Chromatography and Separation Chemistry - American Chemical

12 Manipulation of Stationary-Phase Acid-Base Properties by a Surface-Buffering Effect Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on February 19, 2015 | http://pubs.acs.org Publication Date: January 27, 1986 | doi: 10.1021/bk-1986-0297.ch012

Boronic Acid-Saccharide Complexation C. H . Lochmüller and Walter B. Hill P. M. Gross Chemical Laboratory, Duke University, Durham, NC 27706

The presence of residual amine groups in sur face bound, silica-based phenylboronic acid phases lowers the apparent pK of the acid groups. This "surface buffering" effect permits boronate-saccharide complexation chemistry to occur at much lower pH values than is t y p i c a l l y the case. The broader implications of the de liberate use of such effects are discussed. a

Lochmuller, Wilder and M a r s h a l l ( 1 ) observed an apparent l o w e r i n g of the pKa f o r surface-bound quinazolines using photothermal spectrometric t i t r a t i o n approach and further evidence f o r the mechanism of t h i s change i n acid base behavior has recently been r e p o r t e d by L o c h m u l l e r and H i l l ( 2 ) . It appears that s i t e s i t e i n t e r a c t i o n s (charge repulsion) and "surface buffering" by residual amine groups combine to produce the apparent change. Mat l i n and Davidson (3 ) a l s o observed interactions between the n e u t r a l s p e c i e s p i c r a m i d o p r o p y l and propylamine i n a "mixed", bonded phase by photοthermal spectrometry. One of the problems with s i l i c a - b a s e d chromatographic materials i s t h e i r r e l a t i v e l y high s o l u b i l i t y i n mobile phases of pH higher than 7.5 units. It i s interesting to speculate that at least for some important i o n i c e q u i l i b r i a , the acid-base chemistry of bound molecules might be manipulated by deliberately i n t r o ducing a "surface b u f f e r i n g " e f f e c t . We chose to e x p l o r e t h i s p o s s i b i l i t y by studying the complexation of saccharides by boronic acid anion because of the importance of saccharides and the nor mally high pH conditions required (pH 8-10) Gilham had postulated that the l o c a l p o s i t i v e or n e g a t i v e charges on c e l l u l o s e i n f l u e n c e d the pKa of phenylboronic acid at low mobile phase i o n i c s t r e n g t h (4 ). In a d d i t i o n , Lochmuller and Amoss f i r s t demon s t r a t e d the advantage of "mixed" bonded phases i n systems where the complexation constants are large and contribute to poor trans f e r k i n e t i c s (5 ). Later Karger used this approach to improve the performance of bound metal complex phases ( 6 ) . Partial derivati zation of amine s i l i c a s might be expected to improve the e f f i c i e n cies obtained with boronate phases by the same reasoning. 0097-6156/86/0297-0210$06.00/0 © 1986 American Chemical Society

In Chromatography and Separation Chemistry; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on February 19, 2015 | http://pubs.acs.org Publication Date: January 27, 1986 | doi: 10.1021/bk-1986-0297.ch012

12.

