous s o l i d s " , while a n o n - c r y s t a l l i n e s o l i d produced from a l i q u i d i s r e f e r r e d to as " g l a s s " ( F i g . 1). In the n o n - c r y s t a l l i n e s o l i d s , f r e e energy i n excess over the s t a b l e c r y s t a l l i n e s o l i d i s i n c l u d e d i n t o the system and t h i s may be achieved by a sudden change of a thermodynamic v a r i a b l e l i k e temperature or p r e s s u r e . B . CONCEPTS OF CRYSTALLINITY The c r y s t a l l i n e s t a t e , c h a r a c t e r i z e d by the p e r f e c t l y o r d e r e d c r y s t a l l a t t i c e and the n o n - c r y s t a l l i n e (amorphous) 2 Metastable phases Excess free energy Stable phases Non-crystalline s o l i d s Amorphized s o l i d s Glasses t i l l Energy Energy Energy added added l e f t i n by by by shear r a d i - reac-ation t i o n Energy frozen i n from melt of same composi-t i o n by cooling C r y s t a l l i n e s o l i d s Liquids F i g . 1 The r e l a t i o n s h i p between c r y s t a l l i n e and non-crystalline s o l i d phases. The r e l a t i o n s h i p of l i q u i d s to glasses i s also shown (Roy, 1970) 3 s t a t e r e p r e s e n t e d by a d i s o r d e r e d l a t t i c e , represent two extremes of l a t t i c e order and intermediate s t a t e s are p o s s i b l e . The term degree of c r y s t a l l i n i t y i s u s e f u l i n attempts to q u a n t i t a t e these intermediate s t a t e s . There are broadly two.concepts of c r y s t a l l i n i t y of s o l i d s . 1. Two-state model of c r y s t a l l i n i t y The f o l l o w i n g d i s c u s s i o n i s based on e x t e n s i v e i n v e s t i g a t i o n s of polymer c r y s t a l l i n i t y which were reviewed by M i l l e r (1966a). Many polymers e x h i b i t p r o p e r t i e s a s s o c i a t e d with both c r y s t a l l i n e m a t e r i a l s (e.g., d i f f r a c t i o n of x-rays, e v o l u t i o n of l a t e n t heat on c o o l i n g from the melt) and n o n - c r y s t a l l i n e m a t e r i a l s (e.g., d i f f u s e x-ray s c a t t e r i n g ) . T h i s behavior can be e x p l a i n e d by the two-state (or • f r i n g e d - m i c e l l e ) model a c c o r d i n g to which polymeric m a t e r i a l s c o n s i s t of s m a l l but p e r f e c t c r y s t a l l i n e r e g i o n s ( c r y s t a l l i t e s ) which are embedded w i t h i n a continuous amorphous m a t r i x . I t i s b e l i e v e d that a given molecule of polymer passes from one c r y s t a l l i t e to the next through a d i f f u s e , amorphous region a t the c r y s t a l l i t e ends. The p r o p e r t i e s of each c r y s t a l l i n e and amorphous phase are assumed to be independent of the presence and amount of the o t h e r . An observed p r o p e r t y i s taken as the sum of e x t e n s i v e p r o p e r t i e s of the two phases, i . e . , the p r o p e r t i e s of " c r y s t a l l i n e " and "amorphous" phases are a d d i t i v e . For example, the x-ray d i f f r a c t i o n p a t t e r n would be c o n s i d e r e d as the s u p e r p o s i t i o n of d i f f r a c t i o n from the c r y s t a l l i n e regions and of s c a t t e r i n g 4 from the amorphous r e g i o n s . The percent c r y s t a l l i n i t y , P, i s c a l c u l a t e d from: P"P° P = £ — § - x 100 (1) P C~Pa where p i s a s p e c i f i c p r o p e r t y (e.g., s p e c i f i c volume) of the sample under i n v e s t i g a t i o n , and p° and p°, are the s p e c i f i c p r o p e r t i e s of the completely amorphous and completely c r y s t a l l i n e samples r e s p e c t i v e l y . A c c o r d i n g to the USP (USP XX, 1980a), s o l i d s are e i t h e r c r y s t a l l i n e , n o n - c r y s t a l l i n e or a mixture of the two. The degree of c r y s t a l l i n i t y depends on the f r a c t i o n of c r y s t a l l i n e m a t e r i a l i n a mixture of the amorphous and c r y s t a l l i n e s t a t e s . C l e a r l y , the USP s u b s c r i b e s to the two-state model of c r y s t a l l i n i t y and the d e c r e a s i n g c r y s t a l l i n i t y a c c o r d i n g to t h i s model i s shown s c h e m a t i c a l l y i n F i g . 2. 2. One-state Model As the two-state model was r e f i n e d , the d i v i s i o n of a sample i n t o two i d e a l i z e d r e g i o n s , c r y s t a l l i n e and amorphous, was r e a l i z e d to be u n r e a l i s t i c and a r b i t r a r y . Thus the o v e r s i m p l i f i e d nature of the two-state model became apparent even though the model was capable of e x p l a i n i n g some p r o p e r t i e s of polymers. Moreover, the c r y s t a l l i n i t y values of the same sample obtained u s i n g d i f f e r e n t methods o f t e n f a i l e d to agree and t h i s was a t t r i b u t e d to the departure of the a c t u a l s t r u c t u r e of the polymer from that of the i d e a l i z e d 5 CRYSTALLINE • (long range order) decreasing crystallinity AMORPHOUS (short range order) 2 STATE MODEL 100% crystalline mixture 0 % crystalline 1 STATE MODEL increasing lattice disorder continuous transition F i g . 2 Schematic representation of the two models of c r y s t a l l i n i t y . Squares represent the l a t t i c e s of perfect c r y s t a l s , while i r r e g u l a r shapes represent the amorphous state; no assumptions are implied regarding the structure of the l a t t e r . 6 two-state model ( M i l l e r , 1966b). D i f f e r e n t experimental techniques weigh the spectrum of order present d i f f e r e n t l y and M i l l e r (1966b) concluded that agreement among c r y s t a l l i n i t y d e t e r m i n a t i o n s by d i f f e r e n t techniques was f o r t u i t o u s . I t was suggested t h e r e f o r e that the word " c r y s t a l l i n i t y " should always be m o d i f i e d by an experimental a d j e c t i v e , e.g., x-ray c r y s t a l l i n i t y , - d e n s i t y c r y s t a l l i n i t y , e t c . Moreover, the two-state model was developed u s i n g polymeric m a t e r i a l s . The a p p l i c a t i o n of such a model to most pharmaceuticals i s q u e s t i o n a b l e because of the d i f f e r e n c e s between polymers and other c r y s t a l l i n e m a t e r i a l s . An a l t e r n a t i v e concept has emerged, a c c o r d i n g to which, the degree of c r y s t a l l i n i t y has a value l o c a t e d on a continuous s c a l e which v a r i e s between 100% and 0% depending on the s t a t e of o r d e r / d i s o r d e r i n the l a t t i c e ( M i l l e r , 1966a; Huttenrauch, 1978). The 100% c r y s t a l l i n e m a t e r i a l i s c o n s i d e r e d t o have a p e r f e c t l y ordered l a t t i c e while the 0% c r y s t a l l i n e (or n o n - c r y s t a l l i n e or amorphous) s t a t e i s c h a r a c t e r i z e d by a completely d i s o r d e r e d l a t t i c e . The t r a n s i t i o n from the 100% to 0% c r y s t a l l i n i t y i s brought about by i n c r e a s i n g l a t t i c e d i s o r d e r . Since there i s no sharp d i s t i n c t i o n between the c r y s t a l l i n e and amorphous s t a t e s , t h i s model i s r e f e r r e d to as a one-state model. D i s o r d e r i n a c r y s t a l l a t t i c e c o u l d be produced by i m p e r f e c t i o n s ( d e f e c t s ) i n the l a t t i c e and an i n c r e a s i n g c o n c e n t r a t i o n of i m p e r f e c t i o n s p r o g r e s s i v e l y i n c r e a s e s the d i s o r d e r c ausing a decrease i n the degree of c r y s t a l l i n i t y . C r y s t a l d e f e c t s are broadly c l a s s i f i e d i n t o : 1. Zero-dimensional d e f e c t s . These are l o c a l i z e d i m p e r f e c t i o n s and are r e f e r r e d t o as p o i n t d e f e c t s . D i f f e r e n t types of p o i n t d e f e c t s are produced by (Carstensen, 1973b): a. vacant l a t t i c e s i t e s (vacancies) b. atoms, molecules or ions found i n metastable i n t e r s t i t i a l p o s i t i o n s ( i n t e r s t i t i a l s ) . F o r e i g n s p e c i e s may a l s o occupy i n t e r s t i t i a l p o s i t i o n s . 2. One dimensional d e f e c t s . The most important d e f e c t s i n t h i s category are d i s l o c a t i o n s . They are of two p o s s i b l e types - edge d i s l o c a t i o n s and screw d i s l o c a t i o n s . An edge d i s l o c a t i o n may be thought of as the i n s e r t i o n of an e x t r a plane of atoms t e r m i n a t i n g along the l i n e of d i s l o c a t i o n ( V i c k e r y , 1983). In a screw d i s l o c a t i o n , the atomic planes are j o i n e d together i n such a way as to form a s p i r a l s t a i r c a s e , winding round the l i n e of d i s l o c a t i o n . 3. Two dimensional d e f e c t s . The two types of c r y s t a l d e f e c t s i n t h i s category of p o t e n t i a l pharmaceutical s i g n i f i c a n c e a r e : a. G r a i n boundaries. In many s o l i d s , each c r y s t a l i s composed of numerous c r y s t a l l i t e s . Each of the c r y s t a l l i t e s i s m i s o r i e n t e d with i t s neighbor to a g r e a t e r or l e s s e r extent and the g r a i n boundary i s a t r a n s i t i o n zone between c r y s t a l l i t e s ( F r i e d e l , 8 1964). b. S t a c k i n g f a u l t s . These are planes a c r o s s which the r e g u l a r s t a c k i n g sequence of the c r y s t a l s t r u c t u r e i s a l t e r e d ( H o l t , 1980). The two models s c h e m a t i c a l l y shown i n F i g . 2 r e p r e s e n t , in a h i g h l y s i m p l i f i e d way, the complex t r a n s i t i o n from the c r y s t a l l i n e t o the amorphous s t a t e . I t i s r e c o g n i z e d that other models or combinations of models are p o s s i b l e . A d i r e c t c o r r e l a t i o n between the d e f e c t content of c r y s t a l s and t h e i r degree of c r y s t a l l i n i t y has not yet been e s t a b l i s h e d . T h i s i s because t h e r e are no known experimental techniques t h a t can s i m u l t a n e o u s l y q u a n t i t a t e d i f f e r e n t types of c r y s t a l i m p e r f e c t i o n s . Moreover, the presence of c r y s t a l i m p e r f e c t i o n s alone i s u n l i k e l y to make a m a t e r i a l completely n o n - c r y s t a l l i n e . As d i s c u s s e d e a r l i e r , the amorphous or n o n - c r y s t a l l i n e s t a t e i s c h a r a c t e r i z e d by the absence of long range p e r i o d i c i t y and the i m p e r f e c t i o n s d i s c u s s e d above, even i f present i n overwhelming c o n c e n t r a t i o n , are probably incapable of causing complete l a t t i c e d i s o r d e r . There i s a l s o a l i m i t to the maximum a t t a i n a b l e c o n c e n t r a t i o n of c e r t a i n types of i m p e r f e c t i o n s . For example, the maximum number of d i s l o c a t i o n s p o s s i b l e i s around 1 0 1 2 cm" 2 ( V i c k e r y , 1983). C. METHODS OF QUANTITATING CRYSTALLINITY In order t o f u l l y c h a r a c t e r i z e the s o l i d phase, p r e c i s e methods f o r determining the degree of c r y s t a l l i n i t y are 9 d e s i r e d . Some of the experimental methods f o r q u a n t i t a t i o n of c r y s t a l l i n i t y are d e s c r i b e d below. 1. Powder x-ray D i f f r a c t i o n Powder x-ray d i f f r a c t i o n i s the method most widely used to determine the degree of c r y s t a l l i n i t y of pharmaceuticals (e.g., Black and L o v e r i n g , 1977; Nakai et a l . , 1977, 1982). The t o t a l energy of the d i f f r a c t e d r a d i a t i o n from the c r y s t a l l i n e and amorphous components may be co n s i d e r e d as p r o p o r t i o n a l to the q u a n t i t y of c r y s t a l l i n e and amorphous phases r e s p e c t i v e l y ( C l a r k and T e r f o r d , 1955). The percent c r y s t a l l i n i t y , P x, from powder x-ray d i f f r a c t i o n s t u d i e s can t h e r e f o r e be c a l c u l a t e d a c c o r d i n g to the f o l l o w i n g r e l a t i o n s h i p (Klug and Alexander, 1974b): I P = — C — x 100 (2) x i + i c a where I and I g are r e s p e c t i v e l y the c r y s t a l l i n e and amorphous i n t e n s i t i e s of d i f f r a c t e d x-rays. In the measurement of d i f f r a c t i o n l i n e i n t e n s i t y , i n t e g r a t e d i n t e n s i t y r a t h e r than the maximum i n t e n s i t y must be measured because of p o s s i b l e v a r i a t i o n s i n l i n e shape due to v a r i a t i o n s i n m i c r o s t r a i n ( d i s o r d e r ) and p a r t i c l e s i z e ( C u l l i t y , 1978a). Chaklader (1963) found that even the i n t e g r a t e d i n t e n s i t y v a l u e s of l a r g e p a r t i c l e s ( >43 um diameter) of qua r t z were h i g h l y v a r i a b l e but sm a l l e r p a r t i c l e s e x h i b i t e d r e p r o d u c i b l e peak i n t e n s i t i e s . In the q u a n t i t a t i o n of c r y s t a l l i n i t y from i n t e g r a t i o n of peak areas, the methods of Ruland (Ruland, 10 1961) and of Hermans (Hermans and Weidinger, 1948) have been r e p o r t e d i n the pharmaceutical l i t e r a t u r e (Nakai et a l . , 1977, 1982; Otsuka and Kaneniwa, 1983; M o r i t a and H i r o t a , 1982). The main problem i n the use of i n t e g r a t e d peak i n t e n s i t y i s that the s e p a r a t i o n of amorphous s c a t t e r i n g from the t o t a l d i f f r a c t i o n p a t t e r n i s , at best, a r b i t r a r y (Alexander, 1969a). I f the shape of the d i f f r a c t i o n l i n e s are not a f f e c t e d by v a r i a t i o n s i n d i s o r d e r and p a r t i c l e s i z e , then the maximum i n t e n s i t i e s r a t h e r than i n t e g r a t e d i n t e n s i t i e s may be used as a measure of d i f f r a c t i o n l i n e i n t e n s i t y . The qua r t z content of dusts have been determined with s a t i s f a c t o r y accuracy by simply measuring maximum i n t e n s i t i e s ( C u l l i t y , 1978b) and a s i m i l a r method may be used to determine the degree of c r y s t a l l i n i t y , p r o v i d e d the experimental sample s a t i s f i e s the above s t a t e d c o n d i t i o n . Measurement of absolute i n t e n s i t y would, however, r e q u i r e c o r r e c t i o n f o r background s c a t t e r i n g . T h i s requirement can be overcome by the use of an i n t e r n a l standard. An i d e a l i n t e r n a l standard would, (a) have a d i f f r a c t i o n p a t t e r n which does not i n t e r f e r e with that of the m a t e r i a l being analyzed, (b) be of high c r y s t a l symmetry so tha t strong but few d i f f r a c t i o n peaks are produced, (c) have a d e n s i t y c l o s e t o that of the sample under i n v e s t i g a t i o n so that homogenity i n mixing i s . maintained and (d) be c h e m i c a l l y s t a b l e ( S h e l l , 1963). Two problems a s s o c i a t e d with the powder x-ray d i f f r a c t i o n method are the e f f e c t s of sample o r i e n t a t i o n ( p r e f e r r e d o r i e n t a t i o n ) and p a r t i c l e s i z e on x-ray i n t e n s i t y . Reduction 11 of p a r t i c l e s i z e by prolonged g r i n d i n g i s the s i n g l e most e f f e c t i v e means f o r minimizing p r e f e r r e d o r i e n t a t i o n e r r o r s (Klug and Alexander, 1974c). Since g r i n d i n g reduces the c r y s t a l l i n i t y of many m a t e r i a l s (e.g., Motooka et a l . , 1969; F l o r e n c e et a l . , 1974; Lee and Hersey, 1977; Otsuka and Kaneniwa, 1983), i t cannot be used i n c r y s t a l l i n i t y d e t e r m i n a t i o n s . Moreover, decreased p a r t i c l e s i z e can cause a broadening of x-ray l i n e s and t h i s e f f e c t u s u a l l y becomes apparent when the p a r t i c l e s i z e i s below 100 nm (1000 A ) . The Sch e r r e r formula r e l a t e s the x-ray l i n e breadth, 0 ( t h i s i s the angular width u s u a l l y measured i n radians) t o the s i z e , t , of a c r y s t a l ( C u l l i t y , 1978c): 0.9X t = (3) B c o s t 9 f i where X i s the wavelength of x-rays used and 6- i s the angle of peak d i f f r a c t i o n . Carbon black has been used as a d i s p e r s i n g agent to prevent both agglomeration and o r i e n t a t i o n of p e n i c i l l i n c r y s t a l s but t h i s r e q u i r e d p r i o r g r i n d i n g of the sample with carbon black ( C h r i s t et a l . , 1948). Chaklader (1963) used the f o l l o w i n g procedure t o minimize p r e f e r r e d o r i e n t a t i o n and nonuniform packing of p a r t i c l e s . The powder was packed i n t o the r e c t a n g u l a r c a v i t y of the sample h o l d e r , the s u r f a c e l e v e l l e d with a g l a s s s l i d e and x-ray counts o b t a i n e d . The s u r f a c e of the sample was then d i s t u r b e d and r e l e v e l l e d and x-ray counts a g a i n o b t a i n e d . T h i s procedure was repeated s e v e r a l times and the mean count value was determined. Otsuka and Kaneniwa (1983) used l i t h i u m f l u o r i d e as an x-ray i n t e r n a l standard but a l s o b e l i e v e d that i t acted as a d i l u e n t and r e s i s t e d p r e f e r r e d o r i e n t a t i o n of sample c r y s t a l s . 2. C a l o r i m e t r y 2.1 S o l u t i o n c a l o r i m e t r y The use of s o l u t i o n c a l o r i m e t r y i s based on the o b s e r v a t i o n t h a t , f o r many s o l i d s , the energy of the amorphous form i s s i g n i f i c a n t l y higher than the energy of the c r y s t a l l i n e form ( P i k a l et al . , 1978). The percent c r y s t a l l i n i t y from s o l u t i o n c a l o r i m e t r y , P g, i s d e f i n e d as: AH - AH P = — i §_ x l 0o (4) 5 AH - AH, c a where AH . AH and AH are the heats of s o l u t i o n to i n f i n i t e s a c d i l u t i o n ( i n any f i x e d s o l v e n t ) of the sample, the 0% c r y s t a l l i n e (amorphous) standard, and the 100% c r y s t a l l i n e standard r e s p e c t i v e l y . I f the energy d i f f e r e n c e between the amorphous and c r y s t a l l i n e s t a t e s i s l a r g e , c a l o r i m e t r i c c r y s t a l l i n i t i e s are p o t e n t i a l l y more p r e c i s e than c r y s t a l l i n i t y data d e r i v e d from x-ray d i f f r a c t i o n ( P i k a l et a l . , 1978). The data i n Table I was o b t a i n e d by a v a r i e t y of methods but shows t h a t the d i f f e r e n c e i n heats of s o l u t i o n between the amorphous and c r y s t a l l i n e forms of a compound can be q u i t e l a r g e thus making s o l u t i o n c a l o r i m e t r y a p o t e n t i a l l y u s e f u l t echnique. Table I Heats of s o l u t i o n of some compounds i n both c r y s t a l l i n e and amorphous s t a t e s . Compound P h y s i c a l form Heat of s o l u t i o n (kJ mol" 1) Reference S u l f a t h i a z o l e ' c r y s t a l l i n e , form I x-ray amorphous, c o - p r e c i p i t a t e d Z i n c s u l f a t e , c r y s t a l l i n e monohydrate x-ray amorphous, prepared by vacuum de h y d r a t i o n of heptahydrate Copper s u l f a -te monohydr-^ ate Cefamandole n a f a t e C e p h a l o t h i n sodium P e n i c i l l i n G potassium' c r y s t a l l i n e x-ray amorphous, prepared by vacuum de h y d r a t i o n of pentahydrate c r y s t a l l i n e , Y-form x-ray amorphous, f r e e z e d r i e d c r y s t a l l i n e x-ray amorphous, f r e e z e d r i e d c r y s t a l l i n e x-ray amorphous, f r e e z e d r i e d +38.7 + 3.37 -44.9 -76.7 -42.3 -72.3 + 8.03 -18.4 +7.95 -17.2 - 1.34 -22.6 S i m o n e l l i et a l . , 1976 F r o s t e t a l . , 1951 F r o s t e t a l . , 1951 P i k a l e t a l . , 1978 P i k a l e t a l . , 1978 P i k a l e t a l . , 1978 a C a l c u l a t e d from van't Hoff p l o t s . The heats of s o l u t i o n of s e v e r a l c r y s t a l l i n e z i n c s u l f a t e samples c o n t a i n i n g v a r y i n g percentages of water of c r y s t a l l i z a t i o n were determined, and a l i n e a r r e l a t i o n s h i p between heat of s o l u t i o n and water content was e s t a b l i s h e d . The heat of s o l u t i o n of c r y s t a l l i n e z i n c s u l f a t e monohydrate was o b t a i n e d from t h i s r e l a t i o n s h i p . A somewhat s i m i l a r procedure was used f o r determining the heats of s o l u t i o n o f amorphous z i n c s u l f a t e monohydrate, and c r y s t a l l i n e and amorphous copper s u l f a t e monohydrate. cDetermined d i r e c t l y by s o l u t i o n c a l o r i m e t r y . 14 The use of Eq. (4) r e q u i r e s the determination of the d i f f e r e n t i a l heat of s o l u t i o n which i s i m p r a c t i c a l to measure d i r e c t l y ( D a n i e l s and A l b e r t y , 1967). The d i f f e r e n t i a l heat of s o l u t i o n i s almost constant i n very d i l u t e s o l u t i o n s and under such c o n d i t i o n s the d i f f e r e n t i a l and i n t e g r a l heats of s o l u t i o n are e s s e n t i a l l y equal (Glasstone and Lewis, 1982a). Hence, i n t e g r a l heat of s o l u t i o n v a l u e s can be used to c a l c u l a t e percent c r y s t a l l i n i t y , p r o v i d e d the s o l u t i o n s are very d i l u t e . 