Preparation and characterizetion of well-defined chemically bonded stationary phases for high pressure liquid chromatography

PREPARATION AND CHARACTERIZATION

OF WELL-DEFINED

CHEMICALLY BONDED STATIONARY PHASES

FOR

HIGH PRESSURE LIQUID CHROMATOGRAPHY

G E R T E . B E R E N D S E N

P R E P A R A T I O N A N D C H A R A C T E R I Z A T I O N

O F W E L L - D E F I N E D

C H E M I C A L L Y B O N D E D S T A T I O N A R Y P H A S E S

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PREPARATION AND CHARACTERIZATION

OF WELL-DEFINED

CHEMICALLY BONDED STATIONARY PHASES

FOR

HIGH PRESSURE LIQUID CHROMATOGRAPHY

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Contents

1. WHY CHEMICALLY BONDED STATIONARY PHASES? 1 - H i s t o r y o f HPLC and i n t r o d u c t i o n o f c h e m i c a l l y bonded phases 1

- P r e p a r a t i o n o f c h e m i c a l l y bonded s t a t i o n a r y phases f o r l i q u i d

chromatography 4 S t a t e o f t h e a r t o f RPLC a t t h e s t a r t o f t h e p r e s e n t i n v e s t i g a t i o n 7

Formulation and aim of the present investigation 9

R e f e r e n c e s 11

2. A GEOMETRICAL MODEL FOR CHEMICALLY BONDED TMS AND PDS PHASES 15

- I n t r o d u c t i o n 15 - The s t r u c t u r e o f s i l i c a 17

- Geometry o f t h e bonded phase 24 C o n c l u s i o n s ' 35

R e f e r e n c e s 37

3. PREPARATION AND CHROMATOGRAPHIC PROPERTIES OF SOME CHEMICALLY BONDED

PHASES FOR REVERSED PHASE LIQUID CHROMATOGRAPHY 39

I n t r o d u c t i o n 39 - E x p e r i m e n t a l 43

P r e p a r a t i o n o f c h e m i c a l l y bonded phases 44

R e s u l t s o f e l e m e n t a l a n a l y s i s 45 C h r o m a t o g r a p h i c p r o p e r t i e s 47

Retention on chemically bonded phases with maximum coverage 47

Trimethylsilyl phases with various surface coverage 51

Retention behaviour of homologous series 53

X

4. PREPARATION OF BONDED PHASES FOR HPLC

A r a p i d and s i m p l e c a t a l y t i c h y d r o s i l y l a t i o n o f t e r m i n a l o l e f i n s f o r

the p r e p a r a t i o n o f m o n o c h l o r o s i l a n e s 59

- I n t r o d u c t i o n 59 - E x p e r i m e n t a l 60 - P r e p a r a t i o n o f c h e m i c a l l y bonded p a c k i n g s 61

Synthesis of bonded packings 64

- R e s u l t s and d i s c u s s i o n 65

Surface coverage 65

Structure of chemically bonded packings 70

Geometrical pore model of chemically bonded stationary phases 75

Estimate of surface coverage 77

Thermal stability of bonded packings 80

C o n c l u s i o n s and recommendations 81

R e f e r e n c e s 84

5. ON THE DETERMINATION OF THE HOLD-UP TIME IN REVERSED PHASE LIQUID

CHROMATOGRAPHY 87 - I n t r o d u c t i o n 87

Rough estimates 88

Unretained compounds 88

Static methods 89

Linearization of the net retention time for homologous series 90

- T h e o r y 91

Linearization of homologous series 93

E x p e r i m e n t a l 94

Apparatus and chemicals 94

Standardization 94

Measurements on salts 95

Residence time of mobile phase components 98

Linearization of homologous series 98

- R e s u l t s and d i s c u s s i o n 102

e porosities 102

Residence time of the solvent components 103

Measurements on salts 105

Linearization of homologous series 106

Comparison of n-alkylsilyl bonded phases 107

Other mobile phase systems 109

C o n c l u s i o n s and recommendations 110

x i

6. THE ROLE OF THE CHAIN LENGTH OF CHEMICALLY BONDED PHASES AND THE

RETENTION MECHANISM IN REVERSED PHASE LIQUID CHROMATOGRAPHY 115

- I n t r o d u c t i o n 115 E x p e r i m e n t a l 11 7 Apparatus 117 Chemicals 117 Column packing 117 Hold-up time 117 C h r o m a t o g r a p h i c p r o p e r t i e s o f bonded p a c k i n g s 118

Influence of bonded phase chain length on retention 118

Selectivity 123 - R e t e n t i o n mechanism 129 R e t e n t i o n b e h a v i o u r o f homologous s e r i e s The common i n t e r s e c t i o n p o i n t 133 - C o n c l u s i o n s 135 R e f e r e n c e s 136

7. HAND-TIGHTENED SLURRY VESSEL FOR PACKING HIGH PRESSURE LIQUID

CHROMATOGRAPHY COLUMNS 139 I n t r o d u c t i o n 139 - E x p e r i m e n t a l 140 - R e s u l t s and d i s c u s s i o n 143 R e f e r e n c e s 144 SUMMARY 145 SAMENVATTING 149 REFERENCES/LITERATUUR 153

1

1

Why chemically bonded stationary phases?

HISTORY OF HPLC AND INTRODUCTION OF CHEMICALLY BONDED PHASES

H i g h P r e s s u r e L i q u i d Chromatography, HPLC, i s an a n a l y t i c a l s e p a r a t i o n method w h i c h has been i n t r o d u c e d i n t h e l a t e ' s i x t i e s by L i p s k y | l | and Huber

|2|. Same as o t h e r c h r o m a t o g r a p h i c methods i t s s e p a r a t i o n p r i n c i p l e i s b a s e d on the d i s t r i b u t i o n o f t h e components to be s e p a r a t e d , between a moving phase ( m o b i l e phase) and a s t a t i o n a r y p h a s e . I f t h e components o f t h e sample a r e d i s t r i b u t e d i n d i f f e r e n t r a t i o s between b o t h p h a s e s , t h e y w i l l be t r a n s p o r t e d a t d i f f e r e n t v e l o c i t i e s t h r o u g h t h e c h r o m a t o g r a p h i c system, so t h a t a s e p a r a t i o n w i l l be e f f e c t e d .

The HPLC-method has been i n t r o d u c e d as a complement to Gas Chromatography, GC, w h i c h by t h a t time was a l r e a d y advanced d e v e l o p e d and w i d e l y a p p l i e d . A p a r t

from d i f f e r e n c e s i n i n s t r u m e n t a t i o n , t h e main d i f f e r e n c e between b o t h methods i s t h e use o f a l i q u i d i n s t e a d o f a gaseous m o b i l e phase, w h i c h has two m a j o r c o n s e q u e n c e s . On t h e one hand, t h e r a t e s o f d i f f u s i o n a r e 10,000 times l o w e r i n l i q u i d s t h a n i n g a s e s , so t h a t p a r t i c l e d i m e n s i o n s s h o u l d be s m a l l e r f o r HPLC t h a n f o r GC ( i . e . 5 - 10 Um p a r t i c l e s i n s t e a d o f about 200 ]im), to a c h i e v e comparable e q u i l i b r a t i o n and a n a l y s i s t i m e s . On t h e o t h e r hand, l i q u i d s a r e

2

about 60 times more v i s c o u s than gases. Both f a c t o r s n e c e s s i t a t e s the use of h i g h p r e s s u r e s to l e t the l i q u i d m o b i l e p h a s e ( e l u e n t ) p e r c o l a t e r a p i d l y t h r o u g h the column. To r e s t r i c t the p r e s s u r e t o a p r a c t i c a l l i m i t o f about 20 MPa (y 200 atm) t h e column l e n g t h d e c r e a s e s from some 2 meter i n GC to 10 - 30 cm i n HPLC.