L O C H M U L L E R A N D HILL

Boronic Acid-Saccharide

Complexation

211

Because of t h e i r r e l a t i v e abundance and t h e i r widespread importance i n b i o l o g y and medicine, mixtures of s a c c h a r i d e s , n u c l e o s i d e s and n u c l e o t i d e s have been separated u s i n g h i g h performance l i q u i d chromatography (HPLC) with amino, cyano, n^. -alkane, s i l i c a and ion-exchange columns (7-9 ). Mobile-phase a d d i t i v e s have a l s o been used to enchance the saccharide selec t i v i t y on both normal and r e v e r s e d phases as w e l l as on c a t i o n exchange resins (10-12). Phenylboronic a c i d - d i o l derivatives have a l s o been analyzed by gas chromatography and mass spectroscopy (13) and the interaction between boronic acids and d i o l s has been e x t e n s i v e l y studied (14). Aqueous boric acid mobile phases were i n i t i a l l y used to separate d i o l s by paper chromatography and by e l e c t r o p h o r e s i s (15). The incorporation of phenylboronic acid as a mobile phase a d d i t i v e was shown to increase the Rf values for most saccharides to a g r e a t e r extend than that seen with b o r i c a c i d ( 16 ). P h e n y l b o r o n i c a c i d derivatives also display a large b a c t e r i o s t a t i c e f f e c t compared to boric acid (17). After Gilham i n i t i a l l y demonstrated the effectiveness of boronic acid-substi tuted cellulose i n the separation of complex mixtures of n u c l e o s i d e s ( 4 ) o t h e r boronic acid stationary phases have been prepared u s i n g p o l y s t y r e n e , c e l l u l o s e and s i l i c a gel (18-23). Although these s t a t i o n a r y phases are quite s e l e c t i v e towards d i o l s , they require alkaline mobile phases f o r adequate r e t e n t i o n which se v e r e l y l i m i t s the use of s i l i c a gel matrices. The present study investigates the use of boronic acid-substituted, amine-modified, s i l i c a g e l matrices for the separation of saccharides and nucleo sides under neutral conditions. P h e n y l b o r o n i c a c i d i s a Lewis a c i d (2Λ) whose a c i d i t y i s i n f l u e n c e d by s u b s t i t u e n t s on the a r o m a t i c r i n g and the p h e n y l boronate a n i o n has a t e t r a h e d r a l s t r u c t u r e (see F i g u r e 1 ) . The e q u i l i b r i u m between the p h e n y l b o r o n a t e a n i o n and a d i o l i s shown i n F i g u r e 2. P h e n y l b o r o n i c a c i d s a l s o i n t e r a c t w i t h amines a l t h o u g h t h i s i n t e r a c t i o n i s q u i t e weak (2_5). The v a r i o u s c r y s t a l l i n e s t r u c t u r e s of p h e n y l b o r o n i c a c i d and monosaccharides and n u c l e o s i d e s t h a t have been p o s t u l a t e d (26,27) demonstrate the p h e n y l b o r o n i c a c i d s can form complexes w i t h a v a r i e t y of d i o l s . Although most of these c r y s t a l l i n e p h e n y l b o r o n i c complexes are a i r s t a b l e , the s a c c h a r i d e complexes r a p i d l y h y d r o l y z e i n water or i n a l c o h o l s and the n u c l e o s i d e complexes h y d r o l y z e i n water i n l e s s than 15 minutes. S i n c e t h e s e d e r i v a t i v e s decompose, i n water, the e x i s t e n c e of an a i r - s t a b l e b o r o n a t e - d i o l complex i s not a guarantee of c o m p l e x a t i o n under c h r o m a t o g r a p h i c c o n d i t i o n s (16) . A more a c c u r a t e p r e d i c t o r i s the magnitude of the f o r m a t i o n c o n s t a n t f o r a p o l y o l w i t h p h e n y l b o r o n i c a c i d i n water ( 2 4 ) .

EXPERIMENTAL Materials. The saccharides, 4-bromotoluene, magnesium turnings, n-bromosuccimide, η-propylamine and benzoyl peroxide were purchased from A l d r i c h Chemical Company (Milwaukee, WI, U.S.A.) and the nucleosides were purchased from Sigma Chemical Company (St. Louis, MO, U.S.A.). B o r i c a c i d and n-butanol were obtained from Mallinckrodt Chemical Company (Paris, KY, U.S.A.).



C H R O M A T O G R A P H Y A N D SEPARATION CHEMISTRY

OH

F i g u r e 1· Water.

C

6

H

5 " B

N

2

6

5

V

0H

3

The E q u i l i b r i u m between Phenylboronic Acid and

OH OH OH

OH

Ηθη H 0 3

HO-"

C

6

7

H

5 -

H

B

3°

2H 0 2

On

*3° 6

5

χ

α>

S

H B:J

2H 0 2

5

F i g u r e 2. The E q u i l i b r i u m between the Phenylboronate Anion and a D i o l .



12.