2.2 Fusion c a l o r i m e t r y The percent c r y s t a l l i n i t y from heat of f u s i o n , P ^ , can be c a l c u l a t e d a c c o r d i n g to the f o l l o w i n g r e l a t i o n s h i p (Ke, 1966): heat of f u s i o n of experimental sample P f = . x 100 (5) 1 heat of f u s i o n of 100% c r y s t a l l i n e sample N o n c r y s t a l l i n e (amorphous) compounds are c h a r a c t e r i z e d by the absence of a sharp m e l t i n g endotherm. An advantage of t h i s method i s the p o s s i b i l i t y of q u a n t i t a t i n g c r y s t a l l i n i t y without an amorphous r e f e r e n c e standard and i t has been used i n the e v a l u a t i o n of c r y s t a l l i n i t y of polymers. However, the compound must not decompose before or d u r i n g m e l t i n g and t h e r e f o r e t h e r m o l a b i l e compounds cannot be i n v e s t i g a t e d . 3. D e n s i t y D e n s i t y measurements can p r o v i d e an i n d i c a t i o n of the s t a t e of order of a s o l i d . Although there are e x c e p t i o n s , c r y s t a l l i n e m a t e r i a l s i n general have a higher d e n s i t y than 15 t h e i r amorphous c o u n t e r p a r t s because the atoms i n the c r y s t a l l a t t i c e are l o c a t e d at the minimum p o s s i b l e d i s t a n c e from each other. An i n c r e a s e i n l a t t i c e d i s o r d e r (decreasing c r y s t a l l i n i t y ) w i l l u s u a l l y r e s u l t i n an i n c r e a s e i n volume and t h e r e f o r e a decrease i n d e n s i t y . The f r e e volume of s a l t - t y p e substances i n the l i q u i d s t a t e (which i s a d i s t u r b e d and t h e r m a l l y e x c i t e d s t a t e ) i s about 29% of the t o t a l volume, but i n s o l i d s i t i s only about 26% (Huttenrauch, 1978). Since t h i s a l t e r a t i o n proceeds i n a l i n e a r manner, there i s a l i n e a r r e l a t i o n s h i p between d e n s i t y and degree of o r d e r . I f the d e n s i t y of the p e r f e c t l y c r y s t a l l i n e (100% c r y s t a l l i n e ) s o l i d , Pc, and the d e n s i t y of the same m a t e r i a l i n the amorphous (0% c r y s t a l l i n e ) s t a t e , p_, are taken as the two l i m i t s , the percent c r y s t a l l i n i t y of the sample under i n v e s t i g a t i o n , P^, can be deduced from the f o l l o w i n g r e l a t i o n s h i p : P, = P ~ P a x 100 (6) d 'c " where p i s the d e n s i t y of the sample under i n v e s t i g a t i o n . The d e n s i t y of a p e r f e c t c r y s t a l pQ can be c a l c u l a t e d from (Alexander, 1969b): MZ  ( 7 ) where M i s the molecular weight, Z i s the number of molecules per u n i t c e l l , A i s the Avogadro number and V i s the volume of the u n i t c e l l . The u n i t c e l l parameters are u s u a l l y determined from s i n g l e c r y s t a l x-ray s t u d i e s . The d e n s i t y of the amorphous s t a t e can be approximated from group c o n t r i b u t i o n s to molar volume (Grant, 1983). T h i s method i s however 16 r e s t r i c t e d to those compounds c o n t a i n i n g f u n c t i o n a l groups of known molar volume val u e s (Rheineck and L i n , 1968; Exner, 1967). S e v e r a l techniques are a v a i l a b l e f o r the d e t e r m i n a t i o n of the d e n s i t y of s o l i d s (Bauer and Lewin, 1972), but the suspension d e n s i t y method i s unique because , i t i s p o s s i b l e not only to d i f f e r e n t i a t e between samples having very small d i f f e r e n c e s i n d e n s i t y (Johnston and Hutchison, 1940) but i t may a l s o p r o v i d e a method of d i s t i n g u i s h i n g between the two models of c r y s t a l l i n i t y . A l i q u i d i s chosen which has a d e n s i t y c l o s e to that of the s o l i d and which n e i t h e r r e a c t s with nor d i s s o l v e s the s o l i d . The s o l i d i s d i s p e r s e d i n the l i q u i d and the temperature a l t e r e d u n t i l the s o l i d i s suspended, at which temperature, the d e n s i t y of the s o l i d i s equal to t h a t of the l i q u i d . Since the temperature c o e f f i c i e n t of expansion of a s o l i d i s g e n e r a l l y much l e s s than that of a l i q u i d , the e f f e c t of temperature on the d e n s i t y of the s o l i d i s c o n s i d e r e d to be n e g l i g i b l e (Estermann et al ., 1949). I f the simple two-state model i s v a l i d , then on d i s p e r s i o n i n the suspending l i q u i d , a p a r t i a l l y c r y s t a l l i n e sample would separate i n t o two f r a c t i o n s as a r e s u l t of the d i f f e r e n c e i n d e n s i t y between the c r y s t a l l i n e and amorphous s t a t e s . On the other hand, i f the one-state model i s a p p l i c a b l e , then p r o g r e s s i v e changes i n c r y s t a l l i n i t y must be accompanied by g r a d u a l , p r o g r e s s i v e changes i n d e n s i t y . 17 The suspension d e n s i t y method, i n a h i g h l y r e f i n e d form, was developed and used by Hutchison and Johnston (1940) f o r the p r e c i s e d etermination of the r e l a t i v e d e n s i t i e s of l i t h i u m f l u o r i d e samples w i t h i n an e r r o r l i m i t of about ±5xl0~ 6 g cm" 3 and the a b s o l u t e d e n s i t i e s to w i t h i n an e r r o r about 10 times l a r g e r . Vaughan et al . (1958) s t u d i e d the d e n s i t y changes which accompany p l a s t i c deformation on compressing potassium c h l o r i d e c r y s t a l s . Using the suspension d e n s i t y technique, they were ab l e to q u a n t i t a t e a d e n s i t y decrease of 18.6 x 10" 6 g cm" 3 per percent deformation. In the pharmaceutical l i t e r a t u r e , Huttenrauch and Reiner (1976a) suggested the use of the suspension d e n s i t y method f o r determining the degree of c r y s t a l l i n i t y (degree of o r d e r ) . They used carbon t e t r a c h l o r i d e as the suspending l i q u i d f o r m i c r o c r y s t a l l i n e c e l l u l o s e s i n c e i t s d e n s i t y v a r i e s from 1.584 g cm" 3 at 20°C (Lange's Handbook of Chemistry, 1979b) to 1.496 g cm" 3 at 70°C (Timmermans, 1950) compared with the change i n d e n s i t y from 1.588 g cm" 3 f o r c r y s t a l l i n e c e l l u l o s e to 1.482 g cm" 3 f o r amorphous c e l l u l o s e . The c r y s t a l l i n i t y of c e l l u l o s e was p r o g r e s s i v e l y decreased by g r i n d i n g . The changes i n c r y s t a l l i n i t y of l a c t o s e with g r i n d i n g were a l s o i n v e s t i g a t e d by s i m i l a r methods (Huttenrauch and K e i n e r , 1976b). 4. I n f r a r e d (IR) Spectroscopy There seems to be no p r e d i c t a b l e r e l a t i o n s h i p between the degree of c r y s t a l l i n i t y of a compound and i t s IR a b s o r p t i o n b e h a v i o r . According to K o s s l e r (1967), some IR a b s o r p t i o n 18 bands of polymers may appear only when the m a t e r i a l e x i s t s i n a c r y s t a l l i n e s t a t e . These can be i d e n t i f i e d as t r u l y c r y s t a l l i n e bands i f : (a) x-ray d i f f r a c t i o n data prove the m a t e r i a l i s c r y s t a l l i n e , (b) the band disappears on m e l t i n g and (c) other.IR s t u d i e s (e.g., s o l i d s o l u t i o n s i n isomorphous m a t r i c e s ) show that the band depends on the e x i s t e n c e of a c r y s t a l l a t t i c e ( Z e r b i and C i a m p e l l i , 1964). Y a r i v and M e n d e l o v i c i (1979) found that p o o r l y c r y s t a l l i n e hematite had 3 a b s o r p t i o n bands at 308, 445 and 530 cm"1 which were ass i g n e d as oxygen displacements.The oxygen displacement a b s o r p t i o n bands i n w e l l c r y s t a l l i z e d hematite s h i f t e d t o h i g h e r f r e q u e n c i e s of 333, 468 and 543 cm" 1. Sharp IR bands were observed with anhydrous a m p i c i l l i n while a m p i c i l l i n monohydrate e x h i b i t e d d i f f u s e bands which were i n d i c a t i v e of a low degree of order (Grant and A l b u r n , 1965). S i m i l a r r e s u l t s have been obtained with c a l c i u m g l u c e p t a t e . X-ray amorphous, anhydrous, c a l c i u m g l u c e p t a t e had a few l a r g e and p o o r l y d e f i n e d a b s o r p t i o n bands while the c r y s t a l l i n e c a l c i u m g l u c e p t a t e hydrate (3 1/2 molecules of water of c r y s t a l l i z a t i o n ) had sharp IR bands (Muller et al ., 1979; Suryanarayanan and M i t c h e l l , 1984). C r y s t a l l i n e d i g o x i n was c h a r a c t e r i z e d by peaks at 1775 and 3095 cm"1 which were absent i n amorphous d i g o x i n and the i n t e n s i t y of the above peaks were used t o q u a n t i t a t e the degree of c r y s t a l l i n i t y of d i g o x i n (Black and L o v e r i n g , 1977). Otsuka and Kaneniwa (1983) determined the degree of c r y s t a l l i n i t y of ground c e p h a l e x i n by a somewhat s i m i l a r method. In a l l the f o r e g o i n g s t u d i e s , the 19 e f f e c t of changes i n c r y s t a l l i n i t y on the IR p a t t e r n s have been presented. However, the r e l a t i o n s h i p between the s t a t e of l a t t i c e o r d e r / d i s o r d e r i n a compound and i t s IR behavior has not been d i s c u s s e d m e c h a n i s t i c a l l y . F r e s h l y prepared aluminum hydroxide g e l which was x-ray amorphous was s u b j e c t e d to IR s t u d i e s by N a i l et al . (1975). There was a broad a b s o r p t i o n band i n the 2900 - 3700 cm"1 r e g i o n which i n d i c a t e d hydroxyl groups i n many environments thus c o n f i r m i n g the h i g h l y d i s o r d e r e d nature of the f r e s h g e l . As the g e l aged, peaks appeared at 3520 and 3740 cm"1 with a shoulder at 3612 cm" 1. In the O-H deformation r e g i o n , the f r e s h g e l showed a broad peak at 900 cm"1 and as the g e l aged a shoulder developed at 1020 cm"1 which e v e n t u a l l y became a w e l l r e s o l v e d peak. The powder x-ray d i f f r a c t i o n p a t t e r n , a l s o underwent a s i m i l a r change with the gradual appearance of peaks with a g i n g . However, i t was concluded that IR spectroscopy was more s e n s i t i v e to changes i n c r y s t a l l i n i t y because with IR, c r y s t a l l i z a t i o n was evident a f t e r =«42 days, whereas wi t h x-ray d i f f r a c t i o n , c r y s t a l l i z a t i o n was e v i d e n t o n l y a f t e r =*70 days. In c e l l u l o s e , the hydroxyl groups i n the amorphous region were r a p i d l y c o n v e r t e d to d e u t e r o x y l groups whereas the d e u t e r a t i o n was v e r y slow i n the c r y s t a l l i n e r e g i o n (Nakai et al ., 1977). The comparison of a b s o r p t i o n band due to hydroxyl and d e u t e r o x y l groups i n samples of c e l l u l o s e ground f o r d i f f e r e n t p e r i o d s of time was used to determine t h e i r degree 20 of c r y s t a l l i n i t y . 5. Other Methods Several other techniques for quantitating c r y s t a l l i n i t y in polymers include nuclear magnetic resonance spectroscopy and r e l a t i n g the mechanical properties of polymers to th e i r c r y s t a l l i n i t y ( M i l l e r , 1966c). These techniques may be of limited use in assessing c r y s t a l l i n i t y of pharmaceutical compounds. Polarized l i g h t microscopy i s widely used to assess q u a l i t a t i v e l y whether or not pharmaceutical s o l i d s are c r y s t a l l i n e . C r y s t a l l i n e materials (except those belonging to the cubic c r y s t a l system) are o p t i c a l l y anisotropic and exhibit birefringence when placed and rotated between crossed Nicol prisms (Bunn, 1946; USP XX, 1980b). On the other hand, amorphous materials being o p t i c a l l y isotropic have only one r e f r a c t i v e index value and are not v i s i b l e between crossed Nicol prisms. C r y s t a l l i n e cephalothin sodium did not undergo detectable s o l i d - s t a t e decomposition at 50°C, but i t s x-ray amorphous counterpart did, resulting in the following equation which Pikal et a l . , (1978) used to evaluate the percent c r y s t a l l i n i t y , P t, from s t a b i l i t y : k P. = 100(1 - -£) (8) t k a where k and k are the f i r s t - o r d e r decomposition rate 21 co n s t a n t s f o r the sample and amorphous standard r e s p e c t i v e l y . T h i s method i s u s e f u l f o r determining the percent c r y s t a l l i n i t y of samples i n those s i t u a t i o n s where the 100% c r y s t a l l i n e standard i s not a v a i l a b l e . Since the amount of moisture adsorbed was l i n e a r l y r e l a t e d t o the degree of c r y s t a l l i n i t y of both c e p h a l o t h i n sodium ( P i k a l et al ., 1978) and indomethacin (Imaizumi et al ., 1980), t h i s c o u l d be used as a technique t o e v a l u a t e c r y s t a l l i n i t y . A s i m i l a r q u a n t i t a t i v e r e l a t i o n s h i p between the degree of c r y s t a l l i n i t y of c e l l u l o s e and i t s a d s o r p t i o n of ge n t i a n v i o l e t was found by Huttenrauch and Reiner (1975). The d e n s i t y of d i s l o c a t i o n s ( l i n e d e f e c t s ) p r o v i d e s some i n s i g h t i n t o the s t a t e of order of l a r g e s i n g l e c r y s t a l s . Because of the l o c a l i z e d energy a s s o c i a t e d with d i s l o c a t i o n s , two-dimensional n u c l e a t i o n occurs more r a p i d l y at the s i t e where a d i s l o c a t i o n emerges on a c r y s t a l s u r f a c e than elsewhere. T r e a t i n g a c l e a v e d s u r f a c e with an e t c h i n g s o l u t i o n r e v e a l s the d i s l o c a t i o n s i t e s as e t c h p i t s which can be seen and counted under a microscope (Burt and M i t c h e l l , 1981; F r i e s e n et a l . , 1981). T h i s method has s e v e r a l l i m i t a t i o n s : ( i ) i t i s r e s t r i c t e d t o l a r g e well-formed c r y s t a l s , ( i i ) only up t o about 10 8 d i s l o c a t i o n s / c m 2 can be v i s u a l l y counted, ( i i i ) the method i s r e s t r i c t e d t o q u a n t i t a t i n g d i s l o c a t i o n s ; other types of c r y s t a l i m p e r f e c t i o n s such as p o i n t d e f e c t s are not i n c l u d e d and ( i v ) the q u a n t i t a t i o n of d i s l o c a t i o n s i s r e s t r i c t e d t o the c l e a v e d s u r f a c e s . In a d d i t i o n t o the above 22 l i m i t a t i o n s , a c o r r e l a t i o n between d i s l o c a t i o n d e n s i t y and degree of c r y s t a l l i n i t y has not yet been e s t a b l i s h e d (see s e c t i o n B.2 i n I n t r o d u c t i o n ) . 6. I d e n t i f i c a t i o n of models of c r y s t a l l i n i t y - l i m i t a t i o n s of the methods Most of the methods d i s c u s s e d above measure some change i n the p r o p e r t y of the e n t i r e sample due to changes i n c r y s t a l l i n i t y . The suspension d e n s i t y method does not p r o v i d e any d i r e c t i n f o r m a t i o n as to the s t a t e of l a t t i c e order of the p a r t i c l e s but the d e n s i t y of the i n d i v i d u a l p a r t i c l e s does i n d i r e c t l y i n d i c a t e t h e i r s t a t e of l a t t i c e , o r d e r . When examined m i c r o s c o p i c a l l y using p o l a r i z e d l i g h t , the p a r t i c l e s are e i t h e r b i r e f r i n g e n t ( c r y s t a l l i n e ) or n o n - b i r e f r i n g e n t (amorphous) and t h e r e f o r e q u a n t i t a t i v e i n f o r m a t i o n about in t e r m e d i a t e s t a t e s of l a t t i c e order i s not obt a i n e d . I n c r e a s i n g l a t t i c e d i s o r d e r as w e l l as de c r e a s i n g p a r t i c l e s i z e can cause x-ray l i n e broadening. None of the other techniques provide i n s i g h t i n t o the s t a t e of l a t t i c e o r d e r / d i s o r d e r of the i n d i v i d u a l p a r t i c l e s which c o n s t i t u t e the sample. For example, a decreased enthalpy of f u s i o n and a decreased enthalpy of s o l u t i o n suggest a decrease i n c r y s t a l l i n i t y but gi v e no i n d i c a t i o n of the s t a t e of order of the i n d i v i d u a l p a r t i c l e s i n the sample under i n v e s t i g a t i o n . T h e r e f o r e , these techniques are inca p a b l e of d i s t i n g u i s h i n g between the two models of c r y s t a l l i n i t y (one-state model and two-state model). 23 7. Comparison of q u a n t i t a t i v e c r y s t a l l i n i t y v a l u e s obtained by d i f f e r e n t methods Few workers have determined and compared the c r y s t a l l i n i t y of the same samples by d i f f e r e n t methods. Table II l i s t s some of these s t u d i e s i n which the c r y s t a l l i n i t y of c e l l u l o s e and c e p h a l e x i n was a l t e r e d by g r i n d i n g and c e p h a l o t h i n sodium samples of v a r y i n g c r y s t a l l i n i t y were prepared by f r e e z e d r y i n g and spray d r y i n g . A comparison of the percent c r y s t a l l i n i t y v a l u e s shows t h a t the v a l u e s are o f t e n i n poor agreement. According t o P i k a l et a l . (1978), i f a p a r t i a l l y c r y s t a l l i n e sample were simply a mixture of the amorphous and c r y s t a l l i n e s t a t e s (two-state model), a l l v a l i d measures of c r y s t a l l i n i t y would g i v e i d e n t i c a l r e s u l t s . The l a c k of q u a n t i t a t i v e agreement of c r y s t a l l i n i t i e s obtained by d i f f e r e n t methods was a t t r i b u t e d mainly to the f a i l u r e of the two-state model. D. METHODS OF DECREASING THE DEGREE OF CRY STALLINITY OF SOLIDS F i g . 1 i l l u s t r a t e s the p o s s i b l e ways of p r o v i d i n g excess f r e e energy to c r y s t a l l i n e s o l i d s so as to convert them to t h e i r c o r r e s p o n d i n g amorphous forms. Amorphous pharmaceuticals have been prepared by g r i n d i n g t h e i r c r y s t a l l i n e c o u n t e r p a r t s or from s o l u t i o n s by f r e e z e d r y i n g , spray d r y i n g or p r e c i p i t a t i o n (Table IV). Though a l l of these methods may be used to o b t a i n samples of v a r y i n g c r y s t a l l i n i t y , g r i n d i n g i s a convenient method because of the ease with which the degree of Table I I Comparison of degrees of c r y s t a l l i n i t y o b t a i n e d by d i f f e r e n t methods. Compound Method of sample p r e p a r a t i o n % C r y s t a l l i n i t y determined by Reference X-ray Herm- Using an's inter*-meth- n a l od s t a n -dard IR S o l u - Stab-t i o n i l i t y c a l o - a t 50°C rime- and t r y 31% RH C e l l u l o s e G r i n d i n g Cephalexin G r i n d i n g 0 min 63 59 Nakai e t a l . , 40 min 49 57 1977 8 h 14 38 32 h 0 10 15 min 60 28 12 Otsuka and 30 min 42 28 15 Kaneniwa, 1 h 29 17 15 1983 2 h 5 4 17 Cep h a l o t h i n Commercial l o t s 72 Freeze d r i e d 62 Freeze d r i e d 47 ( d i f f e r e n t batch) Spray d r i e d 37 93 88 54 47 100 100 85 44 P i k a l e t a l . , 1978 I n t e r p o l a t e d from p u b l i s h e d f i g u r e s . 25 c r y s t a l l i n i t y can be a l t e r e d by simply changing the grinding time. A decrease in c r y s t a l l i n i t y with increasing grinding time has been observed with microcrystalline c e l l u l o s e (Nakai et a l . , 1977; Huttenrauch, 1978), lactose (Huttenrauch and Keiner, 1976b; Nakai et a/., 1982) and cephalexin (Otsuka and Kaneniwa, 1983). In the dry grinding of c r y s t a l l i n e materials, considerable energy i s supplied of which only a f r a c t i o n i s used in p l a s t i c deformation and in the formation of new surfaces (Hersey and Krycer, 1979). The remainder i s converted d i r e c t l y into heat or i s stored by the material causing progressive l a t t i c e disorder resulting in decreased c r y s t a l l i n i t y . Prolonged grinding can lead to the formation of an amorphous phase (Lin and Somasundaran, 1972) or the creation of an amorphous surface layer (Khodakov and Rebinder, 1961). Other techniques for decreasing the c r y s t a l l i n i t y of so l i d s are: (a) preparation of s o l i d dispersions where the c r y s t a l l i n e s o l i d i s dispersed in a s o l i d state inert c a r r i e r or matrix (Chiou and Riegelman, 1971) and (b) the addition of non-toxic impurities during c r y s t a l l i z a t i o n (Chow et a l . , 1984). This study i s r e s t r i c t e d to systems consisting of calcium gluceptate and water. 