Because i n the e a r l y days o f modern l i q u i d chromatography columns w i t h the d e s i r e d s m a l l p a r t i c l e s (and w i t h s u f f i c i e n t l y u n i f o r m p a r t i c l e - s i z e d i s t r i b u t i o n ) were n o t a v a i l a b l e , d r y p a c k e d columns, f i l l e d w i t h 30 to 50 ym p a r t i c l e s , were used f o r the i n i t i a l development o f the HPLC-method,

The two main G C - t e c h n i q u e s ( i . e . GLC w i t h a l i q u i d s t a t i o n a r y phase and a mechanism b a s e d on g a s - l i q u i d p a r t i t i o n , and GSC w i t h a s o l i d a d s o r b e n t and, c o n s e q u e n t l y , an a d s o r p t i o n mechanism) were adopted i n l i q u i d c h r o m a t o g r a p h y , r e s u l t i n g i n l i q u i d - l i q u i d p a r t i t i o n c h r o m a t o g r a p h y , LLC, and l i q u i d - s o l i d a d s o r p t i o n chromatography, LSC.

Based on s u r f a c e c h a r a c t e r i s t i c s , s i l i c a ( S i O ^ ) a p p e a r e d t o be the most v e r s a t i l e a d s o r b e n t f o r LSC |3| and support m a t e r i a l f o r the p h y s i c a l l y coated s t a t i o n a r y p h a s e i n LLC |4|.

S i n c e the s u r f a c e a r e a o f the s i l i c a m a t e r i a l i s c o v e r e d w i t h p o l a r s i l a n o l 2 2

groups ( t o an e x t e n t o f about 8 ymol/m , or 4.8 per nm ) on which a d s o r p t i o n of s o l u t e m o l e c u l e s can take p l a c e , n o n p o l a r e l u e n t s a r e needed i n o r d e r t o a c h i e v e s u f f i c i e n t r e t e n t i o n o f the s o l u t e i n a c h r o m a t o g r a p h i c column. Here r e t e n t i o n d e n o t e s the r e t a r d a t i o n o f a s o l u t e due to an " i n t e r a c t i o n " w i t h the s t a t i o n a r y phase i n the column. The r e t e n t i o n i s e x p r e s s e d e.g. i n t h e r e t e n t i o n time, t . However, the a p p l i c a b i l i t y o f t h i s L S C - s y s t e m a p p e a r e d t o be r e s t r i c t e d to r e l a t i v e l y n o n p o l a r s o l u t e s .

I n o r d e r t o change the p o l a r i t y o f the s t a t i o n a r y phase, the s i l i c a s u r f a c e was c o a t e d by p o l a r l i q u i d s ( L L C ) , whereas s t i l l n o n p o l a r e l u e n t s were u s e d . However, the a p p l i c a b i l i t y was a g a i n r a t h e r l i m i t e d due t o the s o l u b i l i t y o f the p h y s i c a l l y c o a t e d s t a t i o n a r y phase i n t h e m o b i l e p h a s e , w h i c h r e s u l t e d i n column i n s t a b i l i t y . A c t u a l l y , one was r e s t r i c t e d t o u s i n g phases ( i . e . m o b i l e and s t a t i o n a r y ) w i t h a r e l a t i v e l y l a r g e d i f f e r e n c e i n p o l a r i t y ( i . e . c o m p l e t e l y i m m i s c i b l e l i q u i d s ) . T h e o r e t i c a l l y , t h i s means t h a t a compound w i l l be r e t a i n e d e i t h e r v e r y l i t t l e o r v e r y s t r o n g l y d e p e n d i n g on i t s f a v o u r e d i n t e r a c t i o n w i t h the m o b i l e o r the s t a t i o n a r y p h a s e . Hence, a d e c r e a s e o f t h e p o l a r i t y d i f f e r e n c e between b o t h phases c o u l d o n l y be o b t a i n e d by s a t u r a t i n g the m o b i l e phase w i t h s t a t i o n a r y p h a s e . A t y p i c a l phase s y s t e m as t r i e t h y l e n e g l y c o l c o a t e d on s i l i c a as s t a t i o n a r y phase and t r i e t h y l e n e - g l y c o l - s a t u r a t e d hexane as m o b i l e phase was used. A w i d e range o f f a i r l y p o l a r s o l u t e s as p h e n o l s , s t e r o i d s |4| and aromatic a l c o h o l s |5J could be separated w i t h t h i s system. Obviously, such systems r e q u i r e

3 e x a c t c o n t r o l o f t e m p e r a t u r e and b e s i d e s l o n g e q u i l i b r a t i o n times t o s t a b i l i z e the column.

F o r t h e same r e a s o n , c o m p l e t e l y r e v e r s e d s y s t e m s , i . e . i n w h i c h a n o n p o l a r s t a t i o n a r y phase ( e . g . s q u a l a n e ) was c o a t e d o n t o a s i l i c a - b a s e d s u p p o r t and the samples e l u t e d w i t h w a t e r - a l c o h o l m o b i l e p h a s e s , a r e a l s o no l o n g e r u s e d . The n o n p o l a r phase was s t r i p p e d from the column caused by the "weak h o l d i n g " o f t h i s phase to even a s i l y l a t e d s i l i c a |6|. S i n c e i n such a s y s t e m t h e p o l a r i t i e s o f the m o b i l e and s t a t i o n a r y phase a r e r e v e r s e d , compared w i t h t h e c o n v e n t i o n a l arrangement o f a p o l a r s t a t i o n a r y phase and a l e s s p o l a r m o b i l e phase, i t was c a l l e d " r e v e r s e d - p h a s e " . The term " r e v e r s e d - p h a s e " chromatography, however, was c o i n e d by Howard and M a r t i n |7|, who c a r r i e d o u t l i q u i d - l i q u i d p a r t i t i o n chroma-t o g r a p h y on p a r a f f i n o i l and n - o c chroma-t a n e as chroma-the s chroma-t a chroma-t i o n a r y phase u s i n g aqueous e l u e n t s . I n the p a s t few y e a r s the emphasis has f u n d a m e n t a l l y s h i f t e d i n f a v o u r o f n o n p o l a r s t a t i o n a r y p h a s e s . T h e r e f o r e , i n g e n e r a l , a s y s t e m i s c a l l e d " R e v e r s e d Phase" ( a b b r e v i a t e d by RP) , when the m o b i l e phase i s more p o l a r t h a n the s t a t i o n a r y phase, and "Normal Phase" o r " S t r a i g h t Phase", when t h e m o b i l e phase i s more n o n p o l a r t h a n t h e s t a t i o n a r y phase.

A n o t h e r d i s a d v a n t a g e o f t h e LLCsystems i s t h a t t e c h n i q u e s such as f l o w -programming, i n w h i c h the f l o w i s changed d u r i n g a c h r o m a t o g r a p h i c r u n , and g r a d i e n t e l u t i o n , where t h e c o m p o s i t i o n o f t h e m o b i l e phase i s g r a d u a l l y v a r i e d d u r i n g the r u n , cannot be a p p l i e d .

Hence, one d i d s t a n d i n need o f a s t a t i o n a r y phase which c o u l d n o t be s t r i p p e d from the s u p p o r t . T h i s e x p l a i n s the development o f the c h e m i c a l l y bonded s t a t i o n a r y phase f o r HPLC i n the b e g i n n i n g o f the ' s e v e n t i e s . H a l a s z and S e b e s t i a n |8,9|, K i r k l a n d | l 0 ] and M a j o r s were the p i o n e e r s , who i n t r o d u c e d t h e c h e m i c a l l y bonded p h a s e . A l l were p r o b a b l y i n s p i r e d by S t e w a r t and P e r r y who f i r s t s u g g e s t e d i n 1968 | l 2 | t h a t the t r e a t m e n t o f a s i l i c e o u s s u p p o r t w i t h o c t a d e c y l c h l o r o s i l a n e w o u l d p o s s i b l y y i e l d a u s e f u l s t a t i o n a r y p h a s e f o r LC.