Complexation

213

3 - a m i n o p r o p y l t r i e t h o x y s i l a n e (AO750) and 3-aminopropyldimethylethoxysilane (A0735) were purchased from Petrarch Systems, Inc. (Briston, PA, U.S.A.) P a r t i s i l 10 S i l i c a Gel (s = 323 m/g; pore diameter = 93A) was obtained from Whatman Chemical Separa tion, Inc. ( C l i f t o n , NJ, U.S.A.) Methanol and water were Omnisolv-HPLC grade from MCB (Cincinnati, OH, U.S.A.), and the 0.05 M phosphate buffers were prepared with Omnisolve - HPLC grade water. S y n t h e s i s . 4 - T o l y l b o r o n i c Acid was prepared from 4-bromotoluene, magnesium turnings and tributylboronic acid according to the pro cedure of Bean and Johnson (28). The y i e l d was 56% after several r e c r y s t a l l i z a t i o n s from water. The addition of N-bromosuccinimide to 4-tolyboronic acid with dibenzoyl peroxide i n anhydrous carbon tetrachloride gave an 85% y i e l d of 4-(co-bromomethyl)phenylboronic acid (29). The s y n t h e s i s of both aminated s i l i c a phases consisted of adding either 3 - a m i n o p r o p y l t r i e t h o x y s i l a n e ( f o r Phase B) or 3a m i n o p r o p y l d i m e t h y l e t h o x y s i l a n e ( f o r Phase D) to dried (130°C) P a r t i s i l 10 and refluxing for eight hours i n sodium-dried toluene with s t i r r i n g over dry n i t r o g e n gas. After Soxhlet-extraction w i t h methanol, the aminated s i l i c a gels were stored i n a vacuum desiccator u n t i l use. The physical c h a r a c t e r i s t i c s of these s t a tionary phases are l i s t e d i n Table I. Both aminated phenylboronic stationary phases (Phases Β and D) were prepared by adding a 20% excess of 4-(w-bromomethyl)phenylboronic acid to the respective aminated P a r t i s i l 10 phase and r e f l u x i n g i n sodium-dried toluene with pyridine (see Figure 3). After Soxhlet-extracting with methanol for forty-eight hours, the phases were stored i n a desiccator u n t i l use. a-(3-Aminopropyldimethylethoxysilane)-4-tolyboronic a c i d was prepared from an equimolar solution of 3-aminopropyldimethylethoxysilane and 4-(u>-bromomethyl)phenylboronic acid at room tempera ture i n sodium-dried t o l u e n e . After several r e c r y s t a l l i z a t i o n s from hexane the structure was confirmed using proton nuclear mag n e t i c resonance and i n f r a r e d spectroscopy (Yield - 65%, m.p. 172°C). Phase Ε was prepared by adding t h i s s i l a n e to d r i e d P a r t i s i l 10 and refluxing for eight hours, the phase was stored i n a desiccator u n t i l use. The p h y s i c a l c h a r a c t e r i s t i c s of a l l of these stationary phases are l i s t e d i n Table I. a-(ii-Propylamino)-tolylboronic acid (the model compound) was s y n t h e s i z e d by adding a 0.1 molar solution of 4-(a>-bromomethyl)phenylboronic acid to a 1.0 molar s o l u t i o n of f r e s h l y d i s t i l l e d n-propylamine at 50°C with s t i r r i n g over dry nitrogen gas. After d i s t i l l i n g off the solvent and the excess reagent, the product was r e c r y s t a l l i z e d several times with i s o p r o p a n o l and the s t r u c t u r e confirmed by proton nuclear magnetic resonance and infrared spec troscopy (Yield - 72%; m.p. - 196°C). The pKa of the model com pound was determined to be 10.4 according to the potentiometric t i t r a t i o n method of T o r s s e l l (30). Chromât ography The chromatographic system incorporated a Varian (Walnut Creek, CA, U.S.A.) Model 5000 l i q u i d chromatograph, a V a r i a n CDS 111L data system and a V a l c o i n j e c t i o n valve f i t t e d with a 10 1. i n j e c t i o n loop. Solute e l u t i o n was monitored with a P e r k i n Elmer



214


Table I: Physical Properties of P a r t i s i l 10 Stationary Phases Phase I.D.

A

I n i t i a l Amine Concentration moles/m ( x l O )

B

C

D

E

3.27

3.27

1.48

1.48

0.815

Phenylboronic Acid Concentration moles/m ( x l O )

—

0.946

—

0.475

0.815

Residual Amine Concentration moles/m ( x l O )

—

2.32

—

1.00

0.00

Residual S i l a n o l Concentration moles/m ( x l O )

4.73

4.73

6.52

6.52

7.19

—

2.46

—

2.11

0.00

40.90

40.90

18.50

18.50

10.20

—

28.90

—

32.10

2

2

2

2

6

6

6

6

Ratio of Residual Amines to Phenylboronic Acid I n i t i a l Amine % Reaction Phenylboronic Acid % Reaction

• I n i t i a l concentration assumed to be 8.00

2

moles/m .



12.



215

Complexation

\

-Si-O-H

/ \

BrCH C h4B(OH)

I

2

6

2

2

3

I

Si-0-Si-(CH ) NH 2

3

\ •Si-0-Si-(C H ) N H C H - ^ ~ ^ - B ( O H )

7

-Si-0-Si-(CH ) NH2-

\

2

3

2

ι

-^SiO-Si-(CH ) NH

2

2

3

2

PHASE A

\ 93 BrCH2C H B(OH) -Si-0-Si-(CH ) NH . CH H

6

2

3

4

2

3

-Si-0-Si-ÎcH ) NH 7

2

CH-a

3

2

v

-SiO-SKCH ) NHCH CH 2

3

\

9 3 -Si-0-SKCH ) NH / CH

2

:

3

H

2

3

2

3

Si-0-Si-(CH ) NHCH - ^ } - B ( O H ) CH 2

3

2

2

3

F i g u r e 3. The S y n t h e s i s of Phyenylboronic A c i d Phases on P a r t i s i l 10.