26 E. EFFECT OF CHANGES IN DEGREE OF CRYSTALLINITY ON VARIOUS PROPERTIES The degree of c r y s t a l l i n i t y of a s o l i d can have a profound i n f l u e n c e on i t s p r o p e r t i e s . Table I I I l i s t s some of the changes i n the p r o p e r t i e s of s o l i d s induced by changes i n the degree of c r y s t a l l i n i t y . Decreasing c r y s t a l l i n i t y c o n f e r s both d e s i r a b l e (eg., i n c r e a s e d apparent s o l u b i l i t y , d i s s o l u t i o n r a t e , improved mixing e t c . ) and u n d e s i r a b l e (eg., decreased s t a b i l i t y , i n c r e a s e d a d s o r p t i o n e t c . ) p r o p e r t i e s t o pharmaceutical s o l i d s . As i s evi d e n t from Table I I I , the bulk of the work i n t h i s area i s by Huttenrauch and h i s co-workers. Two s o l i d s s t u d i e d by these workers are m i c r o c r y s t a l l i n e c e l l u l o s e and l a c t o s e . Huttenrauch and Keiner (1979a) dehydrated a - l a c t o s e monohydrate by h e a t i n g i t at 125°C under vacuum. I n c r e a s i n g the d r y i n g time was s a i d t o decrease the c r y s t a l l i n i t y of anhydrous a - l a c t o s e . T h i s work, however r a i s e s some q u e s t i o n s r e g a r d i n g methodology and i n t e r p r e t a t i o n : 1. The suspension d e n s i t y method ( d i s c u s s e d i n s e c t i o n C.3 i n I n t r o d u c t i o n ) was used f o r e v a l u a t i n g the changes i n c r y s t a l l i n i t y . W i t h . i n c r e a s i n g d r y i n g time, two processes occur s i m u l t a n e o u s l y : (a) p r o g r e s s i v e d e h y d r a t i o n and (b) p r o g r e s s i v e decrease i n c r y s t a l l i n i t y of the anhydrate. The f i r s t process would cause an i n c r e a s e i n d e n s i t y of the p a r t i c l e s because Huttenrauch and Keine r (1979a) gave the x-ray d e n s i t y of a - l a c t o s e monohydrate as 27 Table I I I E f f e c t o f changes i n c r y s t a l l i n i t y on v a r i o u s p r o p e r t i e s . P r o p e r t y Compound(s) E f f e c t Reference A d s o r p t i o n of water A d s o r p t i o n of water A d s o r p t i o n of water A d s o r p t i o n of dye S o l u b i l i t y and d i s s o l u -t i o n r a t e D i s s o l u t i o n r a t e S o l i d - s t a t e s t a b i l i t y Rate and ex t e n t o f water removal Indomethacin C e p h a l o t h i n sodium Two types of c e l l u l o s e C e l l u l o s e Digoxin* Potassium p e r c h l o r a t e Sodium p r a s -terone s u l -f a t e W e t t a b i l i t y ( i ) S u l f a t - . h i a z o l e * ( i i ) L a c t o s e ( i ) C i t r i c ac-i d mono-h y d r a t e 3 ( i i ) L a c t o s e monohy-d r a t e I n v e r s e l y r e l a t e d t o p e r c e n t c r y s t a l -l i n i t y ( l i n e a r ) I n v e r s e l y r e l a t e d t o p e r c e n t c r y s t a l -l i n i t y ( n o n - l i n e a r above 88% c r y s t a l -l i n i t y ) I ncreased adsorp-t i o n i n c e l l u l o s e w i t h lower c r y s t a l -l i n i t y I n v e r s e l y r e l a t e d t o p e r c e n t c r y s t a -l l i n i t y ( l i n e a r ) I ncreased s o l u b i l i -t y and d i s s o l u t i o n r a t e w i t h decreased c r y s t a l l i n i t y I ncreased d i s s o l u -t i o n r a t e w i t h i n c r e a s e d d i s l o c a -t i o n d e n s i t y I n v e r s e l y r e l a t e d t o p e r c e n t c r y s t a -l l i n i t y I n creased w e t t a b i -l i t y w i t h i n c r e a s e d d i s o r d e r i n s o l i d s I ncreased l a t t i c e d i s o r d e r caused i n -cr e a s e d r a t e and ex t e n t o f water removal Imaizumi e t a l . , 1980 P i k a l e t a l . , 1978 Huttenrauch e t a l . , 1976 Huttenrauch and K e i n e r , 1975 F l o r e n c e e t a l . , 1974 Burt and M i t c h e l l , 1981 Nakagawa e t a l . , 1982 Huttenrauch and M o e l l e r , 1983 Huttenrauch and F r i c k e , 1981 M e l t i n g Digoxin* The m e l t i n g beha-v i o r a f f e c t e d by changes i n c r y s t a -l l i n i t y F l o r e n c e e t a l . , 1974; Chiou and K y l e , 1979 2 8 Table I I I (continued) P r o p e r t y Compound (sj E f f e c t Reference M i x i n g of s o l i d s M i x i n g , i n c -o r p o r a t i o n and e f f i c i -ency of l u -b r i c a n t s i n t a b l e t s P r o p e r t i e s of suppo-s i t o r i e s Drug r e l e a s e from o i n t -ment base C o n s i s t e n c y of ointment base N o r e t h i s t e -rone a c e t a t e i n l a c t o s e L a ctose w i t h magnesium s t e a r a t e as l u b r i c a n t P o l y e t h y l e n e g l y c o l 4000 a S a l y c y l i c a c i d r e l e a s e from ointme-n t base con-s i s t i n g o f p o l y e t h y l e n e g l y c o l and m i n e r a l o i l a Two ointment bases: (i) a r t i f i c i -a l p e t -r o l a t u m 3 ( i i ) p o l y e t h y -lene gels Increased homoge-n i t y o f powder w i t h i n c r e a s e d d i s o r d e r i n l a c t o s e ( l i n e a r ) Increased d i s o r d e r of l a c t o s e i n c r e a s -ed the mechanical s t a b i l i t y o f t a b l e t s Increased d e n s i t y , impact s t r e n g t h and d i s s o l u t i o n time with i n c r e a s e d c r y s t a l l i n i t y Increased r e l e a s e r a t e of drug w i t h i n c r e a s e d degree of o r d e r of base Improvement i n c o n s i s t e n c y w i t h i n c r e a s e d c r y s t a -l l i n i t y Huttenrauch and Reiner, 1979b; Huttenrauch e t al.,1979 Huttenrauch, 1977b Huttenrauch and F r i c k e , 1979b Huttenrauch and F r i c k e , 1979a Huttenrauch e t a l . , 1973 Speculation 29 1.520 g cm" 3 compared with 1.547 g cm" 3 f o r a - l a c t o s e . The second process would most l i k e l y decrease the d e n s i t y of the p a r t i c l e s . I t i s not c l e a r how the opposing i n f l u e n c e s of these two processes were d i s t i n g u i s h e d i n the suspension c e l l . 2. I t o h et a l . (1977) r e p o r t that h e a t i n g a - l a c t o s e monohydrate at 111°C under vacuum r e s u l t e d i n the formation of u n s t a b l e anhydrous l a c t o s e t h a t was h i g h l y h y g r o s c o p i c . Since Huttenrauch and Keiner (1979a) used s i m i l a r c o n d i t i o n s f o r dehydrating a - l a c t o s e monohydrate, the formation of an u n s t a b l e phase i s a p o s s i b i l i t y which was not d i s c u s s e d . 3. In a d d i t i o n to the unstable anhydrate, Lerk et a l . (1984) r e p o r t the p r e p a r a t i o n of two more c r y s t a l forms of anhydrous a - l a c t o s e from a - l a c t o s e monohydrate. Though Huttenrauch and Keiner r e p o r t an x-ray d e n s i t y value of 1.547 g cm"3 f o r anhydrous a - l a c t o s e , no d e t a i l s of the c h a r a c t e r i z a t i o n of t h i s m a t e r i a l were g i v e n . F. NON-CRYSTALLINE SOLIDS 1. P r e p a r a t i o n N o n - c r y s t a l l i n e s o l i d s of pharmaceutical i n t e r e s t have been prepared by a v a r i e t y of techniques (Table I V ) . In each case, the f i n a l s o l i d phase was concluded to be n o n - c r y s t a l l i n e from i t s d i f f u s e x-ray d i f f r a c t i o n p a t t e r n . 30 T a b l e IV P r e p a r a t i o n of n o n - c r y s t a l l i n e compounds by d i f f e r e n t methods. Reference Compound Method o f prepa-r a t i o n o f the n o n - c r y s t a l l i n e compound Otsuka and Kaneniwa, C e p h a l e x i n 1983 Lerk e t a l . , 1984 Nakai e t a l . , 1977 a-Lactose monohydrate M i c r o c r y s t a l l i n e c e l l u l o s e By g r i n d i n g the c o r r e s -ponding c r y -s t a l l i n e forms H a l e b l i a n e t a l . , F l u p r e d n i s o l o n e 1971 P i k a l e t a l . , 1977, S e v e r a l g-lactam Freeze d r y i n g 1978 a n t i b i o t i c s Yarwood e t a l . , 1983 Sodium e t h a c r y n a t e C o r r i g a n e t al.,1984 Some t h i a z i d e d i u r e t i c s Sato e t al.,1981 9,3"-Diacetylmi- Spray d r y i n g decamycin Stagner and G u i l l - Iopanoic a c i d or y , 1979 M u l l i n s and Macek, 1.9.6.0 Nov o b i o c i n P r e c i p i t a t i o n from s o l u t i o n Borka, 1974 Indomethacin Formation o f Summers, 1978 S e v e r a l b a r b i t u r a t e s g l a s s e s by c o o l i n g from the m e l t 31 2. Pharmaceutical i m p l i c a t i o n s The e f f e c t of changes i n c r y s t a l l i n i t y on the p r o p e r t i e s of some substances was l i s t e d i n Table I I I . The f o l l o w i n g d i s c u s s i o n i s t h e r e f o r e r e s t r i c t e d t o a comparison of the p r o p e r t i e s of some s o l i d s i n j u s t two d i s c r e t e s t a t e s - c r y s t a l l i n e and amorphous. In a s o l i d , the energy of the amorphous form i s higher than t h a t of the c r y s t a l l i n e form. T h e r e f o r e , the amorphous s o l i d w i l l be more r e a c t i v e than i t s c r y s t a l l i n e c o u n t e r p a r t and w i l l l i k e l y have a higher apparent s o l u b i l i t y and d i s s o l u t i o n r a t e . The use of the n o n - c r y s t a l l i n e form may t h e r e f o r e be p r e f e r r e d . M u l l i n s and Macek (1960) observed t h a t , f o r the same dose, x-ray amorphous novo b i o c i n was r e a d i l y absorbed i n dogs and produced t h e r a p e u t i c a l l y adequate c o n c e n t r a t i o n s while c r y s t a l l i n e n o v o b i o c i n was not absorbed at a l l . T h i s d i f f e r e n c e i n b i o a v a i l a b i l i t y was c o r r e l a t e d to the marked d i f f e r e n c e s i n the d i s s o l u t i o n r a t e and apparent s o l u b i l i t y of the c r y s t a l l i n e and amorphous forms i n O.IN h y d r o c h l o r i c a c i d . Amorphous n o v o b i o c i n i n suspension had a tendency to r e a d i l y c o n v ert t o the s t a b l e c r y s t a l l i n e form. T h i s problem may be encountered whenever the metastable form of a compound i s used and i n such cases steps have to be taken t o sto p or at l e a s t d e c e l e r a t e such a t r a n s i t i o n . A l m i r a n t e et a l . (1960) ad m i n i s t e r e d both c r y s t a l l i n e and amorphous forms of chloramphenicol s t e a r a t e t o r a t s and while the amorphous form was h y d r o l y z e d to l i b e r a t e the a n t i b i o t i c , the c r y s t a l l i n e form was t h e r a p e u t i c a l l y i n e r t . The d u r a t i o n of a c t i o n of 32 i n s u l i n can be c o n t r o l l e d by i t s c r y s t a l l i n i t y . When reac t e d with z i n c c h l o r i d e , i n s u l i n p r e c i p i t a t e s as an i n s o l u b l e complex and depending on the pH, i t p r e c i p i t a t e s e i t h e r as an amorphous or as a c r y s t a l l i n e phase ( H a l l a s - M o l l e r et a l . , 1952). Prompt I n s u l i n Zinc Suspension (USP XX, I980d) i s d e s c r i b e d as a suspension of amorphous i n s u l i n z i n c complex and i t s e f f e c t begins about 1 h a f t e r subcutaneous i n j e c t i o n , reaches a maximum i n 6 h and l a s t s about 12 to 16 h (Ma r t i n d a l e , 1982a). Extended I n s u l i n Zinc Suspension (USP XX, I980d) c o n s i s t s of a predominantly c r y s t a l l i n e s o l i d phase and i t s e f f e c t begins 4 to 6 h a f t e r subcutaneous i n j e c t i o n , reaches a maximum i n about 10 to 19 h and l a s t s f o r 30 to 36 h. Intermediate response time and d u r a t i o n of a c t i o n i s achieved through I n s u l i n Zinc Suspension (USP XX, I980e) which c o n s i s t s of a mixture of c r y s t a l l i n e and amorphous i n s u l i n i n a r a t i o of approximately 7 p a r t s of c r y s t a l s to 3 p a r t s of amorphous m a t e r i a l s . The e f f e c t begins w i t h i n about 2 h, reaches a maximum i n about 8 to 12 h, and l a s t s f o r about 30 h. The i n c r e a s e d r e a c t i v i t y of n o n - c r y s t a l l i n e s o l i d s can le a d t o problems p a r t i c u l a r l y of chemical s t a b i l i t y . In the case of c e p h a l o s p o r i n s ( P f e i f f e r et a l . , 1976; P i k a l et a l . , 1978; O b e r h o l t z e r and Brenner, 1979) and potassium p e n i c i l l i n (Mathews et a l . , 1966 and P f e i f f e r et a l . , 1976) the amorphous forms were q u a l i t a t i v e l y compared t o t h e i r c r y s t a l l i n e c o u n t e r p a r t s , and found to be l e s s s t a b l e . P i k a l et a l . (1977) concluded that the s t a b i l i t y of the amorphous forms of some 33 c e p h a l o s p o r i n s ( c e p h a l o t h i n sodium, cefamandole sodium and cefamandole nafate) were at l e a s t one order of magnitude l e s s than the corres p o n d i n g c r y s t a l l i n e forms. Some drugs are d e t e c t a b l y hygroscopic only i n t h e i r amorphous form. T h i s has been observed i n e p i c i l l i n (Hou and R e s t i v o , 1975) and potassium p e n i c i l l i n (Mathews et a l . , 1966). 3. S t r u c t u r e o f n o n - c r y s t a l l i n e s o l i d s There are broadly two schools of thought about the s t r u c t u r e of n o n - c r y s t a l l i n e s o l i d s - the "random network" theory of Zachariasen (1932) and the " m i c r o c r y s t a l l i t e " theory due t o Lebedev (1921). Zachariasen s p e c i f i c a l l y d i s c u s s e s the atomic arrangement i n g l a s s e s but the arguments are a l s o r e l e v a n t t o other n o n - c r y s t a l l i n e s o l i d s l i k e amorphous s o l i d s . The p e r i o d i c i t y of arrangement of atoms i s the c h a r a c t e r i s t i c f e a t u r e of a c r y s t a l . P e r i o d i c i t y i m p l i e s that a s t r u c t u r a l u n i t c a l l e d a u n i t c e l l e x i s t s which b u i l d s up a c r y s t a l by being repeated i n three d i r e c t i o n s . G l a s s e s are c h a r a c t e r i z e d by an absence of such long range p e r i o d i c i t y . However, i t i s v a l i d to assume that the atoms i n g l a s s are l i n k e d t ogether by f o r c e s e s s e n t i a l l y the same as i n c r y s t a l s , because over long ranges of temperature, the mechanical p r o p e r t i e s of a g l a s s and i t s c r y s t a l l i n e c o u n t e r p a r t are comparable. The i s o t r o p i c c h a r a c t e r o f g l a s s i s a n a t u r a l consequence of the absence of symmetry i n the network ( l a t t i c e ) because the atomic arrangement w i l l be s t a t i s t i c a l l y the same i n a l l d i r e c t i o n s . The network i n g l a s s can be 34 c h a r a c t e r i z e d by an i n f i n i t e l y l a r g e u n i t c e l l c o n t a i n i n g an i n f i n i t e number of atoms. Because of the l a c k of p e r i o d i c i t y , no two atoms are s t r u c t u r a l l y e q u i v a l e n t while i n a c r y s t a l l a t t i c e l i k e t h a t of sodium c h l o r i d e , a l l the sodium ions are s t r u c t u r a l l y e q u i v a l e n t i . e . a l l of the sodium ions have e x a c t l y i d e n t i c a l surroundings ( s u r f a c e c o n d i t i o n s are d i s r e g a r d e d ) . S i m i l a r l y , a l l the c h l o r i d e ions are a l s o s t r u c t u r a l l y e q u i v a l e n t . Since the atoms in g l a s s e s are s t r u c t u r a l l y u nequivalent, the energy r e q u i r e d to detach an atom from the network w i l l be d i f f e r e n t f o r each i n d i v i d u a l atom. With i n c r e a s i n g temperature, an i n c r e a s e d number of atoms are detached so that the breakdown of the network i s a continuous r a t h e r than an abrupt phenomenon ( i . e . the absence of a sharp m e l t i n g p o i n t i s c h a r a c t e r i s t i c of amorphous * s o l i d s ) . On the other hand, a c r y s t a l l a t t i c e c o n s i s t s of s t r u c t u r a l l y e q u i v a l e n t atoms, and when the thermal energy reaches a d e f i n i t e v a l u e , a l l the e q u i v a l e n t atoms are s i m u l t a n e o u s l y detached and the c r y s t a l network breaks down a b r u p t l y . F i g u r e 3a shows, i n two dimensions, the l a t t i c e of a c r y s t a l of composition A 2 X 3 while F i g . 3b r e p r e s e n t s the g l a s s network of the same compound. Warren (1937) worked with v i t r e o u s s i l i c a (fused q u a r t z ) and, from the x-ray r e s u l t s , p i c t u r e d g l a s s y s i l i c a as a random network i n which each s i l i c o n atom was surrounded t e t r a h e d r a l l y by four oxygen atoms, and each oxygen bonded to two s i l i c o n atoms, the two bonds t o an oxygen being roughly d i a m e t r i c a l l y o p p o s i t e . The o r i e n t a t i o n of one t e t r a h e d r a l group with r e s p e c t to a 35 (a) (b) F i g . 3 Schematic representation i n two dimensions of l a t t i c e of a compound of composition A-X^ (• atoms of A, 0 atoms of X) : (a) glass and (b) crystal (Zachariasen, 1932). 36 ne i g h b o r i n g group c o u l d be p r a c t i c a l l y at random. However, there was a d e f i n i t e scheme of s t r u c t u r e i n v o l v e d i n t h a t each atom had a d e f i n i t e number of nearest neighbors at a d e f i n i t e d i s t a n c e , but no u n i t of s t r u c t u r e repeated i t s e l f i d e n t i c a l l y at r e g u l a r i n t e r v a l s i n three dimensions. Hence the m a t e r i a l was not c r y s t a l l i n e . A c c o r d i n g to Lebedev (1921,1940) g l a s s e s are an aggregation of h i g h l y d i s p e r s e d c r y s t a l l i t e s of 0.7 to 1.5 nm (7 to 15 A) s i z e range and i n s p i t e of t h e i r s m a l l s i z e are not g r e a t l y d i s t o r t e d and preserve to a c o n s i d e r a b l e degree t h e i r i n d i v i d u a l p r o p e r t i e s . For example, Wagner et al. (1968) prepared vapor quenched f i l m s of AgCu a l l o y and found them to be m i c r o c r y s t a l l i n e with f a c e - c e n t r e d c u b i c s t r u c t u r e and a p a r t i c l e s i z e of l e s s than 1.6 nm. C r y s t a l s of t h i s s i z e range w i l l have very broad x-ray d i f f r a c t i o n l i n e s and may be c o n s i d e r e d t o be x-ray amorphous ( C u l l i t y , 1978c). Warren (1937) s t u d i e d the x-ray behavior of v i t r e o u s s i l i c a and found evidence r e f u t i n g Lebedev's ' m i c r o c r y s t a l l i t e ' theory. From the x-ray l i n e breadth of fused q u a r t z , he c a l c u l a t e d the average p a r t i c l e s i z e to be 0.77 nm. Since the edge of the u n i t c e l l of c r i s t o b a l i t e (the c r y s t a l l i n e form of quartz) i s =*0.7 nm, i f Lebedev's theory were v a l i d , then each c r y s t a l l i t e should comprise of s c a r c e l y more than one u n i t c e l l and c a l l i n g i t " c r y s t a l l i n e " i s of q u e s t i o n a b l e v a l i d i t y . Moreover, i f the m a t e r i a l d i d c o n s i s t of d i s c r e t e c r y s t a l l i t e s , then s m a l l angle s c a t t e r i n g (at 26 37 l e s s than 10°) can be expected. For example, s i l i c a g e l s t r o n g l y s c a t t e r s x-rays up to 10° 20 due to the e x i s t e n c e of d i s c r e t e p a r t i c l e s of 1-10 nm s i z e range. Warren observed no small angle s c a t t e r i n g with fused q u a r t z and concluded that the scheme of bonding was e s s e n t i a l l y continuous and f i t t e d the model of Zachariasen. N e v e r t h e l e s s , the s t r u c t u r e of n o n - c r y s t a l l i n e m a t e r i a l s i s s t i l l o n l y i ncompletely understood ( Z a r z y c k i , 1977). Since d i f f r a c t i o n s t u d i e s are i n s e n s i t i v e to f i n e d e t a i l s of s t r u c t u r e , they cannot be used to d i s t i n g u i s h between random network and m i c r o c r y s t a l l i t e models. High r e s o l u t i o n e l e c t r o n microscopy c o n s t i t u t e s an improvement over the d i f f r a c t i o n methods, but i t p r o v i d e s only two-dimensional i n f o r m a t i o n and t h e r e f o r e cannot p r o v i d e an u n e q u i v o c a l d i s t i n c t i o n between the two models. Acco r d i n g to Z a r z y c k i (1977), only by m o d e l l i n g can t h r e e - d i m e n s i o n a l i n f o r m a t i o n be o b t a i n e d and thus the s t r u c t u r e s of the d i s o r d e r e d s t a t e understood. Though the c o n t r o v e r s y i s f a r from r e s o l v e d , G a s k e l l (1977) b e l i e v e s that a referendum on the s u b j e c t of m i c r o c r y s t a l l i t e versus random network as the most a p p r o p r i a t e model f o r the s t r u c t u r e of " i d e a l " g l a s s e s would r e s u l t i n an overwhelming vote i n favor of the l a t t e r . 