4

PREPARATION OF CHEMICALLY BONDED STATIONARY PHASES FOR LIQUID CHROMATOGRAPHY

Well-known o r g a n i c r e a c t i o n s have been a p p l i e d to m o d i f y the s i l i c a s u r f a c e , w h i c h e x i s t s o f d i f f e r e n t k i n d s o f p o l a r s i l a n o l groups and u n r e a c t i v e s i l o x a n e bonds. F i g u r e 1 i l l u s t r a t e s the p o s s i b l e hydroxyl group c o n f i g u r a t i o n s on the s i l i c a s u r f a c e |3|. H /

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I.

Surface hydroxyl group configurations

The f o l l o w i n g methods were a p p l i e d f o r t h e p r e p a r a t i o n o f c h e m i c a l l y bonded s t a t i o n a r y p h a s e s .

(i) Reactions with alcohols /8/

The f i r s t bonded phases were p r e p a r e d by r e a c t i n g the s u r f a c e s i l a n o l groups w i t h an a l c o h o l y i e l d i n g an a l k o x y s i l a n e p h a s e , as shown i n f i g u r e 2. s i l i c a surface

%

1

low surface coverage

hydrolytia i n s t a b i l i t y

FIGURE 2

Formation of an ether-bond between alcohol and s i l i c a to give an alkoxy silane phase (R - hydrocarbon chain)

A d i s a d v a n t a g e o f t h i s type o f bonded p h a s e i s t h a t t h e a p p l i c a b i l i t y i s r e s t r i c t e d to a p H - r e g i o n o f 4 - 7, due to h y d r o l y s i s o f the e t h e r bond, and t h a t r e l a t i v e l y low s u r f a c e c o v e r a g e s w i t h the o r g a n i c groups on the s i l i c a s u r f a c e a r e o b t a i n e d .

(ii) Reactions with amines /9/ and Grignard reagents /13,14/

must be c o n v e r t e d to c h l o r i d e groups by t r e a t m e n t w i t h t h i o n y l c h l o r i d e .

The r e a c t i o n o f a p r i m a r y amine w i t h s i l i c a c h l o r i d e ( f i g u r e 3a) y i e l d s an a l k y l a m i n o s i l a n e bond, w h i c h i s a l s o s e n s i t i v e t o h y d r o l y s i s and a p p l i c a b l e o v e r t h e same p H - r e g i o n as the a l k o x y s i l a n e p h a s e s . C o n t r a r y t o t h i s , the G r i g n a r d r e a g e n t y i e l d s a r e l a t i v e l y s t a b l e a l k y l s i l a n e bond ( f i g u r e 3b) a p p l i c a b l e o v e r the pH-range o f 2 - 7 | l 5 , 1 6 | o r 8 |6|. A d i s a d v a n t a g e o f b o t h r e a c t i o n s i s t h a t r e l a t i v e l y low s u r f a c e c o v e r a g e s a r e o b t a i n e d . An e x t r a d i s a d v a n t a g e o f the G r i g n a r d r e a c t i o n i s , t h a t the magnesium s a l t s formed d u r i n g the r e a c t i o n r e m a i n p a r t i a l l y a d s o r b e d onto the p a c k i n g | l 7 | , w h i c h might i n f l u e n c e r e t e n t i o n .

*

i

m

i

I \

stable bond (b)

RMgCl^

1-1

^ ^ h y d r o l y t i c instable bond (a)

^•B low surface coverages (a and b)

^mMgCl^- salts remain adsorbed (b)

FIGURE 3

(a) Formation of an N-bonded alkylamino silane phase

(h) Formation of an alkyl silyl bonded phase using a Grignard reagent

(Hi) Reaction with organo silanes /17-30/

By f a r t h e most w i d e l y a p p l i e d method t o p r e p a r e c h e m i c a l l y bonded p h a s e s i s the r e a c t i o n w i t h d i c h l o r o - and t r i c h l o r o s i l a n e s . B o n d i n g o f t h e s e s i l a n e r e a g e n t s w i t h the s i l i c a s u r f a c e o c c u r s t h r o u g h e l i m i n a t i o n o f HC1.

As shown i n f i g u r e 4a_, d i c h l o r o s i l a n e can r e a c t i n two ways; w i t h one o r w i t h two c h l o r i n e atoms. Due t o s t e r e o c h e m i c a l h i n d r a n c e the t r i c h l o r o s i l a n e c a n o n l y r e a c t w i t h one o r two c h l o r i n e atoms | 3 l | , f i g u r e 4b. I n o t h e r words, t h e r e w i l l be some u n r e a c t i v e S i - C l groups l e f t , w h i c h w i l l be h y d r o l y s e d d u r i n g the w a s h i n g p r o c e d u r e . I n f a c t , i f t r i c h l o r o s i l a n e f a v o u r s t o r e a c t w i t h o n l y one c h l o r i d e g r o u p , more s i l a n o l groups w i l l be i n t r o d u c e d than were p r e s e n t on the s i l i c a s u r f a c e b e f o r e r e a c t i o n . O b v i o u s l y , p r e p a r i n g r e v e r s e d phase m a t e r i a l s means t h e c o n v e r s i o n o f t h e p o l a r s i l i c a m a t e r i a l i n t o a n o n p o l a r bonded p a c k i n g . The aim i n t h a t i s t o c o n v e r t as much as s i l a n o l g r o u p s as p o s s i b l e . Hence,

6 BIFUNCTIONAL REAGENT : S i — 0 — j - H : S i 0 — H + S i C i / \ CH„

TRI FUNCTIONAL REAGENT

| S i 0 H C l

I.

M >

M

• Q i high surface coverage

wm polymerization • (extra) silanol groups

mm surface coverage not well defined

1

••••.I.-'

2

°JIJ K

V

,

/

Reaction of silanol groups to give an alkyl s i l y l bonded phase using dichloro-and trichlorosilanes

The S i - O - S i bond f o r m a t i o n i s a l s o r e l a t i v e l y r e s i s t e n t t o h y d r o l y s i s and a p p l i c a b l e o v e r t h e pH-range between 2 and 7 o r 8.

T h i s t y p e o f b o n d i n g r e a c t i o n , w h i c h y i e l d s r e l a t i v e l y h i g h s u r f a c e

c o v e r a g e s , s h o u l d be c a r r i e d o u t under a b s o l u t e l y anhydrous c o n d i t i o n s . P r e s e n c e of m o i s t u r e causes h y d r o l y s i s o f S i - C l bonds so t h a t p o l y m e r i z a t i o n r e a c t i o n s can o c c u r on the s i l i c a s u r f a c e . T h i s r e s u l t s i n bad d e f i n e d s u r f a c e c o v e r a g e s .

A n o t h e r p r o b l e m w i t h t h i s t y p e o f r e a c t i o n i s t h e c a l c u l a t i o n o f the e x a c t s u r f a c e c o v e r a g e . S i n c e the d e g r e e o f c o n v e r s i o n o f c h l o r i n e atoms i s unknown the m o l e c u l a r w e i g h t o f the bonded o r g a n i c group i s o n l y be a p p r o x i m a t e l y known. T h i s r e s u l t s i n an e s t i m a t e d s u r f a c e c o v e r a g e .

As p r o v e d c h r o m a t o g r a p h i c a l l y by Knox and J u r a n d |321 under r e v e r s e d phase c o n d i t i o n s , a s u b s t a n t i a l p a r t o f s i l a n o l g r o u p s r e m a i n b e h i n d a f t e r r e a c t i o n w i t h n - o c t a d e c y l t r i c h l o r o s i l a n e . P o s t - s i l a n i z a t i o n w i t h a s m a l l s i l y l a t i n g agent

i n o r d e r t o cap some e x t r a s i l a n o l groups p r o v e d t o i n f l u e n c e b o t h r e t e n t i o n and s e l e c t i v i t y m a r k e d l y .

7 I t s h o u l d be n o t e d t h a t the above t y p e o f bond, i . e . the S i - O - S i bond, can a l s o be o b t a i n e d by u s i n g d i a l k o x y - and t r i a l k o x y s i l a n e s i n s t e a d o f c h l o r o s i l a n e s . However, i n g e n e r a l , the s u r f a c e c o v e r a g e s a r e l o w e r .

D e s p i t e some s h o r t c o m i n g s t o be d i s c u s s e d below, the i n t r o d u c t i o n o f c h e m i c a l l y bonded s t a t i o n a r y p h a s e s has r e v o l u t i o n i z e d r e v e r s e d phase l i q u i d c h r o m a t o g r a p h y . T h i s i s c l e a r l y d e m o n s t r a t e d by numerous p u b l i c a t i o n s , r e v i e w a r t i c l e s and books |4,6,33-44|, a p p e a r e d i n t h e l a s t few y e a r s . Today, r e v e r s e d phase l i q u i d c h r o m a t o g r a p h y , RPLC, i s a w i d e l y used t e c h n i q u e f o r the s e p a r a t i o n o f a l a r g e v a r i e t y o f m i x t u r e s . Both p o l a r and n o n p o l a r s o l u t e s can be s e p a r a t e d by a d a p t i n g the m o b i l e p h a s e , w h i c h , i n g e n e r a l , e x i s t s o f a b i n a r y m i x t u r e o f an o r g a n i c s o l v e n t ( m o d i f i e r ) and w a t e r . C h a n g i n g t h e c o m p o s i t i o n o f t h i s b i n a r y m i x t u r e , by i n c r e a s i n g o r d e c r e a s i n g the m o d i f i e r c o n t e n t , t h e c h r o m a t o g r a p h i c

s e p a r a t i o n p r o c e s s can be g r e a t l y i n f l u e n c e d . S i n c e t h e c h r o m a t o g r a p h i c RPLC-s y RPLC-s t e m w i t h c h e m i c a l l y bonded phaRPLC-seRPLC-s i RPLC-s RPLC-s i m p l e and r a p i d l y e q u i l i b r a t e d , the m o d i f i e r c o n t e n t can a l s o be changed d u r i n g a c h r o m a t o g r a p h i c r u n w i t h o u t s t r i p p i n g the s t a t i o n a r y p h a s e . As a l r e a d y n o t e d , t h i s t e c h n i q u e , c a l l e d

g r a d i e n t - e l u t i o n , cannot be a p p l i e d i n c l a s s i c a l L L C - p a r t i t i o n s y s t e m s . F o r t h i s r e a s o n and t h e d i s a d v a n t a g e s enumerated above, RPLC has v i r t u a l l y r e p l a c e d LLC.

From i t s c a p a b i l i t y o f s e p a r a t i n g b o t h r e l a t i v e l y n o n p o l a r s o l u t e s and v e r y p o l a r o r i o n i c compounds by a d a p t i n g the m o b i l e phase c o m p o s i t i o n , i t has a l s o d r o v e o u t t h e L S C - v a r i a n t f o r t h e g r e a t e r p a r t . I t has been e s t i m a t e d t h a t 80% o f the a n a l y t i c a l L C - s e p a r a t i o n s a r e c a r r i e d o u t by u s i n g the R P L C - t e c h n i q u e

| l 5 , 4 5 | . The r e p r o d u c i b i l i t y o f the method i s v e r y good and the columns a r e s t a b l e as l o n g as the pH o f t h e e l u e n t i s k e p t w i t h i n t h e abovementioned r e g i o n .

STATE OF THE ART OF RPLC AT THE START OF THE PRESENT INVESTIGATION

The b r i e f s u r v e y g i v e n above d e s c r i b e s the s t a t e o f the a r t o f r e v e r s e d phase l i q u i d chromatography u s i n g c h e m i c a l l y bonded s t a t i o n a r y phases a t the end o f 1975, when we s t a r t e d the r e s e a r c h r e p o r t e d i n t h i s t h e s i s .

A l t h o u g h a v a r i e t y o f n o n p o l a r bonded phases were c o m m e r c i a l l y a v a i l a b l e | 3 9(4 6 |a u n f o r t u n a t e l y , t h e i r d e t a i l e d s p e c i f i c a t i o n s were n o t d i s c l o s e d (and

t h e y s t i l l a r e n o t ! ) . L a r g e m u t u a l d i f f e r e n c e s were, and s t i l l a r e , o b s e r v e d i n p r a c t i c e w i t h t h e s e bonded p a c k i n g s . From t h a t p o i n t o f v i e w t h e r e was a demand f o r t h e p r e p a r a t i o n and i n v e s t i g a t i o n o f w e l l - d e f i n e d c h e m i c a l l y bonded s t a t i o n a r y p h a s e s .

I r r e s p e c t i v e o f t h e u n d i s c l o s e d s p e c i f i c a t i o n s , i n g e n e r a l , one was d i s -s a t i -s f i e d w i t h

(i) the stability of the bonded packings

8

(even between batch to batch packings from the same supplier)

(Hi) mutual differences in separation for the same type of chemically bonded phase, usually expressed as the selectivity. The selectivityt a, is

defined as the ratio of net retentions, (t„ •- t )/(tT} .- t )3 of two different

solutes i and where t is the hold-up of the column, (iv) the column efficiences

U n f o r t u n a t e l y , the s t a b i l i t y o f the c h e m i c a l l y bonded phase (a g e n e r a l l y used e x p r e s s i o n f o r the bonded o r g a n i c groups and the s i l i c a s u p p o r t t o g e t h e r ) , i s n o t s o l v e d i n f a v o u r o f t h e customer. Bonded p a c k i n g s b a s e d on s i l i c a cannot be used o u t s i d e the pH r e g i o n o f 2 to ^7.5. Below pH 2 the bonded chains are h y d r o l y t i c a l l y s t r i p p e d from the s u p p o r t , whereas o v e r pH ^7.5 t h e s i l i c a -s k e l e t o n -s t a r t -s t o d i -s -s o l v e . I n o t h e r word-s, t i l l now t h e r e i -s no r e v e r -s e d pha-se m a t e r i a l a v a i l a b l e w h i c h can be used o v e r the complete pH-range, a p a r t from some l e s s s u c c e s s f u l a t t e m p t s w i t h p u r e c a r b o n a d s o r b e n t s i n the l a s t few y e a r s |47, 48| .

The v a r i a t i o n i n r e t e n t i o n , t , ( i i ) and t h e m u t u a l d i f f e r e n c e s i n s e p a r a -K

t i o n o r s e l e c t i v i t y , a, (Hi), a r e r e l a t e d t o t h e s u r f a c e c o v e r a g e o f t h e bonded o r g a n i c c h a i n s o n t o t h e s i l i c a s u r f a c e . I n g e n e r a l , the lower t h i s c o v e r a g e , the more t h e r e t e n t i o n d e c r e a s e s . O b v i o u s l y , t h i s d e c r e a s e i s n o t o n l y c a u s e d by the r e d u c e d number o f bonded c h a i n s , as i l l u s t r a t e d i n f i g u r e 5, b u t a l s o by t h e r e l a t i v e i n c r e a s e o f the r e m a i n i n g p o l a r s i l a n o l g r o u p s . HIGH COVERAGE LOW COVERAGE FIGURE 5

Decrease in coverage results in an increase of silanol groups

The e f f i c i e n c y , p l a t e number o r q u a l i t y o f the column (iv), i s s t r o n g l y d e t e r m i n e d by

(j) the particle size

0

I.

1

-I

1

1

-I

I

I.