4."Polymorphism" i n n o n - c r y s t a l l i n e s o l i d s Polymorphism i s the a b i l i t y of a compound to c r y s t a l l i z e as more than one d i s t i n c t s p e c i e s ( H a l e b l i a n and McCrone, 3 8 1969). A c c o r d i n g t o Roy (1970), analogous "polymorphs" w i t h d i f f e r e n t n o n - c r y s t a l l i n e s o l i d s t r u c t u r e s a r e p o s s i b l e . For example, he r e p o r t s the p r e p a r a t i o n of r e a c t i o n amorphized, shear amorphized and r a d i a t i o n amorphized s i l i c o n d i o x i d e as w e l l as i t s p r e p a r a t i o n as a g l a s s , as a d e s i c c a t e d g e l and as a vapor d e p o s i t e d n o n - c r y s t a l l i n e s o l i d phase. Though he s t a t e s t h a t these phases have d i f f e r e n t p r o p e r t i e s , the d i f f e r e n c e s were not d i s c u s s e d . Roy (1970) a l s o s u g g e s t s t h a t n o n - c r y s t a l l i n e s o l i d phases w i t h minor d i f f e r e n c e s i n s t r u c t u r e seem p o s s i b l e . An example i s a g l a s s c o o l e d over a 30 day p e r i o d and a n o t h e r quenched i n 2-3 seconds over the same temperature range. A c c o r d i n g t o F i n n e y (1977) a v a r i e t y of amorphous s t r u c t u r e s a r e p o s s i b l e depending upon the p r e p a r a t i o n method, p r e s e n c e of i m p u r i t i e s and t h e n a t u r e of the m o l e c u l e s c o n c e r n e d . I n s p i t e of the m u l t i t u d e of s t r u c t u r e s , F i n n e y b e l i e v e s t h a t a l l amorphous s u b s t a n c e s can be t r e a t e d w i t h i n the same c o n c e p t u a l framework of a random network of l i n k e d m o l e c u l e s ( Z a c h a r i a s e n ' s m o d e l ) . P i k a l et a l . (1978) o b s e r v e d s i g n i f i c a n t l y d i f f e r e n t heat of s o l u t i o n v a l u e s between s p r a y d r i e d and f r e e z e d r i e d cefamandole n a f a t e . They b e l i e v e d t h a t the s p r a y d r i e d amorphous m a t e r i a l was an a n n e a l e d form of t h e amorphous m a t e r i a l and suggested t h e p o s s i b i l i t y of d i f f e r e n c e s i n s t r u c t u r e between d i f f e r e n t amorphous samples of t h e same compound. A c c o r d i n g t o H u t t e n r a u c h ' s (1978) concept of c r y s t a l l i n i t y of s o l i d s , w h i c h was d i s c u s s e d e a r l i e r , t h e 3 9 t r a n s i t i o n from the c r y s t a l l i n e t o the n o n - c r y s t a l l i n e s t a t e i s due to a gradual and continuous i n c r e a s e i n d i s o r d e r of the l a t t i c e . Thus there c o u l d be samples with e x t e n s i v e but not complete l a t t i c e breakdown. For example, spray d r i e d and f r e e z e d r i e d samples might both have a h i g h l y but not completely d i s o r d e r e d l a t t i c e . Small d i f f e r e n c e s i n t h e i r p r o p e r t i e s (e.g., i n e n t h a l p i e s of s o l u t i o n ) c o u l d be due to d i f f e r e n c e s i n the extent of l a t t i c e d i s o r d e r and i t i s unnecessary to invoke the concept of "polymorphism" i n the amorphous s t a t e . G. INTRODUCTION TO CALCIUM GLUCEPTATE Calcium g l u c e p t a t e i s used i n the treatment of c a l c i u m d e f i c i e n c y ( M a r t i n d a l e , 1982b). Complexed with technetium, i t i s used i n n u c l e a r medicine as an organ scanning agent (Chi et a l . , 1978). I t i s very s o l u b l e i n water (Suryanarayanan and M i t c h e l l , 1984) and i s p a r t i c u l a r l y u s e f u l i n the p r e p a r a t i o n of v e t e r i n a r y products c o n t a i n i n g a h i g h c a l c i u m c o n c e n t r a t i o n 1 . When f i r s t i n t r o d u c e d , c a l c i u m g l u c e p t a t e was an approximately equal mixture of the c a l c i u m s a l t s of D-glycero-D-gulo heptonic a c i d ( F i g . 4a) and D-glycero-D-ido heptonic a c i d ( F i g 4b). The mixture of a and 0 epimers was anhydrous and amorphous t o x-rays (Suryanarayanan and M i t c h e l l , 1981). Aqueous s o l u t i o n s (27%w/w) prepared with t h i s m a t e r i a l were s t o r e d f o r two years and were s t a b l e Product i n f o r m a t i o n of c a l c i u m g l u c e p t a t e , P f a n s t i e h l L a b o r a t o r i e s , Waukegan, IL, USA. 40 * C 0 0 • lcoo -1 1 H - *C - OH OH - * C - H 1 1 H -sc - OH H - S C - OH 1 Ca 1 OH - *c - H OH - "C - H 1 1 H - 5c - OH H - 5 C - OH 1 1 H - c - OH H - C - OH J 1 C H 2 0 H CH 2 OH 2 (a) (b) Ca c a l c i u m D-glycero-D-gulo- c a l c i u m D-glycero-D-ido-heptonate heptonate or c a l c i u m a-D-gluco- or c a l c i u m B-D-gluco-heptonate heptonate or c a l c i u m g l u c e p t a t e USP F i g . 4 S t r u c t u r e s o f (a) c a l c i u m a-glucoheptonate and (b) c a l c i u m B-glucoheptonate. ( P f a n s t i e h l , l o t 7311; Table V ) . Calcium g l u c e p t a t e became o f f i c i a l i n the USP i n 1976 (USP XIX, 1976) where i t was d e s c r i b e d as c a l c i u m a-glucoheptonate ( F i g . 4a). In order to comply with pharmacopeial s p e c i f i c a t i o n s , the manufacturers were r e q u i r e d to change t h e i r s y n t h e t i c and r e c r y s t a l l i z a t i o n procedures i n order to exclude the 0-form. T h i s a p p a r e n t l y r e s u l t e d i n an i n c r e a s e i n the r e l a t i v e p r o p o r t i o n of the a epimer (e.g., Givaudan, l o t R 3679 BA and I t a l s i n t e x , l o t R 1432 TJ, Table V), but the m a t e r i a l s t i l l c o n s i s t e d of a mixture of a and p epimers. S o l u t i o n s prepared with these m a t e r i a l s by Suryanarayanan and M i t c h e l l (1984) were not s t a b l e and p r e c i p i t a t e d w i t h i n 9 days. The p r e c i p i t a t e was found to be c r y s t a l l i n e c a l c i u m g l u c e p t a t e hydrate with 3 1/2 molecules of water of c r y s t a l l i z a t i o n . The 27%w/v aqueous s o l u t i o n s of c a l c i u m g l u c e p t a t e are s u p e r s a t u r a t e d with res p e c t t o c a l c i u m g l u c e p t a t e hydrate r e s u l t i n g i n the p r e c i p i t a t i o n of the hydrate. The Givaudan m a t e r i a l has an apparent water s o l u b i l i t y > 200% w/v ( i t s e q u i l i b r i u m s o l u b i l i t y c o u l d not be determined) while the e q u i l i b r i u m s o l u b i l i t y of the p r e c i p i t a t e d hydrate i s «* 3% w/v. M u l l e r et al . ( 1979) r e p o r t e d that the problem of p r e c i p i t a t i o n from s o l u t i o n was encountered from l a t e 1976 onwards (Givaudan, Table V ) . I t t h e r e f o r e appears t h a t approximately equal p r o p o r t i o n s of the a and 0 epimers are s t a b l e i n s o l u t i o n but that s t a b i l i t y decreases with the i n c r e a s e i n the r e l a t i v e p r o p o r t i o n of the a epimer. I t seems p o s s i b l e t h a t the 0 epimer has a s t a b i l i z i n g e f f e c t on the s o l u t i o n . When pure 42 Table V R e l a t i o n s h i p between the p r o p o r t i o n s of a and B epimers and the s t a b i l i t y o f aqueous s o l u t i o n s of c a l c i u m g l u c e p t a t e (27% w/v) s t o r e d a t room temperature (Suryanarayanan and M i t c h e l l , 1984). Calcium g l u c e p t a t e source Epimers i n c a l - P r e c i p i t a -cium g l u c e p t a t e , t i o n b e g i n s , C%) (days) a 0 P f a n s t i e h l , l o t 7311 52 41 S t a b l e Givaudan, l o t R 3679 BA 72 28 8 I t a l s i n t e x , l o t R 1432 T J 72 28 2 G i v a u d a n 3 77 23 4-6 months P f a n s t i e h l , l o t 12953-D 100 0 <1 a R e p o r t e d by M u l l e r e t a l . , 1979; these are f o r m u l a t i o n s and the i n i t i a l c a l c i u m g l u c e p t a t e c o n c e n t r a t i o n i s not g i v e n . 43 c a l c i u m a - g l u c e p t a t e became a v a i l a b l e from P f a n s t i e h l ( l o t 12953-D, Table V), aqueous s o l u t i o n s were found to be extremely unstable and p r e c i p i t a t e d w i t h i n a day of p r e p a r a t i o n . The r o l e of seed c r y s t a l s i n the p r e c i p i t a t i o n of c a l c i u m g l u c e p t a t e s o l u t i o n s has a l s o been s t u d i e d . E l e c t r o n m i c r o s c o p i c s t u d i e s by M u l l e r et al . ( 1979) r e v e a l e d that s o l u t i o n s prepared from l a t e 1976 onwards c o n t a i n e d seed c r y s t a l s of about 1 Sum s i z e which induced c r y s t a l l i z a t i o n . S o l u t i o n s prepared p r i o r to that time d i d not c o n t a i n the seed c r y s t a l s and were s t a b l e . Suryanarayanan and M i t c h e l l (1984) f i l t e r e d (0.22ym membrane f i l t e r ) c a l c i u m g l u c e p t a t e s o l u t i o n s and found that the time f o r p r e c i p i t a t i o n i n c r e a s e d , suggesting that f i l t r a t i o n excluded some but not a l l of the seed c r y s t a l s . When the s o l u t i o n s were f i l t e r e d through a 0.1 um f i l t e r and then examined i n a l a s e r l i g h t s c a t t e r i n g photon c o r r e l a t i o n s p e c t r o m e t e r 2 , there were no p a r t i c l e s of a measurable s i z e ( i . e . >5 nm i n r a d i u s ) suggesting that a l l the seed c r y s t a l s had been excluded. A u t o c l a v i n g at 121°C f o r 20 min r e s u l t e d i n s t a b l e s o l u t i o n s i n a l l cases, suggesting that a u t o c l a v i n g destroyed the seed c r y s t a l s . I t t h e r e f o r e seemed that both seed c r y s t a l s and the r e l a t i v e p r o p o r t i o n s of the a and p epimers played a r o l e i n the s t a b i l i t y of c a l c i u m g l u c e p t a t e s o l u t i o n s . 2 U n p u b l i s h e d work.. The experiments were conducted by Dr. T. Whateley, Department of Pharmacy, U n i v e r s i t y of S t r a t h c l y d e , Glasgow, UK. 4 4 More r e c e n t l y , c a l c i u m g l u c e p t a t e has been commercially a v a i l a b l e as a c r y s t a l l i n e hydrate (3 1/2 molecules of water of c r y s t a l l i z a t i o n ) . H o l s t e i n (1980) who patented the manufacturing procedure of c a l c i u m g l u c e p t a t e ( H o l s t e i n , 1962), r e p o r t s that p r e p a r a t i o n of c a l c i u m g l u c e p t a t e i n the amorphous form has become impossible due to the presence of seed c r y s t a l s or some other f a c t o r i n i t i a t i n g c r y s t a l l i z a t i o n . Holden and Singer (i960) d e s c r i b e d a s i m i l a r i n c i d e n t i n which ethylenediamine t a r t r a t e c r y s t a l s having been grown i n a f a c t o r y f o r a year without any problems were suddenly contaminated by the simultaneous growth of ethylenediamine t a r t r a t e monohydrate c r y s t a l s . C r y s t a l l i z a t i o n of the monohydrate was a t t r i b u t e d to seed c r y s t a l s of the monohydrate though i t was not known how the seed c r y s t a l s had suddenly formed. In the case of c a l c i u m g l u c e p t a t e , the commercially a v a i l a b l e m a t e r i a l has changed a b r u p t l y from an amorphous anhydrate with an apparent aqueous s o l u b i l i t y > 200% w/v, to a c r y s t a l l i n e hydrate with an e q u i l i b r i u m water s o l u b i l i t y of =*3% w/v. I t i s evident t h a t the d i f f e r e n c e i n c r y s t a l l i n i t y and/or the s t a t e of h y d r a t i o n i s r e s p o n s i b l e f o r t h i s dramatic change i n p r o p e r t i e s . I f c r y s t a l l i n e c a l c i u m g l u c e p t a t e hydrate can be dehydrated and rendered amorphous, i t s apparent s o l u b i l i t y can be expected to i n c r e a s e markedly and approach that of amorphous anhydrous c a l i u m g l u c e p t a t e . 4 5 H. OBJECTIVES The o b j e c t i v e s of t h i s i n v e s t i g a t i o n were: I. To c h a r a c t e r i z e the s o l i d - s t a t e p r o p e r t i e s of c a l c i u m g l u c e p t a t e obtained from d i f f e r e n t sources and at d i f f e r e n t times from the same source. 2 . To study the s o l i d - s t a t e and s o l u t i o n phase t r a n s i t i o n s of c a l c i u m g l u c e p t a t e and to prepare ' s t a b l e ' pharmaceutical s o l u t i o n s . 3 . To e v a l u a t e the v a r i o u s concepts of c r y s t a l l i n i t y of s o l i d s u s ing c a l c i u m g l u c e p t a t e as a model compound. 46 EXPERIMENTAL A. APPARATUS A u t o c l a v e , AMSCO g e n e r a l purpose, American S t e r i l i z e r Company. B i o q u e s t b i o l o g i c a l c a b i n e t , Beckton D i c k i n s o n . B o r o s i l i c a t e g l a s s t u b e s , Kimax, O w e n s - I l l i n o i s w i t h p o l y t e t r a f l u o r o e t h y l e n e - 1 i n e d screw cap. Cahn e l e c t r o b a l a n c e , Gram, V e n t r o n C o r p o r a t i o n . C o n s t a n t temperature b a t h , Magni W h i r l , B l u e M E l e c t r i c Company. D e n s i t y meter, DMA 45, Pa a r . D i f f e r e n t i a l i n t e r f e r e n c e c o n t r a s t m i c r o s c o p e , Model R, N i k o n . D i f f e r e n t i a l s c a n n i n g c a l o r i m e t e r w i t h e f f l u e n t gas a n a l y z e r , DSC-1B, P e r k i n Elmer. F r e e z e r (-76°C), UC 105, K e l v i n a t o r . F r e e z e d r y i n g u n i t , V i r t i s Company. Gas chromatograph w i t h a flame i o n i z a t i o n d e t e c t o r , model 5830 A, H e w l e t t P a c k a r d and a GC t e r m i n a l , model 18850 A, He w l e t t P a c k a r d . H o t - s t a g e , FP2, M e t t l e r . I n c u b a t o r , Isotemp, F i s h e r . M e c h a n i c a l agate mortar and p e s t l e , P u l v e r i s e t t e 2, F r i t s c h . Oven, S h e l - l a b model 22, She l d o n . pH meter, model 26, Radiometer. P o l a r i z i n g m i c r o s c o p e , S t a n d a r d 14, Z e i s s . P r o p o r t i o n a l t e m p e r a t u r e c o n t r o l l e r , model 76, YSI Company; w i t h v i n y l p robe, model 402, YSI Company. R o t a t i n g m i x e r , Dyna-mix, F i s h e r . S o l u t i o n c a l o r i m e t e r , model 1451, P a r r . 47 S t e r i f i l f i l t r a t i o n system, M i l l i p o r e . S urface area a n a l y z e r , Quantasorb S o r p t i o n System, Quantachrome. Syringe f i l t e r , o.22 um Nalgene, Nalge Company. Thermogravimetric a n a l y z e r , model 950, Du Pont, with d i f f e r e n t i a l thermal a n a l y z e r , model 900, Du Pont. Vacuum oven, N a t i o n a l A p p l i a n c e . Vacuum pump, Vac Torr S 35, General E l e c t r i c . V i a l s , R e a c t i - V i a l , P i e r c e . Water pump, R21, Haake. X-ray d i f f T a c t o m e t e r with a xenon p r o p o r t i o n a l counter, wide angle, P h i l i p s . B. MATERIALS A c e t o n i t r i l e , HPLC grade, Caledon. Amberlite IR-120 ion exchange r e s i n , M a l l i n c k r o d t . Ammonium c h l o r i d e , BDH. Calc J ium g l u c e p t a t e , P f a n s t i e h l , l o t 7311. C a l c i turn g l u c e p t a t e , Sigma, l o t 126C-0121. Calc : Lum g l u c e p t a t e , Givaudan,lot R 3679 BA. Cal c : Lum g l u c e p t a t e , I t a l s i n t e x , l o t R 1432 T J . Cal c : L u m g l u c e p t a t e , P f a n s t i e h l , l o t 12953-D. Calc : L u m g l u c e p t a t e , P f a n s t i e h l l o t 13313-E. Calc; L u m g l u c e p t a t e , P f a n s t i e h l , l o t 14772. Calc : L u m g l u c e p t a t e , P f a l t z and Bauer, l o t C01300. Calc : L u m g l u c e p t a t e , P r i n c e s s Margaret H o s p i t a l (PMH). Calc ium g l u c e p t a t e , Merck, ST-16944, RM-45940. Carbon t e t r a c h l o r i d e , ACS grade, BDH. C o l l o i d a l s i l i c o n d i o x i d e ( C a b - O - S i l ) , Cabot C o r p o r a t i o n . C u p r i c c h l o r i d e d i h y d r a t e , Matheson Coleman and B e l l . 3% Cyanopropylphenylmethyl s i l i c o n e (OV-225) on Chromosorb W(HP), 100-120 mesh, Western Chromatograp Ethylene dibromide, BDH. Formic a c i d , ACS grade, F i s h e r . a-D-Glucoheptonic a c i d 7-lactone, A l d r i c h . H y d r o c h l o r i c a c i d , ACS grade, A l l i e d Chemical. Indium, Goodfellow M e t a l s . L i t h i u m f l u o r i d e , F i s h e r . Magnesium c h l o r i d e hexahydrate, BDH. Methanol, HPLC grade, F i s h e r . 49 Methylene c h l o r i d e , HPLC grade, Caledon. Phosphoric a c i d , ACS grade, A l l i e d Chemical. Phosphorus pentoxide, ACS grade, BDH. Potassium a c e t a t e , BDH. Potassium carbonate, BDH. S i l i c a g e l , Davison Chemical. Sodium dichromate d i h y d r a t e , BDH. Sodium hydroxide, ACS grade, F i s h e r . Sodium n i t r i t e , A l l i e d Chemical. T r i m e t h y l s i l y l i m i d a z o l e i n p y r i d i n e (TRI-SIL 'Z'), P i e r c e . Tris(hydroxymethyl)aminomethane, P a r r . Water, d i s t i l l e d . Zinc s u l f a t e heptahydrate, ACS grade, BDH. 50 C. CHARACTERIZATION OF CALCIUM GLUCEPTATE Calcium g l u c e p t a t e ( P f a n s t i e h l , lot-13313 E; c r y s t a l l i n e hydrate c o n t a i n i n g 3 1/2 molecules of water of c r y s t a l l i z a t i o n ) , I, was used as r e c e i v e d or i t was d r i e d at 60°C, under vacuum (pressure < 130 Pa) f o r 16 h (USP XX, 1980c) to y i e l d the anhydrate, I I . For the phase t r a n s i t i o n s t u d i e s , i n a d d i t i o n to I and I I , x-ray amorphous, anhydrous c a l c i u m g l u c e p t a t e , I I I ( P f a n s t i e h l , l o t 12953-D) was a l s o used. The c h a r a c t e r i z a t i o n of I and II i s d e s c r i b e d i n the f o l l o w i n g pages. I l l was c h a r a c t e r i z e d e a r l i e r (Suryanarayanan and M i t c h e l l , 1984). The samples of c a l c i u m g l u c e p t a t e obtained from a l l other sources were used as r e c e i v e d to compare some of t h e i r s o l i d - s t a t e p r o p e r t i e s 1. Gas chromatography (GC) The USP monograph d e s c r i b e s c a l c i u m g l u c e p t a t e as c a l c i u m a-glucoheptonate (USP XX, 1980c) but the a n a l y t i c a l procedures f o r the i d e n t i f i c a t i o n and assay of c a l c i u m g l u c e p t a t e are incapable of d i s t i n g u i s h i n g between the c a l c i u m s a l t s of a and j3-D-glucoheptonic a c i d s . Suryanarayanan and M i t c h e l l (1984) obtained c a l c i u m g l u c e p t a t e samples from d i f f e r e n t commercial sources t h a t c o n s i s t e d of v a r y i n g p r o p o r t i o n s of c a l c i u m a-glucoheptonate and c a l c i u m 0-glucoheptonate. A GC technique was developed to separate and determine the r e l a t i v e p r o p o r t i o n s of these two epimers. T h e i r i d e n t i t y was 51 e s t a b l i s h e d by gas chromatography-mass spectrometry (GC-MS). Calcium g l u c e p t a t e s o l u t i o n was passed through a column of c a t i o n exchange r e s i n i n order to convert i t to a and /3-D-glucoheptonic a c i d s and the elua n t was l y o p h i l i z e d and repe a t e d l y t r e a t e d with c o n c e n t r a t e d h y d r o c h l o r i c a c i d . T h i s caused complete c o n v e r s i o n of the glucoheptonic a c i d s to t h e i r c o r r e s p o n d i n g 7-lactones (1,4-lactones) which were t r i m e t h y l s i l y l a t e d with t r i m e t h y l s i l y l i m i d a z o l e i n p y r i d i n e . There was a b a s e l i n e s e p a r a t i o n of the d e r i v a t i z e d a-D-glucoheptonic a c i d 7-lactone and /3-D-glucoheptonic 7-lactone which had r e t e n t i o n times of 9.2 and 7.7 min r e s p e c t i v e l y . GC-MS showed t h a t the above two compounds had s i m i l a r mass fragmentation p a t t e r n s . A commercially a v a i l a b l e r e f e r e n c e sample of a-D-glucoheptonic a c i d 7-lactone when sub j e c t e d t o GC-MS a n a l y s i s under the same c o n d i t i o n s , had a r e t e n t i o n time of 9.2 min and a l s o the same fragmentation p a t t e r n as the above two compounds. Hence, the i d e n t i t y of the compound e l u t i n g at 9.2 min was confirmed. Since the compound e l u t i n g at 7.7 min had a s i m i l a r mass fragmentation p a t t e r n , i t was assumed t o be the t r i m e t h y l s i l y l d e r i v a t i v e of 0-D-glucoheptonic a c i d 7-lactone. A l l samples of c a l c i u m g l u c e p t a t e were analyzed using t h i s GC procedure. 52 2. Powder x-ray d i f f r a c t i o n Samples were exposed t o N i - f i l t e r e d CuKa r a d i a t i o n (36 kV x 16 mA) at a scanning r a t e of 1°20 min" 1 i n a wide angle x-ray d i f f T a c t o m e t e r over a range of 26 from 10° to 40°. 3. Thermal methods 3.1 Thermogravimetric a n a l y s i s ( T G A ) The weight l o s s on h e a t i n g I up to 140°C at 10°C min" 1 was determined by TGA. 3.2 D i f f e r e n t i a l scanning c a l o r i m e t r y (DSC) Samples of about 1 to 5 mg were weighed on an e l e c t r o b a l a n c e d i r e c t l y i n t o aluminum sample pans. Scans were made at v a r i o u s r a t e s u s i n g standard (open) pans, v o l a t i l e ( c l o s e d ) pans and v o l a t i l e pans with a 0.1 to 0.2 mm p i n h o l e . V a p o r i z a t i o n of the water of c r y s t a l l i z a t i o n from the standard pans and the v o l a t i l e pans with a p i n h o l e was d e t e c t e d u s i n g the e f f l u e n t gas a n a l y z e r and was estimated q u a n t i t a t i v e l y by weighing the pan a f t e r each endothermic peak. The c a l o r i m e t e r was c a l i b r a t e d with samples of indium. In a l l cases, the peak temperature i . e . the p o i n t on the temperature s c a l e of maximum d e v i a t i o n from the b a s e l i n e was r e p o r t e d . The temperatures at which phase t r a n s i t i o n s o c c u r r e d depended on the scanning r a t e and a l l r e p o r t e d t r a n s i t i o n temperatures are f o r a scanning r a t e of 10°C m i n - 1 . 53 3 . 3 Thermomicroscopy Samples were mounted dry or i n m i n e r a l o i l on a g l a s s s l i d e and heated on a hot stage while being observed under a microscope. The temperatures at which t r a n s i t i o n s o c c u r r e d were recorded. 4. E q u i l i b r i u m s o l u b i l i t y Excess of I was added to 20 mL of water c o n t a i n e d i n a 100 mL v o l u m e t r i c f l a s k . A few such f l a s k s were m e c h a n i c a l l y r o t a t e d i n water baths set at 25.5, 31.5, 34.0 and 37°C. A l i q u o t s were drawn p e r i o d i c a l l y i n t o a warm s y r i n g e , membrane f i l t e r e d , weighed and d i l u t e d with a known weight of water. The d e n s i t y of the d i l u t e d s o l u t i o n s was determined i n a d e n s i t y meter t h e r m o s t a t i c a l l y c o n t r o l l e d at 25°C. D e t a i l s of the c a l i b r a t i o n of the d e n s i t y meter are given i n s e c t i o n F.3 (p.65) of Ex p e r i m e n t a l . A standard curve was p l o t t e d by p r e p a r i n g s o l u t i o n s of I i n the c o n c e n t r a t i o n range of 0.009 to 0.055 molal and determining t h e i r d e n s i t i e s , F i g . 5 . The c o n c e n t r a t i o n of I i n the d i l u t e d a l i q u o t s was determined from the standard curve. E q u i l i b r i u m was assumed when s u c c e s s i v e a l i q u o t s showed no d i f f e r e n c e i n the c o n c e n t r a t i o n of I. Because of the observed l i n e a r r e l a t i o n s h i p between c o n c e n t r a t i o n and d e n s i t y , the i n t e r c e p t on the y - a x i s ( F i g . 5) should be equal t o the d e n s i t y of water at 25°C. The 54 0 20 40 60 CONCENTRATION, molal x 1000 F i g . 5 Standard curve r e l a t i n g the c o n c e n t r a t i o n o f aqueous s o l u t i o n s o f I w i t h t h e i r d e n s i t i e s . 55 experimental i n t e r c e p t of 0.9970 g cm" 3 was c l o s e t o the r e p o r t e d d e n s i t y of 0.9971 g cm" 3 f o r water at 25°C (CRC Handbook, 1983). D. TREATMENT OF CALCIUM GLUCEPTATE I and II were s u b j e c t e d to the f o l l o w i n g treatments to study p o s s i b l e phase t r a n s i t i o n s . 1. G r i n d i n g Both I and II were ground i n a mechanical agate mortar and p e s t l e f o r v a r y i n g times. I was ground f o r up to 1 h and II f o r up to 4 h. The temperature r i s e i n the sample duri n g g r i n d i n g was not measured, but f o r long g r i n d i n g times, the machine was stopped i n t e r m i t t e n t l y t o l i m i t any temperature r i s e . 1.1 E f f e c t of g r i n d i n g on apparent s o l u b i l i t y Attempts to determine the maximum s o l u b i l i t i e s of unground and ground II were u n s u c c e s s f u l ( s e c t i o n A.5 of R e s u l t s and D i s c u s s i o n , p.82). Hence, the apparent s o l u b i l i t i e s of unground and ground I and II were determined. Each sample was added to about 0.5 mL of d i s t i l l e d water i n a c u l t u r e tube and d i s s o l v e d by v i g o r o u s shaking a t room temperature (*22°C) using a vortex mixer. At the f i r s t s i g n of p e r s i s t e n t t u r b i d i t y , the a d d i t i o n of s o l i d was stopped and the s o l u t i o n was weighed. The water was evaporated o f f at 60°C 56 under vacuum u n t i l the r e s i d u a l s o l i d reached a constant weight. I t i s recognized that the apparent s o l u b i l i t y i s a k i n e t i c p r o p e r t y and that the values of apparent s o l u b i l i t y o b t a i n e d would depend on the experimental method. While determining the apparent s o l u b i l i t y of I I , the f i r s t s i g n of p e r s i s t e n t t u r b i d i t y c o u l d be due t o : ( i ) the s o l u t i o n becoming s a t u r a t e d with II and excess II remaining u n d i s s o l v e d and/or ( i i ) the s o l u t i o n s becoming s u p e r s a t u r a t e d with r e s p e c t to I r e s u l t i n g i n i t s p r e c i p i t a t i o n . The second p o s s i b i l i t y seems more l i k e l y because the appearence of t u r b i d i t y was f o l l o w e d immediately by copious p r e c i p i t a t i o n of I. 1.2 E f f e c t o f g r i n d i n g on s u r f a c e a r e a About 100 mg of each sample was a c c u r a t e l y weighed i n t o the sample c e l l of a s u r f a c e area a n a l y z e r . The s p e c i f i c s u r f a c e area was determined by the m u l t i p o i n t BET method (Lo w e l l , 1973) using 0.072, 0.104 and 0.184 mol percent krypton (adsorbate) i n helium ( c a r r i e r ) . 1.3 E f f e c t o f g r i n d i n g on powder x - r a y d i f f r a c t i o n p a t t e r n The ground samples were su b j e c t e d to powder x-ray d i f f r a c t i o n s t u d i e s under c o n d i t i o n s d e s c r i b e d i n s e c t i o n C.2 (p.52) of E x p e r i m e n t a l . 57 2. Freeze d r y i n g About 1 g of I was d i s s o l v e d i n 50 mL of water, the s o l u t i o n was f r o z e n at -76°C and d r i e d i n a f r e e z e d r y e r . The f r e e z e d r i e d m a t e r i a l was s t o r e d i n a g l a s s d e s i c c a t o r c o n t a i n i n g phosphorus pentoxide u n t i l used. 3. Constant humidity s t u d i e s Chambers of constant r e l a t i v e humidity (RH) ranging from 9 to 90% were obtained by p r e p a r i n g s a t u r a t e d aqueous s o l u t i o n s of phosphoric a c i d (9% RH), -potassium a c e t a t e (20% RH), magnesium c h l o r i d e hexahydrate (33% RH), potassium carbonate (43% RH), sodium dichromate d i h y d r a t e (52% RH), sodium n i t r a t e (66% RH), c u p r i c c h l o r i d e d i h y d r a t e (68% RH), ammonium c h l o r i d e (79% RH) and z i n c s u l f a t e heptahydrate (90% RH) i n g l a s s chambers ( d e s i c c a t o r s ) . A l l the g l a s s chambers were s t o r e d i n an incubator at 25°C. Phosphorus pentoxide was used to o b t a i n a chamber of 0% RH. A c c u r a t e l y weighed amounts of I, II and I I I were p l a c e d i n each chamber. The weight changes were monitored p e r i o d i c a l l y u n t i l the samples a t t a i n e d constant weight. The percent weight change was then c a l c u l a t e d and the s o l i d phase i d e n t i f i e d by powder x-ray d i f f r a c t i o n . E. PREPARATION OF STABLE CALCIUM GLUCEPTATE SOLUTIONS Table V shows that a 27% w/v s o l u t i o n of c a l c i u m g l u c e p t a t e i n water prepared with one p a r t i c u l a r sample of c a l c i u m g l u c e p t a t e ( P f a n s t i e h l , l o t 7311) was s t a b l e d u r i n g two years of storage. Our o b j e c t i v e was to use I to prepare s t a b l e aqueous s o l u t i o n s of ca l i u m g l u c e p t a t e of s i m i l a r c o n c e n t r a t i o n s . T h i s was not p o s s i b l e because of the low aqueous s o l u b i l i t y of I at room temperature (see s e c t i o n A.5 i n R e s u l t s and D i s c u s s i o n , p.80). However, II c o u l d be s u c c e s s f u l l y used to prepare these s o l u t i o n s ( s e c t i o n A.5 i n R e s u l t s and D i s c u s s i o n ) but the s o l u t i o n s were h i g h l y u n s t a b l e and p r e c i p i t a t e d on storage. Suryanarayanan and M i t c h e l l (1984) r e p o r t e d that membrane f i l t r a t i o n i n c r e a s e d the time before p r e c i p i t a t i o n o c c u r r e d while a u t o c l a v i n g r e s u l t e d i n s t a b l e c a l c i u m g l u c e p t a t e s o l u t i o n s . S e v e r a l s o l u t i o n s c o n t a i n i n g between 20 and 27% w/v II i n water were prepared. Some were f i l t e r e d through a f i l t e r paper (Whatman Number 1) while o t h e r s were membrane f i l t e r e d (0.22 Mm). The c o n t r o l batch was l e f t u n f i l t e r e d . A l l the s o l u t i o n s were a u t o c l a v e d at 121°C f o r 20 min. F. DETERMINATION OF DEGREE OF CRYSTALLINITY OF ANHYDROUS CALCIUM GLUCEPTATE Degree of c r y s t a l l i n i t y d e t e r m i n a t i o n s were c a r r i e d out only on ground samples of I I . For a q u a n t i t a t i v e d e t e r m i n a t i o n of percent c r y s t a l l i n t y , i t i s necessary that 100% c r y s t a l l i n e and 0% c r y s t a l l i n e (amorphous) r e f e r e n c e standards be s e l e c t e d . Unground II was chosen as the c r y s t a l l i n e standard (100% c r y s t a l l i n i t y ) and II ground f o r 4 h was used as the amorphous standard (0% c r y s t a l l i n i t y ) . T h e i r powder x-ray d i f f r a c t i o n p a t t e r n s are shown i n F i g . 6a and F i g . 6b 5 9 r e s p e c t i v e l y . 1. Powder x-ray d i f f r a c t i o n The powder x-ray d i f f r a c t i o n c o n d i t i o n s are d e s c r i b e d i n s e c t i o n C.2 (p.52) of Experimental. Around a 26 value of 20°, II i n t e n s e l y d i f f r a c t s x-rays, and the peak at 20.1°, which e x h i b i t e d the l a r g e s t d e v i a t i o n from b a s e l i n e , was chosen f o r c r y s t a l l i n i t y c a l c u l a t i o n s ( F i g . 6a). V a r i o u s p r o p o r t i o n s of the c r y s t a l l i n e and amorphous standards were mixed to y i e l d r e f e r e n c e samples of known c r y s t a l l i n i t y from 0% to 100%. To an a c c u r a t e l y weighed amount of each mixture, 11% w/w l i t h i u m f l u o r i d e was added as an i n t e r n a l standard, mixed w e l l and the mixture r e d r i e d at 60°C under vacuum to constant weight (Imaizumi et al . , 1980). The r a t i o of the x-ray d i f f r a c t i o n i n t e n s i t y of II at 20.1° 26 to that of l i t h i u m f l u o r i d e at 45.0° 26 was c a l c u l a t e d f o r the re f e r e n c e samples of known c r y s t a l l i n i t y . T h i s r a t i o was p l o t t e d as a f u n c t i o n of percent c r y s t a l l i n i t y and used as a standard curve ( F i g . 7 ) . F i g . 8 i s a r e p r e s e n t a t i v e x-ray d i f f r a c t i o n p a t t e r n of a 20% c r y s t a l l i n e sample prepared by mixing a p p r o p r i a t e weights of the c r y s t a l l i n e and amorphous r e f e r e n c e standards. To i n v e s t i g a t e the e f f e c t of g r i n d i n g time on the degree of c r y s t a l l i n i t y , II was ground f o r v a r y i n g p e r i o d s of time. To an a c c u r a t e l y weighed ground sample, 11% w/w l i t h i u m f l u o r i d e was added, the mixture was r e d r i e d and the r a t i o of x-ray d i f f r a c t i o n i n t e n s i t i e s determined as above. The degree F i g . 6 Powder x-ray d i f f r a c t i o n p a t t e r n s o f (a) I I (100% c r y s t a l l i n e standard) and (b) I I ground f o r 4 h (0% c r y s t a l l i n e s t a n d a r d ) . I 25 50 i 75 100 DEGREE OF CRYSTALLINITY, %w/w S t a n d a r d c u r v e r e l a t i n g t h e degree o f c r y s t a l l i n i t y o f I I and i n t e n s i t y r a t i o o f t h e x - r a y d i f f r a c t i o n peak o f I I a t 2 0 . 1 20 t o t h a t o f l i t h i u m f l u o r i d e a t 4 5 20. Mean ± s t a n d a r d d e v i a t i o n a r e shown f o r s e l e c t e d v a l u e s ; o t h e r v a l u e s a r e averages o f two d e t e r m i n a t i o n s . 62 F i g . 8 Powder x-ray d i f f r a c t i o n pattern of 20% c r y s t a l l i n e II containing 11% w/w lithium f l u o r i d e as the i n t e r n a l standard. 63 of c r y s t a l l i n i t y at each g r i n d i n g time was determined from the standard curve. C a l c u l a t i n g the degree of c r y s t a l l i n i t y from an x-ray d i f f r a c t i o n p a t t e r n u s u a l l y i n v o l v e s measuring the t o t a l area under the curve and s u b t r a c t i n g the c o n t r i b u t i o n due to amorphous s c a t t e r i n g . T h i s method i s a r b i t r a r y because of the inherent d i f f i c u l t y i n s e p a r a t i n g the amorphous s c a t t e r i n g from the c r y s t a l l i n e d i f f r a c t i o n . For example, Black and L o v e r i n g (1977) found t h a t a d i g o x i n sample judged to be 100% c r y s t a l l i n e by p o l a r i z e d - l i g h t microscopy was o n l y 50% c r y s t a l l i n e a c c o r d i n g to the above method of c a l c u l a t i n g c r y s t a l l i n i t y . They a t t r i b u t e d the low value to o v e r l a p of c r y s t a l l i n e peaks and proceeded to assume that the sample was 100% c r y s t a l l i n e . The method of Imaizumi et al. (1980) s u c c e s s f u l l y overcomes the problem by adding an i n t e r n a l standard and c a l c u l a t i n g i n t e n s i t y r a t i o s r a t h e r than the areas under the curve. The e f f e c t s of f a c t o r s l i k e background s c a t t e r i n g need not be c o n s i d e r e d because the experimental c r y s t a l l i n i t y was determined from the standard curve ( F i g . 7 ) . L i t h i u m f l u o r i d e was chosen as an i n t e r n a l standard because: (a) i t s x-ray d i f f r a c t i o n peaks d i d not i n t e r f e r e with those of I I , (b) i t belongs to the h i g h l y symmetrical c u b i c c r y s t a l system and t h e r e f o r e has only few but intense d i f f r a c t i o n peaks and (c) i t s l i n e a r a b s o r p t i o n c o e f f i c i e n t i s 31.3 cm"1 compared to 35.3 cm"1 f o r II ( c a l c u l a t i o n s i n Appendix I ) . From m i c r o s c o p i c examination, p a r t i c l e s of 64 l i t h i u m f l u o r i d e were found to be of l e s s than 5 um s i z e . P a r t i c l e s of II were a l s o extremely small i n s i z e ( c a l c u l a t e d h y p o t h e t i c a l p a r t i c l e s i z e given i n Table XI, p.106). The s i m i l a r l i n e a r a b s o r p t i o n c o e f f i c i e n t values of II and l i t h i u m f l u o r i d e and the f a c t that the p a r t i c l e s i z e of both phases was very small suggested that the i n t e n s i t y of d i f f r a c t e d r a d i a t i o n would be n e g l i g i b l y a f f e c t e d by m i c r o a b s o r p t i o n ( C u l l i t y , 1978d). The d e n s i t y of l i t h i u m f l u o r i d e i s 2.640 g cm" 3 (Merck Index, 1983a) .while that of II i s 1.662 g cm - 3 (Table XI, p.106). I d e a l l y , the compound under i n v e s t i g a t i o n and the i n t e r n a l standard should have s i m i l a r d e n s i t i e s so that mixtures remain homogenous a f t e r mixing. In an attempt to e l i m i n a t e the e f f e c t of p a r t i c l e s i z e on the i n t e n s i t y of d i f f r a c t e d peaks, the samples were i n i t i a l l y s i e v e d and a -100 +250 s i e v e f r a c t i o n used f o r x-ray a n a l y s i s . However, examination using a scanning e l e c t r o n microscope showed that a f t e r g r i n d i n g , the powder c o n s i s t e d of aggregates of p a r t i c l e s and s i e v i n g was d i s c o n t i n u e d . The i n t e n s i t y of the d i f f r a c t i o n peaks was u n a f f e c t e d by the method of packing the powders i n t o the sample h o l d e r , showing t h a t the p a r t i c l e s d i d not e x h i b i t a p r e f e r r e d o r i e n t a t i o n . 2. S o l u t i o n c a l o r i m e t r y Heats of s o l u t i o n were determined at room temperature (=*22°C) u s i n g a s o l u t i o n c a l o r i m e t e r , with d i s t i l l e d water as the s o l v e n t . The energy e q u i v a l e n t of the c a l o r i m e t e r and i t s 65 con t e n t s was determined b y ^ d i s s o l v i n g an a c c u r a t e l y weighed amount of tris(hydroxymethyl)aminomethane i n 100 g of 0.1 M h y d r o c h l o r i c a c i d and measuring the temperature change. The c a l o r i m e t e r was s t a n d a r d i z e d as d e s c r i b e d p r e v i o u s l y (Suryanarayanan and M i t c h e l l , 1984). The c r y s t a l l i n e and amorphous standards were mixed i n v a r i o u s p r o p o r t i o n s t o g i v e samples of known percent c r y s t a l l i n i t y as b e f o r e , and t h e i r heats of s o l u t i o n determined. A standard curve was p l o t t e d of the heat of s o l u t i o n as a f u n c t i o n of percent c r y s t a l l i n i t y ( F i g . 9 ) . Samples of II ground f o r d i f f e r e n t times were r e d r i e d at 60°C under vacuum to constant weight before t h e i r heat of. s o l u t i o n v a l u e s were measured and the percent c r y s t a l l i n i t i e s were determined from the standard curve. The f i n a l s o l u t i o n , c o n c e n t r a t i o n s were normally l e s s than 0.01 M. 3. Suspension d e n s i t y method Carbon t e t r a c h l o r i d e and e t h y l e n e dibromide were chosen as the suspending l i q u i d s because n e i t h e r of them r e a c t e d with nor d i s s o l v e d I I . The d e n s i t y of carbon t e t r a c h l o r i d e was lower than that of I I , while that of ethylene dibromide was h i g h e r . The two l i q u i d s were mixed i n v a r y i n g p r o p o r t i o n s u n t i l a mixture was ob t a i n e d with approximately the same d e n s i t y as I I . Samples of II ground f o r d i f f e r e n t times were r e d r i e d at 60°C under vacuum to constant weight. Each sample was 66 DEGREE OF CRYSTALLINITY, %w/w Fig.9 Standard curve r e l a t i n g the degree of c r y s t a l l i n i t y and the heat of solution of II i n water at room temperature (-22°C). The mean ± standard deviation of 100% and 0% c r y s t a l l i n e standards (n=4) are shown. 67 t r a n s f e r r e d t o a Quantasorb sample c e l l , and a stream of n i t r o g e n passed over i t at room temperature f o r 4 h to dry the powder and remove surface i m p u r i t i e s . The c e l l i s a u t o m a t i c a l l y sealed on d i s c o n n e c t i o n from the Quantasorb S o r p t i o n System so that the contents do not come i n c o n t a c t with the atmosphere. The c e l l was then t r a n s f e r r e d to a "dry chamber" (maintained at < 1% RH with phosphorus p e n t o x i d e ) . Here the s o l i d was d i s p e r s e d i n the carbon t e t r a c h l o r i d e -ethylene dibromide mixture c o n t a i n e d i n a b o r o s i l i c a t e g l a s s tube. The tube was c l o s e d t i g h t l y with a p o l y t e t r a f l u o r o e t h y l e n e - l i n e d screwcap and t r a n s f e r r e d to a jacketed c e l l c o n t a i n i n g water ( F i g . 10). Water was pumped from a t h e r m o s t a t i c a l l y c o n t r o l l e d water bath through the double-wall of the c e l l and then through the outer jack e t surrounding the o s c i l l a t i n g tube of a d i g i t a l d e n s i t y meter. The temperature of the water bath was a l t e r e d u n t i l the d i s p e r s e d sample was suspended. A sample of the pure l i q u i d mixture was immediately i n j e c t e d i n t o the o s c i l l a t i n g tube of the d e n s i t y meter. A f t e r e q u i l i b r a t i o n ( d e n s i t y readings constant t o ±0.0001 g cm' 3), the d e n s i t y of the suspension l i q u i d gave the d e n s i t y of the suspended s o l i d . Before using the suspension c e l l to determine the d e n s i t y of samples of I I , the d e n s i t y meter was c a l i b r a t e d at 20°C with a i r and d i s t i l l e d water and i t s accuracy was checked by measuring the d e n s i t y of a number of l i q u i d s and comparing the experimental with the l i t e r a t u r e v a l u e s . E x p e r i m e n t a l l y determined d e n s i t y v a l u e s ( i n g cm" 3) were: a c e t o n i t r i l e 0.7823 [0.7822 (Lange's 68 WATER BATH WITH PROPORTIONAL TEMPERATURE CONTROLLER TEST TUBE WITH SUSPENDING LIQUID AND SOLD SAMPLE DENSITY METER JACKETED BEAKER CONTAINING DISTILLED WATER OSCILLATING TUBE OF DENSITY METER F i g . 10 Schematic diagram of apparatus for suspension density determinations. Arrows indicate the di r e c t i o n of water flow. 69 Handbook of Chemistry, 1979a)], methanol 0.7920 [0.7915 (Merck Index, 1983b)] and methylene c h l o r i d e 1.3255 [1.3255 (Merck Index, 1983c)], which agree c l o s e l y with the l i t e r a t u r e v a l u e s given i n parentheses. The d e n s i t i e s of the r e f e r e n c e l i q u i d s were f i r s t determined with the flow of water i n the d i r e c t i o n shown i n F i g . 10, then the d i r e c t i o n of flow was r e v e r s e d . The d e n s i t i e s were independent of the d i r e c t i o n of flow, showing that there was no s i g n i f i c a n t d i f f e r e n c e i n the temperature between the suspension c e l l and the d e n s i t y meter. The temperature of the water bath was a c c u r a t e l y c o n t r o l l e d to ±0.005°C by means of a p r o p o r t i o n a l temperature c o n t r o l l e r . P r e c i s e temperature c o n t r o l was necessary because a decrease i n c r y s t a l l i n i t y from 100% to 0% caused a change i n suspension temperature of only about 7°C. The d e n s i t y v a l u e s of II ground f o r v a r y i n g time p e r i o d s were determined and the degree of c r y s t a l l i n i t y c a l c u l a t e d using Eq. (6) d i s c u s s e d i n s e c t i o n C.3 (p.15) of I n t r o d u c t i o n . The r e p o r t e d percent c r y s t a l l i n i t y v a l u e s of ground samples (Table XI) are averages of three d e t e r m i n a t i o n s . 4. D i f f e r e n t i a l scanning calorimetry(DSC) U n l i k e the above three techniques which were used to q u a n t i t a t e the c r y s t a l l i n i t y of ground I I , DSC was used q u a l i t a t i v e l y to determine the g r i n d i n g time that causes the complete disappearance of the m e l t i n g endotherm. For these s t u d i e s , standard pans were used and the he a t i n g r a t e was 10°C m in" 1. 5 . P o l a r i z e d l i g h t microscopy Samples of amorphous anhydrous ca l c i u m g l u c e p t a t e ( I I I ) , unground II and II ground f o r v a r y i n g times were mounted i n m i n e r a l o i l and examined by means of a p o l a r i z i n g microscope f o r b i r e f r i n g e n c e . 