-I

1

I

1

9

(¿3) the particle size distribution

(333) and the column packing procedure

I f t h e p a r t i c l e s a r e t o o l a r g e (j), or o f w i d e l y v a r i a b l e s i z e (33), the

p l a t e number i s s e r i o u s l y r e d u c e d . And i f a column i s l o o s e l y p a c k e d t h i s a l s o r e s u l t s i n a l o w e r number.

In c o n t r a s t t o the l a r g e r p a r t i c l e s used i n GC, the s m a l l p a r t i c l e s a p p l i e d i n HPLC (5 - 10 um) c a n n o t be packed as a d r y powder, due t o e l e c t r o s t a t i c e f f e c t s (333). S i n c e the p a r t i c l e s s t r o n g l y a g g l o m e r a t e , a s u s p e n s i o n ( s l u r r y )

has t o be made f r o m the p a c k i n g m a t e r i a l and a l i q u i d o f a p p r o x i m a t e l y the same d e n s i t y . W i t h a h i g h f l o w o r h i g h p r e s s u r e the s l u r r y i s p r e s s e d i n t o the column w h i c h a t the end i s p r o v i d e d w i t h a 2 um " s i e v e " ( f r i t ) . The optimum composition o f t h e s l u r r y l i q u i d has n o t y e t been e l u c i d a t e d . P r o b a b l y , f o r t h a t r e a s o n i t i s s t i l l v e r y d i f f i c u l t t o pack columns r e a l l y r e p r o d u c i b l y .

Formulation and aim of the present investigation

I n o r d e r t o e l u c i d a t e the d i s c u s s e d b e h a v i o u r o f the v a r i a t i o n i n r e t e n t i o n

( i i ) , and s e l e c t i v i t y (Hi), i n g e n e r a l , the f o r m u l a t i o n o f the i n v e s t i g a t i o n

was t o p r e p a r e c h e m i c a l l y bonded phases w i t h a w e l l d e f i n e d o r g a n i c l a y e r bonded o n t o the s i l i c a s u r f a c e . S i n c e i n GC a l a r g e v a r i e t y o f s t a t i o n a r y p h a s e s w i t h a wide f i e l d o f p o l a r i t y were and s t i l l a r e c o m m e r c i a l l y a v a i l a b l e , i t was t h o u g h t t o p r e p a r e w e l l - d e f i n e d c h e m i c a l l y bonded phases from d i f f e r e n t p o l a r i t y . The r o l e o f t h e s e c h e m i c a l l y bonded phases i n the s e p a r a t i o n p r o c e s s ( i . e . t h e r e t e n t i o n mechanism) was p r o p o s e d as the u l t i m a t e aim o f the r e s e a r c h .

To a v o i d problems s u c h as p o l y m e r i z a t i o n , i n s t a b l e bonds, magnesium s a l t d e p o s i t i o n and e x t r a s i l a n o l g r o u p s , c h e m i c a l l y bonded phases have been p r e p a r e d f r o m m o n o f u n c t i o n a l r e a g e n t s , i . e . from d i m e t h y l m o n o c h l o r o - and dimethylmono-a l k o x y s i l dimethylmono-a n e s . The r e dimethylmono-a c t i o n scheme i s i l l u s t r dimethylmono-a t e d i n f i g u r e 6.

, CB

r

13

'••Si 0 - p H + Cl-j-Si R

i

j 1

»• l&jStf 0 Si R

en

d

4

ÏSi O-j-E + R0-j-S'i—R JUDGEMENT

L 1

I

s t a b l e bond

h i g h s u r f a c e c o v e r a g e

J L w e l l d e f i n e d s u r f a c e c o v e r a g e FIGURE 6

Reaction of silanol groups to give an alkyl silyl bonded phase using

monofunc-tional reagents

10

Unger 131,37,491 was the f i r s t who has drawn a t t e n t i o n to the use o f mono-f u n c t i o n a l r e a g e n t s . However, t i l l now o n l y a mono-few s t u d i e s r e p o r t the use o mono-f t h e s e r e a g e n t s f o r the p r e p a r a t i o n o f c h e m i c a l l y bonded p h a s e s f o r HPLC [44,50—521 , T h i s c l e a r l y i l l u s t r a t e s the u n d e r e s t i m a t i o n o f the advantages o f m o n o f u n c t i o n a l r e a g e n t s o v e r d i - and t r i f u n c t i o n a l r e a g e n t s .

A v a r i e t y o f w e l l - d e f i n e d c h e m i c a l l y bonded phases has been p r e p a r e d f r o m m o n o f u n c t i o n a l r e a g e n t s . The p r e p a r a t i o n method i s v e r y s i m p l e , y i e l d i n g h i g h

(maximum) s u r f a c e c o v e r a g e s . E v e n a t room-temperature t h i s p r o c e d u r e y i e l d s h i g h c o v e r a g e s ( s l i g h t l y l o w e r t h a n maximum c o v e r a g e ) . The m o n o f u n c t i o n a l r e a g e n t s i t s e l v e s , can e a s i l y be s y n t h e s i z e d by an o n e s t e p c a t a l y t i c h y d r o s i l y l a -t i o n r e a c -t i o n o f -t e r m i n a l o l e f i n s , w h i c h has been d e s c r i b e d i n C h a p -t e r 4.

The p r e p a r e d bonded p a c k i n g s can be s u b d i v i d e d i n t o t h r e e g r o u p s , i n w h i c h t h e R-group o f f i g u r e 6 p r e s e n t s a

(k) purely hydrocarbon chain, i.e. R - ^y^^n+l

T h i s c h a i n was v a r i e d i n l e n g t h from 1 to 22 c a r b o n atoms, i n o r d e r t o i n v e s t i g a t e the i n f l u e n c e o f c h a i n l e n g t h on t h e c h r o m a t o g r a p h i c s e p a r a t i o n p r o c e s s .

The p r e p a r a t i o n o f t h e s e phases has been d e s c r i b e d i n C h a p t e r 4. I n g e n e r a l , t h e s u r f a c e c o v e r a g e o f t h e s e p h a s e s d e c r e a s e s w i t h i n c r e a s i n g c h a i n l e n g t h f r o m

2 2 some 4 |imol/m f o r the s h o r t e s t c h a i n w i t h one c a r b o n atom (RP-1), to 3.0 limol/m

f o r the l o n g e s t one (RP-22).

S i n c e the o r i g i n a l s i l a n o l c o n c e n t r a t i o n on the s i l i c a s u p p o r t i s about 2

8 ymol/m , the o b t a i n e d s u r f a c e m o d i f i c a t i o n o f l e s s t h a n 50% o f t h e t o t a l number o f s i l a n o l groups might s u g g e s t t h a t the s u r f a c e c o v e r a g e i s r a t h e r i n c o m p l e t e . To a n a l y s e t h i s p o i n t , two g e o m e t r i c a l models have been c o n s t r u c t e d , b a s e d on m o l e c u l a r d i m e n s i o n s . In the f i r s t model, d e s c r i b e d i n C h a p t e r 2, i t has been d e m o n s t r a t e d t h a t the t o p o l o g y o f the e x p e r i m e n t a l s u r f a c e c o v e r a g e o f about

2

4 pmol/m a g r e e s c l o s e l y w i t h t h e t h e o r e t i c a l l y e x p e c t e d maximum c o v e r a g e . I n t h e second model, d e s c r i b e d i n C h a p t e r 4, the i n f l u e n c e o f c h a i n l e n g t h on d e c r e a s i n g s u r f a c e c o v e r a g e has been i n v e s t i g a t e d . H e r e , i t has been d e m o n s t r a t e d t h a t the d e c r e a s e i n s u r f a c e c o v e r a g e f o r l o n g e r c h a i n s i s l a r g e l y d e t e r m i n e d by the r a d i u s o f the p o r e s , p r e s e n t i n the s i l i c a s u p p o r t .