71 RESULTS AND DISCUSSION A. CHARACTERIZATION OF CALCIUM GLUCEPTATE 1. Gas chromatographic a n a l y s i s GC a n a l y s i s of I by the method of Suryanarayanan and M i t c h e l l ( 1 9 8 4 ) gave a s i n g l e compound with a r e t e n t i o n time of 9.2 min. I t t h e r e f o r e c o n s i s t e d of c a l c i u m a-glucoheptonate only and thus complied with the USP XX s p e c i f i c a t i o n s . The same r e t e n t i o n time was ob t a i n e d f o r II showing that d r y i n g at 60°C under vacuum f o r 16 h d i d not cause any d e t e c t a b l e sample d e g r a d a t i o n . 2. Powder x-ray d i f f r a c t i o n The powder x-ray d i f f r a c t i o n p a t t e r n s of I and II are shown i n F i g . 15a (p.90) and 6a (p.60) r e s p e c t i v e l y . II was obt a i n e d from I by de h y d r a t i o n (see s e c t i o n C i n E x p e r i m e n t a l ) . A c c o r d i n g to Carstensen (1973a), there are three p o s s i b l e s o l i d - s t a t e s a f t e r d e h y d r a t i o n : ( i ) the c r y s t a l l a t t i c e i s i d e n t i c a l to t h a t of the o r i g i n a l hydrate, ( i i ) the r e s i d u e has a d i f f e r e n t c r y s t a l l a t t i c e and ( i i i ) the dehydrated m a t e r i a l i s amorphous. Since I and II d i f f r a c t x-rays at n e a r l y the same 26 v a l u e s (Table V I ) , i t i s apparent that the c r y s t a l l a t t i c e of c r y s t a l l i n e c a l c i u m g l u c e p t a t e remains e s s e n t i a l l y unchanged on d e h y d r a t i o n . T a b l e VI Powder x-ray d i f f r a c t i o n data l i s t i n g the e i g h t most i n t e n s e l i n e s o f I f the i n t e n s i t i e s o f the corresponding l i n e s of I I and the e i g h t most i n t e n s e l i n e s o f the p r e c i p i t a t e o b t a i n e d from a s o l u t i o n of I I . I I P r e c i p i t a t e from a solu-t i o n o f I I I n t e r p l - R e l a t i v e I n t e r p l - R e l a t i v e I n t e r p l - R e l a t i v e anar i n t e n s i t y , anar i n t e n s i t y , anar i n t e n s i t y , s p a c i n g , d(A) I / I 0 ( % ) s p a c i n g , d(A) I / I 0 ( % ) s p a c i n g , d(A) I / I 0 ( % ) 2.43 31 2.43 28 2.43 28 2.98 27 2.96 23 2.97 22 3.35 40 3.34 34 3.35 34 3.85 44 3.83 56 3.83 46 3.90 31 3.88 23 3.90 18 4.13 38 4.11 47 4.13 34 4.33 100 4.31 100 4.31 100 5.34 27 5.37 36 5.33 33 a The e i g h t most i n t e n s e l i n e s o f I I do not correspond w i t h the e i g h t most i n t e n s e peaks of I but, except f o r minor d i f f e r e n c e s , the powder x-ray d i f f r a c t i o n p a t t e r n s o f I and I I are v e r y s i m i l a r (see Suryanarayanan and M i t c h e l l , 1984) . 73 3. Thermogravimetric a n a l y s i s The weight l o s s on h e a t i n g I up to 140°C was 11.3% w/w. Ac c o r d i n g to the USP (USP XX, 1980c), c a l c i u m g l u c e p t a t e can e x i s t as an anhydrate or as a hydrate c o n t a i n i n g 2 molecules or 3 1/2 molecules of water of c r y s t a l l i z a t i o n . The observed weight l o s s was c l o s e t o the t h e o r e t i c a l value of 11.4% w/w f o r the complete dehydration of c a l c i u m g l u c e p t a t e hydrate c o n t a i n i n g 3 1/2 molecules of water of c r y s t a l l i z a t i o n . Attempts to determine the water content of I and II by the K a r l F i s c h e r method were u n s u c c e s s f u l . A number of d i f f e r e n t s o l v e n t s ( e . g . , dimethyl s u l f o x i d e , p y r i d i n e ) were used t o d i s s o l v e the s o l i d s but a s a t i s f a c t o r y t i t r a t i o n end po i n t was not obtained with any of them. 4. D i f f e r e n t i a l scanning c a l o r i m e t r y and thermomicroscopy When I was heated i n standard pans, there were two endotherms at * 121 °C and =*144°C with a weight l o s s a f t e r the f i r s t endotherm of ==11.2% w/w which was c l o s e to the weight l o s s o b t a i n e d by TGA ( F i g . 11a). Since t h i s endotherm was accompanied by a peak i n the e f f l u e n t gas a n a l y z e r , i t must be due t o simultaneous d e h y d r a t i o n and v a p o r i z a t i o n of water (Scheme l a ) . The second endotherm caused no change i n e f f l u e n t gas c o n c e n t r a t i o n and was most l i k e l y due t o m e l t i n g of the anhydrate. Thermomicroscopy of I mounted dry or i n m i n e r a l o i l , confirmed that m e l t i n g o c c u r r e d at =*145°C. Based on GC, x-ray and thermal analyses (TGA, DSC and thermomicroscopy), I was i d e n t i f i e d as c r y s t a l l i n e c a l c i u m g l u c e p t a t e hydrate c o n t a i n i n g 3 1/2 molecules of water of c r y s t a l l i z a t i o n . Heating I i n v o l a t i l e pans with or without a p i n h o l e r e s u l t e d i n thermal curves that were markedly d i f f e r e n t from those obtained by h e a t i n g i n standard pans. Because of the u n r e s t r i c t e d nature of standard pans, a hydrate can l o s e water at any temperature where the vapor p r e s s u r e of water surrounding the s o l i d i s l e s s than the e q u i l i b r i u m vapor pressure of the hydrate at that temperature. The d r i f t i n g b a s e l i n e of the dehydration peak ( F i g . 11a) suggests a gradual water l o s s when I i s heated i n standard pans. When I was heated i n a v o l a t i l e pan, there was only one endotherm at =<117°C . T h i s i s c l o s e to the temperature of dehydration of I i n standard pans. The endotherm c o u l d t h e r e f o r e be due to d e h y d r a t i o n . The water l i b e r a t e d on dehydration cannot escape from the v o l a t i l e pans, and probably d i s s o l v e s some of the dehydrated s o l i d . Since the d i s s o l u t i o n of the anhydrate i n water i s an endothermic r e a c t i o n (see F i g . 9 and Table X I ) , the endotherm at <*117°C w i l l a l s o i n c l u d e the heat of s o l u t i o n of the reaction(Scheme l b ) . The pan now c o n t a i n s the anhydrate as w e l l as an aqueous s o l u t i o n of the anhydrate. The presence of the l a t t e r seems to prevent the anhydrate from m e l t i n g , due t o some unknown reason. 7 5 SO 130 LU I -f X LU 0 . >-o DC LU Z H I LU I 170 (b) mmm t I 90 130 170 (d) 60 90 130 170 TEMPERATURE CO F i g . 11 R e p r e s e n t a t i v e thermal curves o f I (a) standard pan, (b) v o l a t i l e pan, (c) v o l a t i l e pan w i t h a p i n h o l e and (d) v o l a t i l e pan w i t h a p i n h o l e under reduced p r e s s u r e . 76 peak tempe-r a t u r e a standard CaGl-3%H 20 ^ C a G l ( s ) + 3JsH 20(g) 119-122°C CaGlCs) - C a G l ( l ) 144-145°C b v o l a t i l e CaGl'3JsH 20 ^ C a G l ( s ) + C a G l ( s o ) 116-119°C pan + H 20(g) c v o l a t i l e CaGl«3JjH 20 ^ C a G l ( s ) + 3JjH 20(g) 115-124°C pan w i t h p i n h o l e d v o l a t i l e CaGl-3%H 20 ^ C a G l ( s ) + 3JsH 20(g) 107-118°C pan w i t h C a G 1 ( s ) - C a G l ( l ) 145-148°C p i n h o l e (under vacuum) Scheme I Dehydration r e a c t i o n s and t r a n s i t i o n s o f I i n the d i f f e r e n t i a l scanning c a l o r i m e t e r ; CaGl = c a l c i u m g l u c e p t a t e , s = s o l i d , g = gas, 1 = l i q u i d and so = s o l u t i o n . The range of the peak temperatures i s based on 3 or more DSC sample runs. 77 When I was heated i n a v o l a t i l e pan with a p i n h o l e , there was an endothermic peak at =*119°C accompanied by a weight l o s s of =*11% w/w which suggests dehydration accompanied by the simultaneous v a p o r i z a t i o n of most of the dehydrated water ( F i g . 11c, Scheme I c ) . However, s i n c e no endotherm was observed at =*145°C i t seems l i k e l y t h a t a small amount of the l i b e r a t e d water i s adsorbed by the anhydrate and prevents m e l t i n g . In v o l a t i l e pans with a p i n h o l e , the r a t e of water vapor l o s s becomes a p p r e c i a b l e only when the i n t e r n a l pressure w i t h i n the pan i s equal to the atmospheric p r e s s u r e . The r e s t r i c t e d escape route (0.1 t o 0.2 mm p i n h o l e ) c o u l d b r i n g the water vapor and the dehydrated m a t e r i a l i n t o i n t i m a t e c o n t a c t , f a c i l i t a t i n g some a d s o r p t i o n of water by the anhydrate. I t i s not known how the adsorbed moisture prevents the m e l t i n g of the anhydrate. When a v o l a t i l e pan with a p i n h o l e was used under reduced p r e s s u r e 1 , the thermal behavior was s i m i l a r to that seen with standard pans i n that two endotherms o c c u r r e d at =*113°C and =*147°C ( F i g . 11d). Because of the reduced p r e s s u r e , the water vapor r e l e a s e d on dehydration (at =*113°C) w i l l be r a p i d l y evaporated. Hence the anhydrate does not adsorb moisture and melts s h a r p l y at =*147°C (Scheme I d ) . I was a l s o heated under atmospheric p r e s s u r e i n a v o l a t i l e pan with a p i n h o l e up to the b a s e l i n e departure of ^ o r these s t u d i e s , a model c a l o r i m e t e r , Du Pont with a used. 910 d i f f e r e n t i a l scanning s e r i e s 99 thermal a n a l y z e r was 78 the f i r s t endotherm at 115°C. The he a t i n g was then stopped and vacuum a p p l i e d f o r 5 min. The run was continued under reduced p r e s s u r e and a melting endotherm was observed a t «*145°C. T h i s confirmed the r o l e of adsorbed water i n p r e v e n t i n g the me l t i n g of the anhydrate s i n c e the a p p l i c a t i o n of vacuum at the de h y d r a t i o n endotherm w i l l remove the water vapor before i t can be adsorbed by the anhydrate. In an attempt to q u a n t i t a t e the enthalpy of the t r a n s i t i o n s o c c u r r i n g on h e a t i n g I, a DSC 2 with a data s t a t i o n 3 was used. In both standard pans and v o l a t i l e pans with a p i n h o l e , dehydration and v a p o r i z a t i o n o c c u r r e d s i m u l t a n e o u s l y and i t was not p o s s i b l e to q u a n t i t a t e the enthalpy of de h y d r a t i o n . Moreover, i n v o l a t i l e pans with a p i n h o l e , the l o s s of water f o l l o w i n g dehydration was incomplete p o s s i b l y due to a d s o r p t i o n of some water vapor. The m e l t i n g of the anhydrate, I I , was accompanied by decomposition and so the enthalpy of m e l t i n g was not determined. When II was su b j e c t e d t o DSC immediately a f t e r p r e p a r a t i o n from I by deh y d r a t i o n , an endotherm o c c u r r e d at =<140°C, i r r e s p e c t i v e of the type of the pan used ( F i g . 12a). There was no a p p r e c i a b l e l o s s i n the weight of the sample. Thermomicroscopy showed that t h i s endotherm corresponded t o m e l t i n g . Since d r y i n g I at 60°C under vacuum f o r 16 h caused a weight l o s s of <*11.3% w/w, II was i d e n t i f i e d as anhydrous c a l c i u m g l u c e p t a t e . Drying f o r p e r i o d s longer than 16 h d i d 2Model DSC-2C, Perk i n Elmer 3Model 3600, P e r k i n Elmer 79 1 • ' 50 90 130 170 TEMPERATURE (°C) F i g . 12 Representative thermal curves of II i n v o l a t i l e pans (a) freshly prepared sample and (b) sample exposed to ambient conditions for 5 min. 80 not cause any f u r t h e r weight l o s s . When II was exposed to ambient c o n d i t i o n s (=35% RH, 23°C) f o r 5 min, there was a small i n c r e a s e i n weight (=2% w/w) suggesting a d s o r p t i o n of moisture and on heating i n v o l a t i l e pans, there was a s i n g l e endotherm at =*118°C, F i g . 12b. The i n c r e a s e i n weight on exposure suggests a d s o r p t i o n of moisture which c o u l d lead to d i s s o l u t i o n of some of the anhydrate on the s u r f a c e s of the p a r t i c l e s . However, at room temperature, the anhydrate has a much higher apparent water s o l u b i l i t y than the hydrate (1.3 molal compared with 0.07 molal, see F i g . 14 on p.88). T h e r e f o r e the s o l u t i o n of the anhydrate would be s u p e r s a t u r a t e d with respect to the hydrate and r e s u l t i n p r e c i p i t a t i o n . The anhydrate would now have a s u r f a c e l a y e r of the, hydrate and i t s dehydration c o u l d be r e s p o n s i b l e f o r the endotherm at =*118°C. The water l i b e r a t e d at =118°C cannot escape from the v o l a t i l e pans and probably r e d i s s o l v e s some of the anhydrate. The anhydrate peak at =*118°C w i l l a l s o i n c l u d e the heat of s o l u t i o n of t h i s r e a c t i o n . I t i s hypothesized that the presence of an aqueous s o l u t i o n of anhydrate prevents the m e l t i n g of the anhydrate. 5. E q u i l i b r i u m s o l u b i l i t y Table VII c o n t a i n s the e q u i l i b r i u m s o l u b i l i t i e s of I determined at d i f f e r e n t temperatures. A van't Hoff p l o t of l o g s o l u b i l i t y a g a i n s t 1/T where T i s the temperature i n °K r e s u l t e d i n the l i n e a r r e l a t i o n s h i p shown in F i g . 13. The heat Table VII Equilibrium s o l u b i l i t y of I at d i f f e r e n t temperatures. Temperature S o l u b i l i t y (°C) (molal) 25.5 0.0581 + 0.0006b 31.5 0.0791 + 0.0010b 34.0 0.0922 + 0.0003b 37.0 0.1048 + 0.0008° aMean ± S.D.; n=6; Cn=3. F i g . 13 The van't Hoff plo t for I i n water. of s o l u t i o n of I c a l c u l a t e d from t h i s p l o t was 40.0 kJ mol" 1. Grant et a l . (1984) have r e c e n t l y shown that van't Hoff p l o t s c o u l d be n o n - l i n e a r p a r t i c u l a r l y when determined over a wide temperature range of 50 degrees or more. Hence the s o l u b i l i t i e s of I were determined over a narrow temperature range (11.5°C) and the van't Hoff p l o t was assumed to be l i n e a r . Attempts to determine the maximum s o l u b i l i t y of II were u n s u c c e s s f u l . I t had a h i g h i n i t i a l s o l u b i l i t y at room temperature (=22°C) but the s o l u t i o n s were unstable r e s u l t i n g i n r a p i d p r e c i p i t a t i o n . The powder x-ray d i f f r a c t i o n p a t t e r n of t h i s p r e c i p i t a t e was i d e n t i c a l to that of I (see Table VI on p. 72) . Suryanarayanan and M i t c h e l l (1984) observed that on d i s s o l u t i o n i n water„ amorphous anhydrous c a l c i u m g l u c e p t a t e p r e c i p i t a t e d as the c r y s t a l l i n e hydrate. A s i m i l a r s o l u t i o n phase t r a n s f o r m a t i o n from an anhydrate to a hydrate has been observed i n s e v e r a l compounds i n c l u d i n g c h o l e s t e r o l , t h e o p h y l l i n e , g l u t e t h i m i d e ( S h e f t e r and H i g u c h i , 1963) and mercaptopurine (Huang and N i a z i , 1977). In s e v e r a l i n s t a n c e s , the e q u i l i b r i u m s o l u b i l i t y of the hydrate and the apparent s o l u b i l i t y of the c o r r e s p o n d i n g anhydrate have been determined. For example, i n the case of t h e o p h y l l i n e , d e h y d r a t i o n of the monohydrate caused a 1.8-fold i n c r e a s e i n apparent aqueous s o l u b i l i t y at 35°C (Shefter and H i g u c h i , 1963). The apparent aqueous s o l u b i l i t y at 37°C, of anhydrous mercaptopurine was c a l c u l a t e d to be 1.6 times that of the e q u i l i b r i u m s o l u b i l i t y of mercaptopurine monohydrate (Huang 84 and N i a z i , 1977; N i a z i , 1978). The apparent s o l u b i l i t y of II at ~22°C was found to be 1.29 molal while that of I was 0.07 molal ( F i g . 14). Thus dehydration of c a l c i u m g l u c e p t a t e hydrate produces an 1 8 - f o l d i n c r e a s e i n i t s apparent s o l u b i l i t y . We are not aware of any other hydrate t h a t undergoes such a dramatic i n c r e a s e i n i t s apparent water s o l u b i l i t y on d e h y d r a t i o n . 6. C h a r a c t e r i z a t i o n of d i f f e r e n t samples of c a l c i u m g l u c e p t a t e Calcium g l u c e p t a t e obtained from d i f f e r e n t sources was c h a r a c t e r i z e d (Table V I I I ) . The r e l a t i v e p r o p o r t i o n s of the a and 0 epimers were determined by the GC method ( s e c t i o n C.1 i n E x p e r i m e n t a l ) . A c c o r d i n g to the USP XX (1980c), c a l c i u m g l u c e p t a t e can be an anhydrate or a hydrate with e i t h e r 2 molecules (6.9% w/w water) or 3 1/2 molecules (11.4% w/w water) of water of c r y s t a l l i z a t i o n . I r r e s p e c t i v e of the commercial source, a l l samples were e i t h e r an amorphous anhydrate or a c r y s t a l l i n e hydrate with 3 1/2 molecules of water of c r y s t a l l i z a t i o n (Table V I I I ) . In the l a t t e r case, the weight l o s s on h e a t i n g i n the DSC ( i n both standard pans and v o l a t i l e pans wi t h a p i n h o l e ) was =*11% w/w. A l l the amorphous, anhydrous samples were manufactured p r i o r t o J u l y , 1980 w i t h the e x c e p t i o n of the m a t e r i a l T a b l e V I I I . Some s o l i d - s t a t e p r o p e r t i e s o f v a r i o u s s a m p l e s o f c a l c i u m g l u c e p t a t e . S a m p l e D a t e o f R e l a t i v e p r o - P o w d e r x - r a y N o . o f m o l e c u l e s w a t e r M a n u f a c t u r e p o r t i o n s o f a a n d 0 e p i m e r s p a t t e r n o f w a t e r o f c r y s t a l 1 i z a t i on s o l u b i l i t y P f a n s t i e h l ; l o t 7311 a 1966 52%o;48%0 A 0 v e r y s o l u b l e * 5 S i g m a ; l o t 126C-0121 1 9 7 6 ° 100%a A 0 v e r y s o l u b l e 1 5 G i v a u d a n ; l o t R 3679 B A a 1979 c 72%o;28%8 A 0 v e r y s o l u b l e * 5 I t a l s i n t e x ; l o t R 1432 T J a 1979c 72%c;28%B A 0 v e r y s o l u b l e * 5 P f a n s t i e h l ; l o t 12953-0(111)' a J a n . 1980 100%a A 0 v e r y s o l u b l e 1 3 P f a n s t i e h l ; l o t 13313-E(I) J u l y 1980 100%a C 3 1/2 s p a r i n g l y s o l u b l e * * P f a n s t i e h l ; l o t 14772 B l e n d o f m a t e r i a l s made i n D e c . 1980 and M a r . 1982 ; 100%a C 3 1/2 s p a r i n g l y s o l u b l e ^ P f a l t z and B a u e r ; l o t C01300 1 9 8 2 ° 100%a C 3 1/2 s p a r i n g l y s o l u b l e ' ' PMH 1982c 100%a C 3 1/2 s p a r i n g l y s o l u b l e ^ M e r c k , ST-16944; RM-45940 1984 100%a A 0 v e r y s o l u b l e ' 5 S a m p l e s u s e d 1n e a r l i e r i n v e s t i g a t i o n s ( S u r y a n a r a y a n a n and M i t c h e l l , 1984). b E q u i l i b r i u m s o l u b i l i t y c a n not be d e t e r m i n e d ; a p p a r e n t s o l u b l i t y >100% w/v a t room t e m p e r a t u r e ( a p p r o x . 22 ° C ) f o r a l l amorphous s a m p l e s ; 27% w/v s o l u t i o n s o f a l l t h e s a m p l e s ( e x c e p t P f a n s t i e h l ; l o t 7311) p r e c i p i t a t e d on s t o r a g e . c Y e a r o b t a i n e d ; a c t u a l m a n u f a c t u r i n g d a t e not k n o w n . d E q u i l i b r i u m s o l u b l i l t y a t 2 5 . 5 ° C was 3.2% w / v ; a l l c r y s t a l l i n e s a m p l e s had s i m i l a r s o l u b i l i t y v a l u e s . A = amorphous t o x - r a y s . C = c r y s t a l l i n e ; a l l s a m p l e s had i d e n t i c a l x - r a y d i f f r a c t i o n p a t t e r n s . manufactured by Merck F r o s s t Canada i n 1984. Calcium g l u c e p t a t e manufactured from J u l y , 1980 onwards was a c r y s t a l l i n e hydrate i r r e s p e c t i v e of the commercial source (again the m a t e r i a l from Merck F r o s s t Canada was an e x c e p t i o n ) . H o l s t e i n i n 1980 had observed that at P f a n s t i e h l L a b o r a t o r i e s , p r e p a r a t i o n of c a l c i u m g l u c e p t a t e i n the amorphous form had become impossible due to the presence of seed c r y s t a l s (presumably of c a l c i u m g l u c e p t a t e hydrate) or some other f a c t o r i n i t i a t i n g c r y s t a l l i z a t i o n of c a l c i u m g l u c e p t a t e hydrate. I t appears that a s i m i l a r problem was encountered by other manufacturers of c a l c i u m g l u c e p t a t e with the c u r i o u s e x c e p t i o n of Merck F r o s s t Canada. B. TREATMENT OF CALCIUM GLUCEPTATE 1. G r i n d i n g I ground f o r 1 h and II ground f o r 4 h were su b j e c t e d to GC a n a l y s i s (see s e c t i o n C.I i n E x p e r i m e n t a l ) . Both the samples c o n t a i n e d a s i n g l e compound with a r e t e n t i o n time of 9.2 min. T h i s compound was e a r l i e r i d e n t i f i e d to be c a l c i u m a-glucoheptonate ( s e c t i o n A.1 i n R e s u l t s and D i s c u s s i o n ) . The absence of a d d i t i o n a l peaks proved that g r i n d i n g I and II f o r 1 and 4 h r e s p e c t i v e l y caused no d e t e c t a b l e sample decomposition. 87 1.1 Effect on apparent solubility The e f f e c t of g r i n d i n g time on the s o l u b i l i t y of I and II i s shown in Fig.14. G r i n d i n g had a marked e f f e c t only on the apparent s o l u b i l i t y of I I . The apparent s o l u b i l i t y of unground II was 1.3 molal while that of II ground f o r 2 h was 5.4 molal i . e . a 4 . 2 - f o l d i n c r e a s e i n apparent s o l u b i l i t y . Table IX l i s t s the e f f e c t of g r i n d i n g on the d i s s o l u t i o n behavior of some other drugs. In one sample of d i g o x i n , g r i n d i n g caused an-=2.2-fold (118%) in c r e a s e i n apparent s o l u b i l i t y . Thus the apparent s o l u b i l i t y of II i s extremely s e n s i t i v e to g r i n d i n g time. Attempts to determine the apparent s o l u b i l i t y of II ground f o r longer than 2 h were u n s u c c e s s f u l because of the extreme i n s t a b i l i t y of these s o l u t i o n s which caused a very r a p i d p r e c i p i t a t i o n of I from s o l u t i o n . 