The i n f l u e n c e o f the R P - c h a i n l e n g t h on r e t e n t i o n and s e l e c t i v i t y has been e x t e n s i v e l y i n v e s t i g a t e d . T h i s i s r e p o r t e d i n C h a p t e r 6. From t h e r e t e n t i o n b e h a v i o u r as a f u n c t i o n o f R P - c h a i n l e n g t h , i t c o u l d be c o n c l u d e d t h a t o n l y the e x t e r i o r p a r t o f the c h e m i c a l l y bonded c h a i n s t a k e s p a r t i n the s e p a r a t i o n p r o c e s s and not the complete c h a i n l e n g t h . I t a p p e a r e d t h a t the i n f l u e n c e o f the a l k y l c h a i n l e n g t h on b o t h r e t e n t i o n and s e l e c t i v i t y i s by f a r s u b o r d i n a t e to the

11 i n f l u e n c e o f the m o b i l e p h a s e . From t h e s e d a t a , a " c o m p u l s o r y a b s o r p t i o n " mechanism i s p r o p o s e d f o r the r o l e o f the c h e m i c a l l y bonded s t a t i o n a r y phase i n r e v e r s e d phase l i q u i d chromatography.

(kk) hydrocarbon chain containing a functional end-group, i.e. R - C

B.-X

The p r e p a r a t i o n o f t h e s e phases has been d e s c r i b e d i n C h a p t e r 4. An a v e r a g e 2

s u r f a c e c o v e r a g e o f 3.3 pmol/m was o b t a i n e d f o r t h e s e p h a s e s , w h i c h were p r e p a -r e d a t -room-tempe-ratu-re.

(kkk) functional group, i.e. R - X

The s y n t h e s i s o f t h e s e p h a s e s has been r e p o r t e d i n C h a p t e r 3. Maximum s u r f a c e c o v e r a g e s were o b t a i n e d and t h e p r e s e n c e o f t h e s e bonded f u n c t i o n a l groups on the s i l i c a s u r f a c e c o u l d be c o n f i r m e d by i n f r a r e d s p e c t r o s c o p y . T h i s c h a p t e r emphasizes the i m p o r t a n c e o f a c o r r e c t l y c a l c u l a t e d s u r f a c e c o v e r a g e .

B e f o r e b e i n g a b l e t o i n t e r p r e t the a b s o l u t e r e t e n t i o n d a t a , a v e r y fundamen-t a l p r o b l e m had fundamen-t o be s o l v e d , i . e . fundamen-t h e e x a c fundamen-t h o l d - u p fundamen-time i n r e v e r s e d phase l i q u i d chromatography. C h a p t e r 5 p r e s e n t s a c o m p a r i s o n o f s e v e r a l methods a p p l i e d i n r e v e r s e d phase chromatography t o d e t e r m i n e the h o l d - u p t i m e . A recommendation i s g i v e n f o r the method t h a t a g r e e s b e s t w i t h the t h e o r e t i c a l l y e x p e c t e d h o l d - u p v a l u e .

F i n a l l y , C h a p t e r 7 d e s c r i b e s the column p a c k i n g p r o c e d u r e w h i c h was u s e d t o p a c k a l l bonded p a c k i n g s .

As c o n c l u s i o n , i t can be s t a t e d t h a t (I) w e l l - d e f i n e d c h e m i c a l l y bonded s t a t i o n a r y phases can be e a s i l y p r e p a r e d f r o m m o n o f u n c t i o n a l r e a g e n t s , (11) the maximum s u r f a c e c o v e r a g e c a n be r e p r o d u c i b l y o b t a i n e d when the same b a t c h o f s i l i c a i s used, and (III) when, i n t h e n e a r f u t u r e , the t e c h n o l o g y o f making r e p r o d u c i b l e s i l i c a b a t c h e s w i l l be a v a i l a b l e , t h e n the way w i l l be c l e a r e d f o r r e p r o d u c i b l e RPLC-chromatography i n b o t h r e t e n t i o n and s e l e c t i v i t y .

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Y o r k , 1968

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6. Knox, J.H., High Performance Liquid Chromatography, E d i n b u r g h U n i v e r s i t y P r e s s , E d i n b u r g h , 1979

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17. M a j o r s , R.E. and Hopper, H.J., J, Chromatogr. Sci., 12, 767, 1964

18. G i l p i n , R.K., K o r p i , J.A. and J a n i c k i , C.A., Anal. Chem., £ 6 , 1314, 1974 19. Knox, J.H. and P r y d e , A., J, Chromatogr., 112, 171, 1975

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21. K i n g s t o n , D.G.I, and G e r h a r t , B.B., J. Chromatogr., 116, 182, 1976 22. K i k t a , E . J . and G r u s h k a , E . , Anal. Chem., 48, 1098, 1976

23. K a r c h , K., S e b e s t i a n , I . and H a l a s z , I . , J. Chromatogr., 122, 3, 1976

24. H e i n e t s b e r g e r , H., M a a s f e l d , W. and R i c k e n , H., Chromatographia, £ , 303, 1976 25. H e m e t s b e r g e r , H., K e l l e r m a n n , M. and R i c k e n , H., Chromatographia,10,726,1977 26. L i t t l e , C . J . , D a l e , A.D. and E v a n s , M.B., J. Chromatogr., 153, 381, 1978;

153, 543, 1978

27. H e n n i o n , M.C., P i c a r d , C. and Caude, M., J. Chromatogr. Sci., 166, 21, 1978 28. L i t t l e , C . J . , D a l e , A.D., Evans, M.B. and W h a t l e r , J.B., J. Chromatogr.,

171, 431, 1978; 171, 435, 1978

29. H e n n i o n , M.C., P i c a r d , C., Caude, M. and R o s s e t , R., Analusis, 6_, 369, 1978 30. H e m e t s b e r g e r , H., Behrensmeyer, P., H e n n i n g , J . and R i c k e n , H.,

Chromatographia, 12, 71, 1979

31. Unger, K.K., B e c k e r , N. and R o u m e l i o t i s , P., J. Chromatogr., 125, 115, 1976 32. Knox, J.H. and J u r a n d , J . , J. Chromatogr., 142, 651, 1977

33. G r u s h k a , E . ( e d . ) , Bonded Stationary Phases in Chromatography, Ann A r b o r S c i e n c e , Ann A r b o r , M i c h . , 1974

34. S n y d e r , L.R. and K i r k l a n d , J . J . , An Introduction to Modern Liquid

Chromato-graphy, W i l e y I n t e r s c i e n c e , New Y o r k , 1975

35. D e y l , Z., Macek, K. and Janak ( e d s . ) , Liquid Column Chromatography, A Survey

of Modern Techniques and Applications, E l s e v i e r , Amsterdam, 1975

1 3 U.K., 1976

37. Unger, K.K., Porous Siliea, J . C h r o m a t o g r . L i b r . , V o l . 1 6 , E l s e v i e r , Amsterdam, 1979

38. P r y d e , A., J. Chromatogr. Soi.3 VI, 486, 1974

39. M a j o r s , R.E., Analusis, _3, 549, 1975

40. Rehäk, V. and SmolkovS, E . , Chromatographia, 9_, 219, 1976 41. G r u s h k a , E . and K i k t a J r . , E . J . , Anal. Chem., 49_, 1004A, 1977 42. M a j o r s , R.E., J. Chromatogr. Sei., J_5, 334, 1977

43. C o l i n , H. and G u i o c h o n , G., J. Chromatogr., _14j_, 289, 1977 44. K a r g e r , B.L. and G i e s e , R.W., Anal. Chem., 50, 1048A, 1978

45. S n y d e r , L.R., D o l a n , J.W. and G a n t , J.R., J. Chromatogr., 165, 3, 1979 46. M a j o r s , R.E. , Intl. Lab., (Nov./Dec. 1975) , 11

47. G u i o c h o n , G. and C o l i n , H., Speetra-Physies Chrom. Review, 4 ( 2 ) , 2, 1978 48. C o l i n , H. and G u i o c h o n , G., J. Chromatogr., 141, 289, 1977

49. R o u m e l i o t i s , P. and Unger, K.K., J. Chromatogr., 149, 211, 1978

50. Tanaka, N., G o o d e l l , H. and K a r g e r , B.L., J. Chromatogr., 158, 233, 1978 51. Moure'y , T.H. and S i g g i a , S., Anal. Chem., 5j_, 763, 1979

52. Venne, J.L.M. v a n d e , Thesis, E i n d h o v e n U n i v e r s i t y o f T e c h n o l o g y , Wibro, Helmond, The N e t h e r l a n d s , 1979

15

2

A geometrical model for chemically bonded

TMS and PDS phases.

ABSTRACT

A g e o m e t r i c a l model i s d e v e l o p e d f o r monomeric TMS and PDS phases t h a t a r e c h e m i c a l l y bonded t o s i l i c a . U s i n g e x p e r i m e n t a l d a t a f o r maximum s u r f a c e c o v e r a g e and c o n s i d e r i n g amorphous s i l i c a as a c o l l e c t i o n o f d i s t o r t e d c r y s t a l s , we c a l c u -l a t e t h a t each nm^ o f t h e s i -l i c a s u r f a c e c o n t a i n s 2.3 m o d i f i e d h y d r o x y -l g r o u p s , 1.3 f r e e h y d r o x y l groups and 0.6 p a i r s o f bonded h y d r o x y l g r o u p s , r e s p e c t i v e l y . From t h e d i m e n s i o n s o f t h e s i l a n e m o l e c u l e i t i s c o n c l u d e d t h a t f o r maximum c o v e r a g e t h e TMS and PDS m o l e c u l e s a r e r i g i d l y a t t a c h e d t o t h e s i l i c a s u r f a c e w i t h an S i - O - S i bond a n g l e between 120 and 140 d e g r e e s . The u n r e a c t e d h y d r o x y l groups a r e n o t c o m p l e t e l y s c r e e n e d b u t w i l l be q u i t e i n a c c e s s i b l e on e i t h e r p h a s e . V e r y l i t t l e f r e e s u r f a c e a r e a remains on the s i l i c a s u r f a c e .

INTRODUCTION

The s t a t e o f t h e a r t o f c h e m i c a l l y bonded phases f o r l i q u i d chromatography has been d e s c r i b e d i n two r e c e n t r e v i e w s . I n h i s g e n e r a l s u r v e y o f s t a t i o n a r y p h a s e s f o r HPLC M a j o r s | l | c o n c l u d e s t h a t f o r l i q u i d - l i q u i d chromatography phy-s i c a l l y c o a t e d phy-s t a t i o n a r y phaphy-sephy-s appear t o have been a l l b u t r e p l a c e d by

chemi-16

c a l l y bonded p h a s e s . C o l i n and G u i o c h o n [ 2 ] n o t e t h a t d e s p i t e t h e i r w i d e s p r e a d use i n r e v e r s e d p h a s e l i q u i d c h r o m a t o g r a p h y the s t r u c -t u r e and -t h e r e -t e n -t i o n mechanism o f c h e m i c a l l y bonded phases a r e s t i l l a m a t t e r o f d i s p u t e . We a g r e e w i t h t h e i r c o n c l u s i o n t h a t " t h e most i m p o r t a n t r e a s o n f o r t h e c u r r e n t c o n t r o v e r s y r e g a r d i n g the s t r u c t u r e and p r o p e r t i e s o f bonded phases and e s p e c i a l l y t h e r e t e n t i o n mechanism a r i s e s from the g r e a t d i f f i c u l t i e s e n c o u n t e r e d i n p r e p a r i n g b a t c h e s o f s i l i c a g e l s and m o d i f i e d s i l i c a g e l s which a r e r e p r o d u c i b l e o r even r e p e a t a b l e " .

To improve o u r i n s i g h t i t i s , t h e r e f o r e , a p r e r e q u i s i t e t o p o s s e s s w e l l - d e f i n e d bonded phases f o r w h i c h the s u r f a c e c o v e r a g e - e x p r e s s e d as t h e number o f m o d i f i e d h y d r o x y l g r o u p s [3] - can be c a l c u l a t e d w i t h a minimum o f a d d i t i o n a l a s s u m p t i o n s . I n t h i s r e s p e c t a l k o x y s i l a n e s o r d i c h l o r o and t r i c h l o r o s i l a n e s a r e l e s s s u i -t a b l e , b e c a u s e -t h e y can r e a c -t w i -t h one o r -two h y d r o x y l g r o u p s and a r e s u b j e c t t o p o l y m e r i z a t i o n r e a c t i o n s . Such problems a r e a v o i d e d i f we use m o n o c h l o r o s i l a n e s , such as t r i m e t h y l c h l o r o s i l a n e (TMS) or p h e n y l d i m e t h y l c h l o r o s i l a n e (PDS). Even t h e n two a d d i t i o n a l d a t a a r e needed t o c o n v e r t the c a r b o n c o n t e n t of the m o d i f i e d s i l i c a to the number o f h y d r o x y l g r o u p s removed, i . e . the s p e c i f i c s u r f a c e a r e a o f the p a c k i n g m a t e r i a l and t h e s u r f a c e d e n s i t y o f h y d r o x y l groups o r i g i n a l l y p r e s e n t . L i t e r a t u r e d a t a , c o n f i r m e d i n the p r e -sent s t u d y a g r e e t h a t m o d i f i c a t i o n o f h y d r o x y l groups i s n e v e r c o m p l e t e [2, 3 ] , so t h a t the c h r o m a t o g r a p h i c a c t i v i t y o f r e m a i n i n g h y d r o x y l g r o u p s r e m a i n s d e b a t a b l e . C h r o m a t o g r a p h i c r e t e n t i o n d a t a can be u s e f u l , but c a r e f u l e x p e r i m e n t s a r e r e q u i r e d t o d i f f e r e n -t i a -t e be-tween -the i n f l u e n c e o f -the m o b i l e phase, -the a c -t i v i -t y o f u n m o d i f i e d h y d r o x y l groups and the p r o p e r t i e s o f the c h e m i c a l l y bonded p h a s e . Such e x p e r i m e n t s w i l l be r e p o r t e d i n a f o l l o w i n g paper [ A ] , w h i c h a l s o d e s c r i b e s the p r e p a r a t i o n o f w e l l - d e f i n e d monomeric p h a s e s .

I n t h i s s t u d y we e x p l o r e a n o t h e r a p p r o a c h . E x c e p t f o r a non-v a l i d a t e d s t a t e m e n t by Unger and c o w o r k e r s [3,5] t h a t t h e maximum c o v e r a g e by TMS a g r e e s w i t h v a l u e s c a l c u l a t e d from m o l e c u l a r s i z e s ,

I 7 the m o l e c u l a r geometry o f c h e m i c a l l y bonded phases appears t o have been i g n o r e d . I n t h i s i n v e s t i g a t i o n we s h a l l p r e s e n t a g e o m e t r i c a l model b a s e d on m o l e c u l a r d i m e n s i o n s , t h a t p r o v i d e s us w i t h a b e t t e r i n s i g h t i n t o the s t r u c t u r e o f the bonded phase and the s c r e e -n i -n g o f r e m a i -n i -n g h y d r o x y l g r o u p s .