1.2 Effect on surface area Only the s u r f a c e areas of unground and ground II were determined (Table XI, p. 106). Even a f t e r g r i n d i n g f o r 4 h there was l e s s than a 3 - f o l d i n c r e a s e i n s u r f a c e a r e a . 1.3 Effect on x-ray diffraction The powder x-ray d i f f r a c t i o n p a t t e r n s of I ground f o r d i f f e r e n t times shown i n F i g . 15 suggest that g r i n d i n g f o r 1 h, makes i t x-ray amorphous. To produce a s i m i l a r e f f e c t i n I I , a longer g r i n d i n g time of 4 h was r e q u i r e d ( F i g . 6 ) . Scheme II c o n t a i n s the apparent s o l u b i l i t i e s as w e l l as the 88 F i g . 14 E f f e c t of g r i n d i n g on the apparent s o l u b i l i t y o f I (•—•—•>-) and I I (-A A--) i n water a t room temperature (-22°C) . Inset: Apparent solubility values of I plotted on an expanded scale. A 1 1 values are averages of two determinations. 89 Table IX E f f e c t of g r i n d i n g on the d i s s o l u t i o n behavior of some drugs. Drug Type of G r i n d i n g E f f e c t g r i n d e r time ( i n comparison w i t h unground drug) Reference D i g o x i n Mortar Not and known p e s t l e In s i m ulated g a s t r -i c f l u i d , 66% of the d i g o x i n d i s s o l -ved i n 2 h compared to the d i s s o l u t i o n of 52% of unground d i g o x i n 3 Shah e t a l . , 1974 Di g o x i n from d i f f e r -ent sources B a l l 3%-8 h Apparent aqueous m i l l s o l u b i l i t i e s i n c r e a s e d between 7 and 118% F l o r e n c e and S a l o l e , 1976 G r i s e o - B a l l f u l v i n m i l l 24 h No i n c r e a s e i n d i s s o l u t i o n r a t e ( i n D i s i n t e g r a t i o n Medium No.l of Jap-anese Pharmacopeia X) Sawayanagi e t a l . , 1982 Cephal- Shaker e x i n m i l l 4 h Approximately 100% i n c r e a s e i n appare-nt e q u i l i b r i u m so-l u b i l i t y i n water Otsuka and Kaneniwa, 1984 a These r e s u l t s may be u n r e l i a b l e because of the r a p i d d e g r a d a t i o n o f d i g o x i n from such h i g h l y a c i d i c d i s s o l u t i o n media (Sonobe e t a l . , 1980). 5 0 r CO > » L_ cc cc > CO z LU 2 5 r 2 5 r (a) (b) (c) (d) 3 0 2 0 1 0 2 0 , degrees 15 Powder x-ray d i f f r a c t i o n patterns of I (a) unground; (b), (c) and (d) ground 30 and 60 min, respectively. 91 method of p r e p a r a t i o n of some s o l i d phases of c a l c i u m g l u c e p t a t e . 2. F r e e z e d r y i n g The p r o d u c t o b t a i n e d on f r e e z e d r y i n g an aqueous s o l u t i o n of I had the f o l l o w i n g p r o p e r t i e s : 1. i t d i d not d i f f r a c t x - r a y s and was t h e r e f o r e x - r a y amorphous. 2. the DSC thermogram, showed no endothermic or e x o t h e r m i c peaks between 30 and 180°C i n d i c a t i n g t h a t i t was anhydrous and a l s o t h a t t h e r e a r e no phase t r a n s i t i o n s i n t h i s t e m p e r a t u r e range. The absence of a m e l t i n g p o i n t was a l s o i n d i c a t i v e of i t s n o n - c r y s t a l l i n e n a t u r e (Ke, 1966). 3. GC a n a l y s i s (see s e c t i o n C.1 i n E x p e r i m e n t a l ) r e s u l t e d i n the e l u t i o n of a s i n g l e compound a t 9.2 min which i s due t o c a l c i u m a - g l u c o h e p t o n a t e . Hence the f r e e z e d r i e d p r o d u c t was amorphous anhydrous c a l c i u m g l u c e p t a t e . 3 . Constant h u m i d i t y s t u d i e s S t o r i n g I , I I and I I I a t a range of r e l a t i v e h u m i d i t i e s r e s u l t e d i n t h e phase t r a n s i t i o n s shown i n Scheme I I I . W h i l e the t r a n s i t i o n of the c r y s t a l l i n e a n h y d r a t e ( I I ) t o t h e c r y s t a l l i n e h y d r a t e ( I ) o c c u r r e d a t RH > 66%, the t r a n s i t i o n of the amorphous anh y d r a t e ( I I I ) t o I o c c u r r e d a t RH > 52%, I ( h y d r a t e ) • c r y s t a l l i n e w / s 0 . 0 7 m o l a l N d r i e d a t 60°C x vacuum x 16 h aqueous s o l u t i o n g r i n d i n g x 1 h h y d r a t e — amorphous t o x - r a y s — w / s 0 . 1 1 m o l a l aqueous s o l u t i o n II ( a n h y d r a t e ) c r y s t a l l i n e w / s 1 .3 m o l a l g r i n d i n g x 2 h a n h y d r a t e — v e r y r e d u c e d c r y s t a l l i n i t y — w / s 5 . 4 m o l a l Scheme I I . Some phases of calcium gluceptate and th e i r aqueous apparent s o l u b i l i t i e s (w/s = apparent water s o l u b i l i t y ) , at room temperature (approx. 22°C) (II) crystalline anhydrate Phase tr a n s i t i o n s of calcium gluceptate. 94 showing that the more e n e r g e t i c amorphous form r e q u i r e d a lower r e l a t i v e humidity f o r the t r a n s i t i o n . Huttenrauch (1977a) observed that the h y g r o s c o p i c i t y of sucrose was r e l a t e d to i t s c r y s t a l l i n i t y . While the moisture a d s o r p t i o n of amorphous sucrose i n c r e a s e d e x p o n e n t i a l l y with i n c r e a s i n g RH s t a r t i n g at 50% RH, c r y s t a l l i n e sucrose d i d not adsorb moisture up to 70% RH. The t r a n s i t i o n of I to II and v i c e v ersa gave some i n t e r e s t i n g r e s u l t s ( F i g . 16). I c o u l d be dehydrated e i t h e r by the USP method (drying at 60°C under vacuum f o r 16 h) or by storage at 0% RH. However, the anhydrous form, I I , d i d not undergo a t r a n s i t i o n to the hydrate, I, u n t i l the RH was r a i s e d above 66%. Between 0% and 66% RH both I and II seemed to be s t a b l e and n e i t h e r underwent a phase t r a n s i t i o n . T h i s o b s e r v a t i o n i s c o n t r a r y to the phase r u l e a c c o r d i n g to which both the anhydrous form and the hydrate of a compound cannot be s t a b l e at one temperature over a range of vapor p r e s s u r e s . Th e r e f o r e i n the 0% to 66% RH range, the r a t e of c o n v e r s i o n of the anhydrate to hydrate or v i c e v ersa must be a very slow p r o c e s s . M i t r e v e j and Hollenbeck (1983) showed the e x i s t e n c e of a hydrophobic f i e l d generated by a s p i r i n c r y s t a l s which i n h i b i t e d the condensation of water from an high humidity environment i n the v i c i n i t y of the c r y s t a l s . When a s p i r i n was mixed with c e r t a i n h y d r o p h i l i c e x c i p i e n t s such as c o l l o i d a l s i l i c o n d i o x i d e , water r e a d i l y condensed on the c r y s t a l s . I t 95 100 r RH, % moles water/mole calcium gluceptate F i g . 16 The hydrate ( I ) - anhydrate(II) t r a n s i t i o n s of c a l c i u m g l u c e p t a t e at 25 C. i s p o s s i b l e that c r y s t a l s of II may generate a s i m i l a r f i e l d . T h i s c o u l d e x p l a i n i t s s t a b i l i t y up to 66% RH, while i f , at higher RH v a l u e s , the water vapor pressure s u c c e s s f u l l y overcomes t h i s f i e l d the t r a n s i t i o n from II to I c o u l d occur. In order to t e s t t h i s h y p o t h e s i s , II was mixed with 1% and 5%w/w c o l l o i d a l s i l i c o n d i o x i d e and s t o r e d at v a r i o u s h u m i d i t i e s . The t r a n s i t i o n from II to I continued to occur only above 66% RH i n d i c a t i n g t h a t II d i d not generate a hydrophobic f i e l d . Another p o s s i b i l i t y i s that of an energy b a r r i e r to the dehydration (I to II) and r e h y d r a t i o n (II to I) r e a c t i o n s . To i n v e s t i g a t e t h i s , the energy of a c t i v a t i o n , E . f o r the dehydration of I to II was c a l c u l a t e d u sing the f o l l o w i n g r e l a t i o n s h i p ( K i s s i n g e r , 1957): d ( l n «/T m) E = - _a (9) d d / T m ) R where <t> i s the heating r a t e (°C m i n " 1 ) , T m i s the temperature (°K) at which peak e n t h a l p i c d e f l e c t i o n occurs and R i s the gas c o n s t a n t . A p l o t of In <t>/^m versus 1/T g i v e s the a c t i v a t i o n energy without any assumptions about the r e a c t i o n mechanism. Samples of I were heated at 2.5, 5, 10, 20 and 40 °C m i n - 1 i n a DSC and the temperature of peak e n t h a l p i c d e f l e c t i o n (the peak due to dehydration and v a p o r i z a t i o n of water at =121°C, Scheme l a ) was noted. The energy of a c t i v a t i o n f o r dehydration of I c a l c u l a t e d from the K i s s i n g e r p l o t was 92.5 kJ m o l " 1 ( F i g . 17). Table X 97 F i g . 17 K i s s i n g e r p l o t f o r c a l c u l a t i o n o f the a c t i v a t i o n energy f o r d e h y d r a t i o n of I [T =temperature of peak (°K); c h e a t i n g r a t e (°C m i n " 1 ) ] . 98 l i s t s the energy of a c t i v a t i o n f o r dehydration of some pharmaceutical h y d r a t e s . The energy of a c t i v a t i o n f o r dehydration of I i s not un u s u a l l y high when compared with the values l i s t e d i n Table X and suggests t h a t an energy b a r r i e r f o r the dehydration of I to II i s u n l i k e l y . The energy of a c t i v a t i o n f o r the t r a n s i t i o n of II to I was a l s o determined. The heat of s o l u t i o n of I c a l c u l a t e d from the van't Hoff p l o t was +40.0 kJ m o l " 1 ( s e c t i o n A . 5 i n R e s u l t s and D i s c u s s i o n , p.80) and that of II determined by s o l u t i o n c a l o r i m e t r y was +13.8 kJ m o l " 1 ( T a b l e XI, p.106). The d i f f e r e n c e i n the heats of s o l u t i o n , AH^, i s the heat change accompanying the de h y d r a t i o n r e a c t i o n and has a value of +26.2 kJ mol"'(Scheme I V ) . The energy of a c t i v a t i o n f o r dehydration of I, E . c a l c u l a t e d from the K i s s i n g e r p l o t was 92.5 kJ a mol" 1. Since the change from I to II i s a r e v e r s i b l e process, the energy of a c t i v a t i o n f o r the t r a n s i t i o n of II to I, E^, can be c a l c u l a t e d from (Glasstone and Lewis, 1982b): E a ~ E b " ^ d ( 1 0 ) E^ was c a l c u l a t e d to be 66.3 kJ mol" 1 and from the low value of E^, an energy b a r r i e r f o r t r a n s i t i o n of II to I seems u n l i k e l y . The d e s o l v a t i o n of c e r t a i n hydrates may be i n i t i a t e d by i n o c u l a t i o n with the d e s o l v a t e d m a t e r i a l , and t h i s has been demonstrated i n c y t o s i n e hydrate (Byrn, 1982). I f i t were s i m i l a r l y p o s s i b l e to i n i t i a t e the dehydration of I, the 99 Table X A c t i v a t i o n e n e r g i e s f o r some dehydration r e a c t i o n s . Compound Energy of a c t i v a t i o n (kJ mol" 1) Reference Cefamandole sodium monohydrate •+• anhydrate A m p i c i l l i n monohydrate -»• anhydrate Sodium prasterone s u l f a t e d i h y d r a t e anhydrate T h e o p h y l l i n e monohydrate •+ anhydrate S u l f a g u a n i d i n e monohydrate anhydrate Mercaptopurine monohydrate anhydrate 71 95 131 140 Between 67 and 168 de-pending on c r y s t a l l i n -i t y o f the samples as w e l l as en-viro n m e n t a l f a c t o r s Between 191 and 264 de-pending on the method of d e t e r m i -n a t i o n P i k a l e t a l . , 1983 Sh e f t e r e t a l . , 1973 Nakagawa e t a l . , 1981 S h e f t e r e t a l . , 1973 Se k i g u c h i e t a l . , 1984 N i a z i , 1978 100 A c t i v a t e d s t a t e I i E =92.5 k J mol Si - I I R e a c t a n t ( I ) I I P r o d u c t ( I I ) 1 I I AH,=26.2 k J mol I _L -1 Scheme IV Some thermodynamic v a l u e s o f d e h y d r a t i o n o f I t o I I . These v a l u e s were used t o c a l c u l a t e t h e energy o f a c t i v a t i o n , E, , f o r t r a n s i t i o n o f I I t o I . 101 r e a c t i o n may proceed at RH valu e s higher than 0%. To t e s t t h i s , samples of I were i n i t i a l l y s t o r e d at 0% RH. As soon as they began to dehydrate to II (which was evident from the samples l o s i n g weight), they were t r a n s f e r r e d to chambers at 9, 33 and 52% RH. The samples d i d not continue to l o s e weight, so both I and II were capable of c o e x i s t i n g at these h u m i d i t i e s . S i m i l a r l y , the c o n v e r s i o n of II to I by i n o c u l a t i o n with I was a l s o attempted. Samples of II were i n i t i a l l y s t o r e d at 79% RH and when they began to gain weight and change to I, they were t r a n s f e r r e d to chambers at 9, 33 and 52% RH. The samples d i d not continue to gain weight, again c o n f i r m i n g that II and I c o u l d c o e x i s t at these h u m i d i t i e s . Though samples of II s t o r e d i n the range of 0-66% RH d i d not undergo a t r a n s i t i o n to I, samples s t o r e d above 0% RH gained weight due to the a d s o r p t i o n of moisture. The i n c r e a s e i n weight ranged from 0.84% w/w at 9% RH to 3.3% w/w at 66% RH. If the adsorbed moisture d i s s o l v e s some of the anhydrate, the s o l u t i o n would be s u p e r s a t u r a t e d with respect t o the s t a b l e hydrate and c o u l d r e s u l t i n p r e c i p i t a t i o n of the hydrate on the su r f a c e of the anhydrous c r y s t a l s . I t i s suggested t h a t t h i s hydrated l a y e r a c t s as a b a r r i e r to the d i f f u s i o n of water vapor and thus prevents the co n v e r s i o n of II to I up to 66% RH. E a r l i e r work with DSC ( s e c t i o n A.4 i n R e s u l t s and D i s c u s s i o n ) showed how the thermal behavior of II was a f f e c t e d by the a d s o r p t i o n of a small amount of moisture. When II was s t o r e d at RH > 66%, the water vapor p r e s s u r e was presumably h i g h enough to overcome the r e s i s t a n c e of the 102 h y d r a t e d s u r f a c e l a y e r t o the p e n e t r a t i o n of water vapor and t r a n s i t i o n t o I o c c u r r e d ( F i g . 1 6 ) . C. STABILIZATION OF CALCIUM GLUCEPTATE SOLUTIONS Aqueous s o l u t i o n s c o n t a i n i n g between 20 and 27% w/v I I were p r e p a r e d . Some s o l u t i o n s were l e f t u n f i l t e r e d w h i l e o t h e r s were f i l t e r e d e i t h e r t h r o u g h a f i l t e r paper (Whatman No. 1)or a membrane f i l t e r (0.22Mm). Immediately f o l l o w i n g t h i s , the s o l u t i o n s were a u t o c l a v e d a t 121°C f o r 20 min. A l l the s o l u t i o n s were s t a b l e showing t h a t n e i t h e r the c o n c e n t r a t i o n of I I nor the f i l t r a t i o n p r o c e d u r e a f f e c t e d s o l u t i o n s t a b i l i t y . The u n f i l t e r e d s o l u t i o n s c o n t a i n e d some u n d i s s o l v e d s o l i d , but t h i s d i d not induce n u c l e a t i o n and c r y s t a l l i z a t i o n i n the a u t o c l a v e d s o l u t i o n s . Suryanarayanan and M i t c h e l l (1984) h y p o t h e s i s e d t h a t the p r e c i p i t a t i o n of c a l c i u m g l u c e p t a t e s o l u t i o n s was ind u c e d by seed c r y s t a l s of c a l c i u m g l u c e p t a t e h y d r a t e and t h a t a u t o c l a v i n g d e s t r o y e d t h e s e seed c r y s t a l s . These r e s u l t s s u pport t h i s h y p o t h e s i s . When one sample was exposed t o f i l t e r e d a i r i n a l a m i n a r f l o w c a b i n e t or i n a " c l e a n " room which housed t h e l a m i n a r f l o w c a b i n e t , i t c o n t i n u e d t o be s t a b l e . When the same s o l u t i o n was exposed t o the atmosphere i n t h e "P h a r m a c e u t i c s L a b o r a t o r y " , p r e c i p i t a t i o n s t a r t e d w i t h i n 24 h. A l l the p r e v i o u s e x p e r i m e n t a l work w i t h c a l c i u m g l u c e p t a t e was done i n the P h a r m a c e u t i c s L a b o r a t o r y and the p r e c i p i t a t i o n was presumably due t o the n u c l e a t i o n by seed c r y s t a l s d i s p e r s e d t h r o u g h o u t t h i s l a b o r a t o r y . The " c l e a n " room i s i n r e l a t i v e l y c l o s e 1 03 p r o x i m i t y to the Pharmaceutics Laboratory and some of the seed c r y s t a l s are l i k e l y to d r i f t to this" room. I t i s suggested that the high e f f i c i e n c y p a r t i c u l a t e a i r f i l t e r (HEPA f i l t e r ) i n the laminar a i r flow c a b i n e t e f f e c t i v e l y removes the seed c r y s t a l s from the a i r both w i t h i n the hood and the " c l e a n " room. I t t h e r e f o r e seems t h a t a u t o c l a v e d s o l u t i o n s of II w i l l be s t a b l e when exposed to a i r as long as there are no seed c r y s t a l s of c a l c i u m g l u c e p t a t e hydrate i n the atmosphere.' Calcium g l u c e p t a t e USP i s commercially a v a i l a b l e as a hydrate with a very low e q u i l i b r i u m water s o l u b i l i t y of =3% w/v. However, ' s t a b l e ' (no p r e c i p i t a t i o n d u r i n g two years of storage) s o l u t i o n s with a high c a l c i u m g l u c e p t a t e c o n c e n t r a t i o n (=25% w/v) can be prepared from t h i s m a t e r i a l . Dehydration of the commercial m a t e r i a l d r a m a t i c a l l y i n c r e a s e s i t s apparent water s o l u b i l i t y and aqueous s o l u t i o n s of the r e q u i r e d c o n c e n t r a t i o n can be prepared with the anhydrate, prov i d e d , the s o l u t i o n s are a u t o c l a v e d immediately a f t e r p r e p a r a t i o n . T h i s method i s s u i t a b l e f o r the p r e p a r a t i o n of s e a l e d p a r e n t e r a l f o r m u l a t i o n s but probably not f o r multi-dose o r a l p r e p a r a t i o n s where exposure to the atmosphere may l e a d to seeding and r e c r y s t a l l i z a t i o n . D. DETERMINATION OF DEGREE OF CRYSTALLINITY OF CALCIUM GLUCEPTATE The degree of c r y s t a l l i n i t y d e t e r m i n a t i o n s were c o n f i n e d to ground II because of the s i g n i f i c a n t e f f e c t of g r i n d i n g on 104 i t s apparent water s o l u b i l i t y . 1 . S e l e c t i o n of c r y t a l l i n e and amorphous r e f e r e n c e standards II was produced by d r y i n g I a t 60°C under vacuum f o r 16 h and t h i s was used as the c r y s t a l l i n e (100% c r y s t a l l i n i t y ) r e f e r e n c e standard. When i t was ground f o r 4 h, i t became amorphous to x-rays ( F i g . 6). Examination by p o l a r i z e d l i g h t microscopy re v e a l e d that unground II c o n s i s t e d of long t h i n c r y s t a l s which were h i g h l y b i r e f r i n g e n t . The s i z e of the c r y s t a l s decreased with g r i n d i n g time, but a f t e r about 30 min g r i n d i n g , the p a r t i c l e s s t a r t e d to form aggregates which were r e a d i l y r e d i s p e r s e d i n the m i n e r a l o i l mounting l i q u i d . The deaggregated m a t e r i a l r e t a i n e d i t s b i r e f r i n g e n t c h a r a c t e r f o r up to 2 h of g r i n d i n g , but a f t e r 4 h g r i n d i n g most of the p a r t i c l e s were n o n - b i r e f r i n g e n t . I t i s apparent that p o l a r i z e d l i g h t microscopy was a more s e n s i t i v e i n d i c a t o r of c r y s t a l l i n i t y than x-ray d i f f r a c t i o n , s i n c e some p a r t i c l e s s t i l l showed b i r e f r i n g e n c e . Nakamachi et al . (1981) dehydrated mercaptopurine monohydrate and a l s o obtained an x-ray amorphous intermediate which e x h i b i t e d b i r e f r i n g e n c e when examined m i c r o s c o p i c a l l y under p o l a r i z e d l i g h t . Since the p a r t i c l e s are e i t h e r b i r e f r i n g e n t or n o n - b i r e f r i n g e n t , p o l a r i z e d l i g h t microscopy only g i v e s a q u a l i t a t i v e i n d i c a t i o n of the p r o g r e s s i v e decrease i n c r y s t a l l i n i t y with g r i n d i n g . On the other hand, decreasing c r y s t a l l i n i t y was r e a d i l y q u a n t i t a t e d from powder x-ray d i f f r a c t i o n p a t t e r n s from the gradual decrease i n the i n t e n s i t y of the d i f f r a c t i o n peaks. 105 With i n c r e a s i n g g r i n d i n g time, the DSC of II ( i n standard pans) showed a p r o g r e s s i v e decrease i n the area of i t s melting endotherm at =144°C (DSC of unground II i s d i s c u s s e d i n s e c t i o n A.4 of R e s u l t s and D i s c u s s i o n ) . The sample ground for 4 h d i d not melt at a l l and the absence of a sharp m e l t i n g p o i n t i s a c h a r a c t e r e s t i c f e a t u r e of n o n - c r y s t a l l i n e m a t e r i a l s (Ke, 1966). Based on x-ray and DSC s t u d i e s , II ground f o r 4 h was s e l e c t e d as the 0% c r y s t a l l i n e (amorphous) standard. 2 . Comparison of c r y s t a l l i n i t y v a l u e s obtained by d i f f e r e n t methods The c r y s t a l l i n i t y of II decreased with g r i n d i n g time and Table XI l i s t s the percent c r y s t a l l i n i t y v a l u e s obtained by d i f f e r e n t methods. The values are i n poor, agreement. t In the pharmaceutical l i t e r a t u r e , the degree of c r y s t a l l i n i t y has u s u a l l y been c a l c u l a t e d assuming that the two-state model i s a p p l i c a b l e (Black and L o v e r i n g , 1977; P i k a l et a/.,1978; Nakai et a l . ,1982). The x-ray c r y s t a l l i n i t y i s o f t e n c a l c u l a t e d a c c o r d i n g to Eq. 2 (see s e c t i o n C.1 i n I n t r o d u c t i o n , p.9) and c a l o r i m e t r i c c r y s t a l l i n i t y a c c o r d i n g to Eq. 4 (see s e c t i o n C.2.1 i n I n t r o d u c t i o n , p.12). In t h i s work, percent c r y s t a l l i n i t y by both x-ray d i f f r a c t i o n and c a l o r i m e t r y was determined from standard curves obtained by mixing v a r i o u s p r o p o r t i o n s of the c r y s t a l l i n e and amorphous re f e r e n c e standards ( F i g . 7 and 9). Eq. 2 and 4 are based on a two-state model of c r y s t a l l i n i t y of polymers a c c o r d i n g to T a b l e XI E f f e c t o f g r i n d i n g on some p r o p e r t i e s o f I I . G r i n d i n g A p p a r e n t S u r f a c e D e n s i t y P a r t i c l e d i a m e t e r Hea t o f % C r y s t a l l i n i t y by t i m e s o l u b i l i t y a r e a (assuming s p h e r i c a l s o l u t i o n x - r a y h e a t o f d e n s i t y p a r t i c l e s ) s o l u t i o n (m in ) ( m o l a l ) (m 2 g " 1 ) (g c m - 3 ) Cnm(A)] ( k J m o l " 1 ) 0 1 .29 5 .45 1.6621 662(6620) +13.77 1 0 0 a 1 0 0 a I 0 0 a 15 1.67 8 . 1 6 1 .6720 440(4400) +5.91 7 2 . 4 6 1 . 8 6 8 . 3 30 2 .41 8 . 0 5 1.6782 438(4380) - 1 . 4 6 3 2 . 2 3 1 . 3 4 8 . 4 60 2 . 9 6 10 .51 1.6822 340(3400) - 6 . 0 9 2 4 . 8 1 2 . 2 3 5 . 6 240 ND 1 4 . 0 8 1 .6933 250(2500) - 9 . 1 3 0 . 0 a 0 . 0 a 0 . 0 a a assumed ND = n o t d e t e r m i n e d 107 which, small but p e r f e c t c r y s t a l l i n e regions are embedded w i t h i n a continous amorphous matrix ( f o r more d e t a i l s see s e c t i o n B.1 i n I n t r o d u c t i o n , p.3). However, even in polymers, such a model i s recognized as a gross o v e r s i m p l i f i c a t i o n and i t s relevance to most pharmaceuticals i s q u e s t i o n a b l e because of the d i f f e r e n c e s between polymers and other c r y s t a l l i n e m a t e r i a l s . Density measurements of ground II were undertaken both as an a l t e r n a t i v e method of measuring the degree of c r y s t a l l i n i t y and as a method of t e s t i n g the models of c r y s t a l l i n i t y . Contrary to e x p e c t a t i o n s , the r e s u l t s i n Table XI show that the d e n s i t y g r a d u a l l y i n c r e a s e d with g r i n d i n g time, suggesting that the c r y s t a l s i n i t i a l l y have an open l a t t i c e which g r a d u a l l y c o l l a p s e s under mechanical s t r e s s . According to the two-state model i l l u s t r a t e d i n F i g . 2, the decrease i n c r y s t a l l i n i t y with g r i n d i n g i s due to a p r o g r e s s i v e c o n v e r s i o n of c r y s t a l l i n e m a t e r i a l to the amorphous s t a t e . However, d i s p e r s i o n of ground II i n the carbon t e t r a c h l o r i d e - e t h y l e n e dibromide mixture d i d not r e s u l t i n s e p a r a t i o n i n t o two f r a c t i o n s corresponding to the c r y s t a l l i n e and amorphous s t a t e s . Instead, there was a gradual and p r o g r e s s i v e change i n the d e n s i t y of II with i n c r e a s i n g g r i n d i n g time, which suggests the one-state model. However, as d i s c u s s e d e a r l i e r , other models of c r y s t a l l i n i t y are p o s s i b l e . For example, the s u r f a c e of a c r y s t a l may become amorphous on g r i n d i n g (Khodakov and Rebinder, 1961) and the t h i c k n e s s of the amorphous l a y e r may i n c r e a s e with g r i n d i n g time u n t i l the 108 whole p a r t i c l e i s amorphous. When d i s p e r s e d i n the suspending l i q u i d , the s o l i d would not separate i n t o two f r a c t i o n s even though each p a t i c l e c o n t a i n s both amorphous and c r y s t a l l i n e phases. Hence, by i t s e l f , the suspension d e n s i t y method does not provide unequivocal evidence f o r a p a r t i c u l a r model. M i c r o s c o p i c a l examination using p o l a r i z e d l i g h t showed that a l l the p a r t i c l e s were b i r e f r i n g e n t even a f t e r 2 h of g r i n d i n g . I f the s u r f a c e amorphization model were c o r r e c t , b i r e f r i n g e n c y would be expected to disappear very q u i c k l y on g r i n d i n g . Hence, formation of an amorphous l a y e r surrounding a c r y s t a l l i n e core does not d e s c r i b e the d e c r e a s i n g c r y s t a l l i n i t y of II as g r i n d i n g c o n t i n u e s . Rather than the s u r f a c e l a y e r s of a p a r t i c l e becoming completely amorphous, i t i s probable that g r i n d i n g causes more d i s o r d e r i n the s u r f a c e l a y e r s than i n the bulk, and that the d i s o r d e r p r o g r e s s i v e l y decreases towards the core of each p a r t i c l e . The r e s u l t s from p o l a r i z e d l i g h t microscopy can a l s o be used to r e f u t e the two-state model. I f the two-state model were c o r r e c t , t h e m a j o r i t y of the p a r t i c l e s should be n o n - b i r e f r i n g e n t a f t e r 1 h of g r i n d i n g because the c r y s t a l l i n i t y value i s l e s s than 50% (Table X I ) . Although the d e n s i t y of a s o l i d i s independent of p a r t i c l e s i z e , i t was important to v e r i f y t h a t the x-ray l i n e broadening and changes i n the heat of s o l u t i o n v a l u e s were not simply due to decrease i n p a r t i c l e s i z e on g r i n d i n g . From the s u r f a c e area determined by krypton a d s o r p t i o n and the d e n s i t y , 109 a h y p o t h e t i c a l p a r t i c l e s i z e was determined f o r each sample (Appendix I I ) , assuming that a l l the p a r t i c l e s were s p h e r i c a l i n shape and of uniform s i z e (Table X I ) . The e f f e c t of p a r t i c l e s i z e on x-ray l i n e breadth u s u a l l y becomes apparent only when the c r y s t a l s i z e i s below 100 n m ( C u l l i t y , 1978c). Since a f t e r 4 h g r i n d i n g , the h y p o t h e t i c a l p a r t i c l e diameter of II was about 250 nm (2500 A ) , i t can be concluded that the x-ray l i n e broadening i s mainly a consequence of d i s t o r t i o n of the c r y s t a l l a t t i c e rather than p a r t i c l e s i z e r e d u c t i o n . Brunauer et al . (1956a) observed that a 2 6 - f o l d i n c r e a s e i n s u r f a c e area of c a l c i u m oxide (from 0.3 to 7.8 m2 g" 1) produced a decrease in t o t a l enthalpy of 0.56 kJ mol" 1 (from -198.19 kJ mol" 1 to -198.75 kJ m o l " 1 ) . F u r t h e r work by Brunauer et a l . (1956b, 1959) confirmed that very l a r g e i n c r e a s e s i n s u r f a c e area produced only small changes i n enthalpy. The r e s u l t s in Table XI show that c r y s t a l l i n e II with an endothermic heat of s o l u t i o n , was rendered x-ray amorphous on g r i n d i n g with an accompanying exothermic heat of s o l u t i o n , i . e . , a very l a r g e enthalpy change f o r a small i n c r e a s e i n s u r f a c e area. D i a l e r and Kuessner (1973) observed a s i m i l a r e f f e c t on m i l l i n g c r y s t a l l i n e sucrose, which r e s u l t e d i n i t s t r a n s f o r m a t i o n i n t o a g l a s s - l i k e m a t e r i a l . The accompanying change i n enthalpy c o u l d not be accounted f o r by the i n c r e a s e i n s u r f a c e area a l o n e . According to C a l v e t and Prat (1963), c r y s t a l d i s s o l u t i o n i s preceded by the exothermic a d s o r p t i o n of s o l v e n t molecules on the s o l u t e s u r f a c e , f o l l o w e d by an endothermic breakup of the c r y s t a l l a t t i c e . 110 Amorphous s o l i d s are c h a r a c t e r i z e d by the absence of long range order i n t h e i r c r y s t a l l a t t i c e . When they d i s s o l v e i n a s o l v e n t , l e s s energy i s r e q u i r e d to break up the l a t t i c e and the o v e r a l l heat of s o l u t i o n g e n e r a l l y becomes exothermic. Thus the change from the endothermic to exothermic heat of s o l u t i o n on g r i n d i n g II i s a t t r i b u t e d to a p r o g r e s s i v e change from an ordered l a t t i c e to a h i g h l y d i s o r d e r e d l a t t i c e c o n t a i n i n g excess f r e e energy. The r e l a t i o n s h i p between degree of c r y s t a l l i n i t y and apparent s o l u b i l i t y i s shown i n F i g . 18. The i n c r e a s e i n apparent s o l u b i l i t y of II on g r i n d i n g i s a l s o a t t r i b u t e d to the i n c r e a s e i n l a t t i c e d i s o r d e r . Because of the method by which the apparent s o l u b i l i t y was determined, the experimental value w i l l depend on the d i s s o l u t i o n r a t e of II as w e l l as e q u i l i b r i u m s o l u b i l i t y of I. Burt and M i t c h e l l (1981) showed that d i f f e r e n c e s i n d i s l o c a t i o n d e n s i t y i n potassium p e r c h l o r a t e c r y s t a l s , induced by changes i n c r y s t a l growth r a t e caused a s i g n i f i c a n t i n c r e a s e i n the d i s s o l u t i o n r a t e c o n s t a n t . F r i e s e n et a l . (1981) i n c r e a s e d the number of d i s l o c a t i o n s from an i n i t i a l value of 3.5 x 10 3 cm" 2 up to =2.5 x 10 5 cm" 2 by mechanically s t r e s s i n g s i n g l e c r y s t a l s of potassium p e r c h l o r a t e , and produced a 40% i n c r e a s e i n the d i s s o l u t i o n r a t e constant. The much g r e a t e r s t r e s s of g r i n d i n g can be expected to introduce much l a r g e r numbers of d i s l o c a t i o n s and other d e f e c t s i n t o a c r y s t a l , and i t i s suggested that the r e s u l t i n g decrease i n c r y s t a l l i n i t y i s r e s p o n s i b l e f o r the i n c r e a s e i n apparent s o l u b i l i t y of I I . J I 1 —I 25 50 75 100 DEGREE OF CRYSTALLINITY, % F i g . 18 R e l a t i o n s h i p between the degree o f c r y s t a l l i n i t y and apparent s o l u b i l i t y of II i n water a t room temperature (~22°C). The i n d i v i d u a l c r y s t a l l i n i t y v a l u e s are averages o f the values determined by x-ray, c a l o r i m e t r y and d e n s i t y f o r each g r i n d i n g time (Table X I ) . In a d d i t i o n to the c r e a t i o n of l a t t i c e d i s o r d e r , g r i n d i n g may i n c r e a s e s o l u b i l i t y both as a r e s u l t of p a r t i c l e s i z e r e d u c t i o n and by exposing more r e a c t i v e c r y s t a l faces to the d i s s o l u t i o n medium. The e f f e c t of p a r t i c l e s i z e r e d u c t i o n on s o l u b i l i t y can be c a l c u l a t e d from the Ostwald-Freundlich equation ( F l o r e n c e and Attwood, 1982), but the 2 . 3 - f o l d i n c r e a s e i n apparent s o l u b i l i t y of II a f t e r g r i n d i n g f o r 60 min i s g r e a t e r than c o u l d be accounted by the 2 . 0 - f o l d r e d u c t i o n i n h y p o t h e t i c a l p a r t i c l e s i z e (see Appendix III f o r c a l c u l a t i o n s ) . Since unground II occurred as long t h i n c r y s t a l s , the s u r f a c e s c r e a t e d on f r a c t u r e may be more r e a c t i v e than s u r f a c e s p a r a l l e l to the long a x i s . The e f f e c t s of c r y s t a l a n i s o t r o p y and h a b i t m o d i f i c a t i o n i n n i c k e l s u l f a t e hexahydrate c r y s t a l s have been s t u d i e d by Burt and M i t c h e l l (1979., l'980),and although s i g n i f i c a n t e f f e c t s on d i s s o l u t i o n r a t e constants were observed, the c o n t r i b u t i o n of d i s s o l u t i o n a n i s o t r o p y towards the o v e r a l l i n c r e a s e i n apparent s o l u b i l i t y found i n t h i s work i s l i k e l y t o be minimal compared with the e f f e c t due to i n c r e a s e d d i s o r d e r . A major d i f f i c u l t y with c a l c u l a t i n g percent c r y s t a l l i n i t y i s the s e l e c t i o n of a p p r o p r i a t e c r y s t a l l i n e and amorphous standards. The p e r f e c t c r y s t a l does not e x i s t , and the m a t e r i a l used as the 100% c r y s t a l l i n e standard (unground II) w i l l c o n t a i n numerous d e f e c t s (reduced c r y s t a l l i n i t y ) as a r e s u l t of i t s p r e p a r a t i o n from I by d e h y d r a t i o n . S i m i l a r l y , II ground f o r 4 h, s e l e c t e d as the 0% c r y s t a l l i n e standard because i t was x-ray amorphous, does not represent a t r u e 113 amorphous s t a t e s i n c e some p a r t i c l e s s t i l l showed b i r e f r i n g e n c e when examined by means of p o l a r i z e d l i g h t microscopy. Degree of c r y s t a l l i n i t y values obtained using one set of standards and a p a r t i c u l a r experimental method are not l i k e l y to agree with values obtained using e i t h e r other standards or another method, and Otsuka and Kaneniwa (1983) have shown that even using two x-ray d i f f r a c t i o n methods r e s u l t e d i n d i f f e r e n t v a l u e s f o r the c r y s t a l l i n i t y of c e p h a l e x i n . Hence, too much importance should not be attached to the numerical v a l u e s of percent c r y s t a l l i n i t i e s . N e v e r t h e l e s s , they provide a u s e f u l i n d i c a t i o n of the s t a t e of order of a s o l i d and can be c o r r e l a t e d with other p r o p e r t i e s of the s o l i d s t a t e which are profoundly i n f l u e n c e d by changes in the s t a t e of ord e r . SUMMARY 1. Calcium g l u c e p t a t e e x i s t s i n both c r y s t a l l i n e and n o n - c r y s t a l l i n e forms and i n each of these i t can be an anhydrate or a hydrate c o n t a i n i n g 3 1/2 molecules of water of c r y s t a l l i z a t i o n . 2. Up u n t i l the e a r l y p a r t of 1980, c a l c i u m g l u c e p t a t e was marketed as the amorphous anhydrate ( I I I ) but since t h i s time the m a t e r i a l a v a i l a b l e commercially has been the c r y s t a l l i n e h y d r a t e d ) . 3. Dehydration of I r e s u l t e d i n a c r y s t a l l i n e anhydrate ( I I ) . The apparent aqueous s o l u b i l i t y of III was found to be > 2 molal while the apparent s o l u b i l i t i e s of I and II were 0.07 and 1.29 molal r e s p e c t i v e l y . 4. S o l u t i o n s prepared with II or III were unstable and p r e c i p i t a t e d on storage as I. .5. S t a b l e c a l c i u m g l u c e p t a t e s o l u t i o n s (=25% w/v) were prepared from I by f i r s t d e h y d r a t i n g i t to II and using II to prepare the s o l u t i o n s . The s o l u t i o n s were s t a b i l i z e d by a u t o c l a v i n g immediately a f t e r p r e p a r a t i o n . 6. Above 66% r e l a t i v e humidity (RH), I was the s t a b l e form and II was s t a b l e at 0% RH. The v a r i o u s s o l i d phases can be i n t e r c o n v e r t e d by the processes of d r y i n g , g r i n d i n g and storage at c o n t r o l l e d RH. 115 7. I and I I c o e x i s t a t RH above 0% and below 67%. The a d s o r p t i o n of a s m a l l q u a n t i t y of water vapor by I I i n h i b i t s the t r a n s i t i o n t o I by p r e v e n t i n g f u r t h e r water u p t a k e . T h i s a d s o r p t i o n a l s o p r e v e n t s the m e l t i n g of I I . 8. G r i n d i n g I I i n c r e a s e d i t s apparent water s o l u b i l i t y . G r i n d i n g d e c r e a s e d both p a r t i c l e s i z e and the degree of c r y s t a l l i n i t y , but the l a t t e r was the major f a c t o r w i t h r e s p e c t t o the i n c r e a s e s i n apparent water s o l u b i l i t y . 9. The degree of c r y s t a l l i n i t y of I I was a s s e s s e d by x - r a y , heat of s o l u t i o n and d e n s i t y measurements, but the v a l u e s were not i n good agreement. 10. I t i s suggested t h a t the d e c r e a s e i n c r y s t a l l i n i t y w i t h g r i n d i n g i s due t o an i n c r e a s e i n l a t t i c e d i s o r d e r throughout the e n t i r e sample ( o n e - s t a t e model) r a t h e r than t o a p r o g r e s s i v e i n c r e a s e i n amorphous m a t e r i a l i n a c r y s t a l l i n e - a m o r p h o u s m i x t u r e ( t w o - s t a t e model). 1 16 REFERENCES Alexander, L.E., X-ray D i f f r a c t i o n Methods i n Polymer Science, Wiley, New York, NY, USA, 1969, (a) p. 176 and (b) p. 409. 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S o c , 54 ( 1 932) 3841-3851. Z a r z y c k i , J . , i n The S t r u c t u r e of N o n - C r y s t a l l i n e M a t e r i a l s , Ed. G a s k e l l , P.H., T a y l o r and F r a n c i s , 1977, pp. 259 and 260. Z e r b i , G. and C i a m p e l l i , F., C l a s s i f i c a t i o n of c r y s t a l l i n i t y bands i n the i n f r a r e d s p e c t r a of polymers. J . Polymer S c i . , Part C , 7 ( 1964) 141-151 . 130 APPENDIX I C a l c u l a t i o n of l i n e a r a b s o r p t i o n c o e f f i c i e n t s of anhydrous c a l c i u m g l u c e p t a t e and l i t h i u m f l u o r i d e The l i n e a r a b s o r p t i o n c o e f f i c i e n t , uc, of a compound i s given by the formula (Bragg, 1962): M c = p I P e ' M e / p e (11 where p i s the d e n s i t y of the compound and p g i s the p r o p o r t i o n by weight of each c o n s t i t u e n t element with mass a b s o r p t i o n c o e f f i c i e n t M e / p e ( i ) Anhydrous c a l c i u m g l u c e p t a t e (II) M o l e c u l a r formula of II i s C 1 0 H 2 6 C a O 1 6 (USP XX, 1980c) Weight f r a c t i o n s of c a l c i u m , carbon, hydrogen and oxygen are 0.082,0.343,0.053 and 0.522 r e s p e c t i v e l y . The d e n s i t y of II i s 1.66 g cm - 3 (Table XI) The mass a b s o r p t i o n c o e f f i c i e n t s of c a l c i u m , carbon, hydrogen and oxygen f o r CuKa r a d i a t i o n are 171, 4.22, 0.391 and 11.0 cm 2 g _ 1 r e s p e c t i v e l y ( C u l l i t y , I978e). The l i n e a r a b s o r p t i o n c o e f f i c i e n t of II = 1.66 {(0.082 x 171) + (0.343 x 4.22) + (0.053 x 0.391) + (0.522 x 11.0)} =35.3 cm"1 ( i i ) L i t h i u m f l u o r i d e Weight f r a c t i o n s of f l u o r i n e and l i t h i u m i n l i t h i u m f l u o r i d e ( L i F ) a r e 0.733 and 0.268 r e s p e c t i v e l y . D e n s i t y of l i t h i u m f l u o r i d e i s 2.64 g cm" 3 (Merck Index, 1983a). The mass a b s o r p t i o n c o e f f i c i e n t s of f l u o r i n e and l i t h i u m f o r CuKa r a d i a t i o n a re 16.0 and 0.477 cm 2 g" 1 r e s p e c t i v e l y ( C u l l i t y , I 9 7 8 e). The l i n e a r a b s o r p t i o n c o e f f i c i e n t of l i t h i u m f l u o r i d e = 2.64{(0.733 x 16.0) + (0.268 x 0.477)} = 31.3 cm" 1 132 APPENDIX II C a l c u l a t i o n of h y p o t h e t i c a l p a r t i c l e s i z e of unground II and II ground f o r 1 h The c a l c u l a t i o n of h y p o t h e t i c a l p a r t i c l e s i z e i s based on the assumption that a l l the p a r t i c l e s were s p h e r i c a l i n shape and of uniform s i z e . ( i ) Unground 11 Surface area = 5.45 m2 g" 1 (Table XI) = 5.45 x 10" cm 2 g" 1 D e n s i t y =1.6621 g cm"3 (Table XI) Volume of 1 g of II = 0.6017 cm 3 Let the number of p a r t i c l e s i n 1 g of powder be n,. The s u r f a c e area of 1 g of powder would be 4 7 r r 2 n 1 and i t s volume 4/3irr n , where r , i s the r a d i u s of the p a r t i c l e s . 2 2 4 Area of 1 g of powder(cm ) 4 i r r 1 n 1 5.45 x 10 Volume of 1 g of powder (cm ) 4/37rr*j in 1 0.6017 r, = 3.31 x IO' 5 cm diameter = 662 nm (6620 A) ( i i ) II ground f o r 1 h Surface area = 10.51 m2 g - 1 ( T a b l e XI) = 10.51 x 10V cm 2 g- 1 D e n s i t y = 1.6822 g cm" 3(Table XI) Volume of 1 g = 0.5945 cm 3 133 Let n 2 be the number of p a r t i c l e s i n 1 g of powder and r 2 be the r a d i u s of each p a r t i c l e . 2 Area of 1 g of powder(cm ) 1 0 5 1 x 1 0 ^ Volume of 1 g of powder(cm^) D T S 9 T 5 r 2 = 1.7 x 1 0 ~ 5 cm diameter = 3 4 0 nm ( 3 4 0 0 A ) 134 APPENDIX III C a l c u l a t i o n of the e f f e c t of p a r t i c l e s i z e on the s o l u b i l i t y The O s t w a l d - F r e u n d l i c h equation r e l a t e s the s o l u b i l i t y of a compound with i t s p a r t i c l e s i z e and i s given by (Florence and Attwood, 1982): where S, i s the s o l u b i l i t y of p a r t i c l e s of r a d i u s r , and S 2 i s the s o l u b i l i t y of p a r t i c l e s of r a d i u s r 2 at a temperature T (°K); M, 7 and p are the molecular weight, s u r f a c e energy and d e n s i t y of the s o l i d r e s p e c t i v e l y . The r a d i u s r 2 of 1 h ground p a r t i c l e s was 1.7 x 10" 5 cm and r a d i u s r , of unground p a r t i c l e s was 3.3 x 10" 5 cm (Table XI) . Molecular weight of II = 490.43 De n s i t y of II = 1.6621 g cm'3 Temperature = 25°C = 298°K (assumed) Since the s u r f a c e energy of II i s not known, a r b i t r a r y values ranging from 1 x 10' 6 J cm - 2 to 5 x 10" 5 J cm" 2 have been c o n s i d e r e d . The c a l c u l a t e d s o l u b i l i t y r a t i o of the 1 h ground to unground sample would range from 1.01 : 1.00 where the s u r f a c e energy i s 1 x 10" 6 J cm" 2 to 1.41 : 1.00 where the s u r f a c e energy i s 5 x 10" 5 J cm* 2. of II (12) 

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