THE STRUCTURE OF SILICA

T h e r e a r e two b a s i c t y p e s o f s i l i c a : c r y s t a l l i n e and amor-phous, the l a t t e r b e i n g e x c l u s i v e l y used i n chromatography. Both t y p e s p o s s e s s the same s t r u c t u r a l u n i t c o n s i s t i n g o f s i l i c o n o x i d e t e t r a h e d r o n s . The e s s e n t i a l d i f f e r e n c e between e i t h e r t y p e i s the o r i e n t a t i o n o f the t e t r a h e d r o n s i n s p a c e . The s t r u c t u r e o f c r y s -t a l l i n e s i l i c a c o n s i s -t s of a -t h r e e d i m e n s i o n a l ne-twork of p a r a l l e l p l a n e s of s i m i l a r u n i t s , whereas i n t h e amorphous s i l i c a t h e pa-r a l l e l p l a n e s a pa-r e m i s s i n g a l t h o u g h t h e t h pa-r e e d i m e n s i o n a l s t pa-r u c t u pa-r e p e r s i s t s [ 6 ] .

The s t r u c t u r e o f the v a r i o u s c r y s t a l l i n e forms o f s i l i c a i s w e l l - k n o w n from X - r a y d i f f r a c t i o n s t u d i e s . Some r e l e v a n t d a t a a r e c o l l e c t e d i n T a b l e 1. T h r e e main c r y s t a l l i n e forms can be d i s t i n g u i s h e d : q u a r t z , t r i d y m i t e and c r i s t o b a l i t e , each e x i s t i n g i n s e -v e r a l m o d i f i c a t i o n s [ 7 , 8 ] . A l t h o u g h t h e s e main t y p e s a r e s t a b l e o v e r w e l l d e f i n e d t e m p e r a t u r e r a n g e s ( q u a r t z up t o 8 7 0 ° C, t r i d y -m i t e between 870 and 1470° C and c r i s t o b a l i t e above 1470° C ) , t h e y a r e n o t r e a d i l y i n t e r c o n v e r t i b l e , as i s shown by the f a c t t h a t a l l t h r e e a r e f o u n d as m i n e r a l s , though t r i d y m i t e and c r i s t o b a l i t e a r e r a r e i n c o m p a r i s o n w i t h q u a r t z . The t r a n s i t i o n p o i n t s o f the a and S m o d i f i c a t i o n s o f t r i d y m i t e and c r i s t o b a l i t e a r e a t r e l a t i -v e l y low t e m p e r a t u r e s , 120 - 160° and 200 - 275° C, r e s p e c t i -v e l y . The f a c t t h a t t h e s e t r a n s i t i o n s c a n be s t u d i e d a t t e m p e r a t u r e s a t w h i c h the forms a r e m e t a s t a b l e i s a f u r t h e r i n d i c a t i o n o f t h e d i f -f i c u l t y o -f c o n v e r t i n g one o -f t h e t h r e e v a r i e t i e s o -f s i l i c a i n t o a n o t h e r [ 9 ] . C o n s e q u e n t l y , a l l c r y s t a l l i n e forms can e x i s t under

TABLE 1

MODIFICATIONS OF S I L I C A *

S i 02~ f o r m c r y s t a l S i - O - S i s p e c i f i c m o l a r S i atoms number o f h y d r o x y l s

s y s t e m bond g r a v i t y volume per p e r 2 nm a n g l e (g/cm3) [29] ( c m3) [29] 2 nm c a l c u l a t e d from d e n s i t y l i t e r a t u r e d a t a a - q u a r t z h e x a g o n a l 142° [9] 2 .65 22.69 0 - q u a r t z h e x a g o n a l 150° [14] 2.53 23.72 8.6 4.3 4.3 [11,41] o r t r i d y m i t e rhombic

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o r d i n a r y c o n d i t i o n s e n c o u n t e r e d i n c h r o m a t o g r a p h i c columns. In t u r n , each of t h e s e c r y s t a l s can be c o n s i d e r e d as a s t a r t i n g p o i n t to f o r m u l a t e a model f o r the amorphous s i l i c a used i n c h r o m a t o g r a -phy.

Indeed, by n a t u r e o f i t s amorphous c h a r a c t e r , the s i l i c a s o f c h r o m a t o g r a p h i c i n t e r e s t cannot be a n a l y s e d by X - r a y d i f f r a c t i o n or by s u r f a c e t e c h n i q u e s , such as ESCA. C o n s e q u e n t l y , s e v e r a l a u t h o r s c o n s i d e r the s t r u c t u r e of amorphous s i l i c a as a d i s t o r t e d c r y s t a l . S t b b e r [ 1 0 ] took the q u a r t z s t r u c t u r e as a s t a r t i n g p o i n t , whereas I l e r [ 1 1 ] , Hockey and coworkers [12, 1 3 ] and B o k s a n y i e t a l . [14] argue t h a t amorphous s i l i c a s h o u l d c l o s e l y r e s e m b l e the B - m o d i f i c a t i o n s of c r i s t o b a l i t e or t r i d y m i t e . A c c o r d i n g t o t h e d a t a i n T a b l e 1 t h e r e i s a d i f f e r e n c e i n d e n s i t y and S i - O - S i bond a n g l e between q u a r t z on the one hand and B t r i d y m i t e and ( 3 c r i s t o -b a l i t e on the o t h e r hand.

F o r the p r e s e n t s t u d y i t i s more i m p o r t a n t t o n o t e the d i f -f e r e n c e between the t h r e e c r y s t a l l i n e -forms w i t h r e s p e c t to the number o f f r e e h y d r o x y l groups a t the s u r f a c e . O b v i o u s l y , t h i s number depends upon the c r y s t a l f a c e c o n s i d e r e d , b u t i t i s a p p r e -c i a b l y h i g h e r f o r g - -c r i s t o b a l i t e than f o r q u a r t z or B_t r i d y m i t e .

T h i s i s due t o the f a c t t h a t the o u t e r s u r f a c e s i l i c o n atoms i n B - c r i s t o b a l i t e a r e bound to two oxygen atoms and hence p o s s e s s two h y d r o x y l g r o u p s , whereas i n q u a r t z and B t r i d y m i t e the s u r f a c e s i

-l i c o n atoms a r e bound t o t h r e e oxygen atoms and hence p o s s e s s o n -l y one h y d r o x y l group. Now i n a l l c r y s t a l l i n e forms the s u r f a c e l a y e r c o n s i s t s a l t e r n a t i n g l y o f a t r u e s u r f a c e s i l i c o n atom w i t h one o r two oxygen bonds r u p t u r e d and a s l i g h t l y lower s i t u a t e d s i l i c o n atom c o n n e c t e d t o f o u r oxygen atoms (compare f i g . 1). C o n s e q u e n t l y , i n B " c r i s t o b a l i t e the number o f f r e e h y d r o x y l groups i s e q u a l t o the number o f s i l i c o n atoms i n the s u r f a c e l a y e r , whereas i n q u a r t z and B - t r i d y m i t e the number o f h y d r o x y l groups i s e q u a l t o h a l f the number o f s i l i c o n atoms i n the s u r f a c e l a y e r .

G i v e n the o r d e r e d s t r u c t u r e o f a c r y s t a l , the number o f hy-d r o x y l groups can be r e a hy-d i l y c a l c u l a t e hy-d f o r any c r y s t a l f a c e . Some

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b) A d e r i v e d a m o r p h o u s s t r u c t u r e r e p r e s e n t e d a s a d i s t o r t e d / i - t r i d y m i t e

FIGURE 1

i n d i c a t i v e d a t a a r e i n c l u d e d i n T a b l e 1. However, such f i g u r e s a r e of l i t t l e use f o r amorphous s i l i c a , where t h e r e g u l a r s t r u c t u r e

i s l o s t . I n a d d i t i o n t o the w i d e l y spaced f r e e h y d r o x y l groups found a t the c r y s t a l s u r f a c e amorphous s i l i c a a l s o c o n t a i n s more c l o s e l y spaced h y d r o x y l g r o u p s . S i n c e t h e d i s t a n c e between the