Bonded


2


216


(Norwalk, Conn., U.S.A.) LC-85B UV/Vis detectro ( c e l l volume = 1.4μ1; response time = 20 mS.) and a L a b o r a t o r y Data C o n t r o l ( R i v i e r a Beach, FL, U.S.A.) R e f r a c t o M o n i t o r Model 1107. The stationary phases were upward s l u r r y packed at 9000 p s i g . w i t h 90:10 (v/v) methanol: water i n t o a 25 cm. χ 4.6 mm. I.D. 316 stainless s t e e l column f i t t e d with 2μπι f r i t s . The average p l a t e number, N, for each column was calculated from the mean values of multiple injections of mannitol, s o r b i t o l and sucrose w i t h a t o t a l l y aqueous mobile phase at 1.0 ml./min. The average Ν for Phases B, D and Ε were 340, 1812, 1500 p l a t e s , r e s p e c t i v e l y . Chromatographic conditions were 25°C, 1.0 ml./min. and 50-100 atm. H 2 Û was used as a measure of the column dead volume. A l l of the solutes were freshly prepared i n the mobile phase. 2

Results and Discussion The i n t e n t of these s t u d i e s i s to i n v e s t i g a t e the influence of neighboring, unreacted amine groups on the apparent pKa of bound, phenylboronic acid stationary phase models. Amine s i l i c a s can be prepared using reagents which result i n either polymer (or "bulk") or monomeric ("brush") phases. Boronate phases prepared from such d i f f e r e n t materials might have s i g n i f i c a n t l y d i f f e r e n t l o c a l en vironments. In addition, i t was considered important to prepare a phase i n which only phenylborate would be present by u s i n g a phenylboronic acid s i l a n e . Three s t a t i o n a r y phases were prepared on P a r t i s i l 10: an a - ( n - p r o p y l a m i n o ) - 4 - t o l y l b o r o n i c acid stationary phase prepared from a "bulk" or polymeric amine s i l i c a (Phase B), an a-(ii-prop y l a m i n o ) - 4 - t o l y l b o r o n i c a c i d s t a t i o n a r y phase prepared from a "brush" or a monomeric amine phase (Phase D), and a phase prepared d i r e c t l y u s i n g an «-(dimethylethoxypropylaminosilane)-4-tolylb o r o n i c a c i d (Phase E ) . The p h y s i c a l c h a r a c t e r i s t i c s of the phases are l i s t e d i n Table IV. The c a p a c i t y f a c t o r s ( k ) for selected saccharides and nucleosides on these columns with water as the mobile phase are l i s t e d i n Table I I . The capacity factors ( k ) for these s o l u t e s on Phase Β and D are much g r e a t e r than those on Phase Ε whereas the amino stationary phases themselves (Phases A and C) have only minimal capacity f o r these s a c c h a r i d e solutes. The order of elution for interacting solutes i s i d e n t i c a l to that observed i n other phenylboronic a c i d chromatographic systems (19,20) although saccharides with unfavorable d i o l forma t i o n s and the 2'-deoxyribose derivatives have l i t t l e capacity on any of these columns. The chromâtograms of a mixture of three interacting saccharides on Phases D and Ε are shown i n Figure IV. Comparison of the intercolumn capacity f a c t o r s i s made more meaningful by n o r m a l i z a t i o n for the boronic acid surface concen t r a t i o n (moles/m2) of the phase (see Table I I I ) . These r e s u l t s i n d i c a t e a l a r g e percentage increase i n normalized capacity fac tors for Phases Β and D w i t h r e s p e c t to Phase Ε which c l e a r l y demonstrates the influence of residual amines on solute retention. The residual amines (found only i n Phase Β and D) are promoting phenylboronate-solute interactions by lowering the apparent pKa of the phase either by direct interaction with boronic acid m o i e t i e s or by indirect buffering of the surface environment. (See F i g . 5.) f

1


12.



Complexation

217


Table I I : The Capacity Factors Of Saccharides And Nucleosides In Water

2

k',100% H 0 Solute

Phase A

Phase Β

Phase C

Phase D

Phase £

0.01 0. 01 0. 02 0. 02 0. 05 0. 02 0. 08 0. 02 0. 02 0. 02 0. 02 0. 02 0..02 0. 02 0..02 0..02 0..06 0..04 0..01 0..02

0. 42 0. 01 0. 15 2. 79 3.07 0. 14 0. 36 0. 14 0.04 0. 09 0. 01 0.01 2. 33 0. 22 0. 27 0. 01 1. 61 4. 14 0. 01 0. 44

0 .02 0..01 0..02 0..01 0,.03 0..02 0..03 0..02 0..02 0. 03 0..02 0..02 0..02 0. 02 0..02 0. 02 0. 04 0. 05 0. 01 0. 02

0..42 0..05 0..16 3. 34 4..14 0. 14 0.,12 0. 05 0. 08 0. 12 0. 05 0. 04 3. 99 0. 21 0. 09 0. 07 2. 22 7. 15 0. 05 0. 08

0. 08 0. 01 0. 10 0. 26 0. 26 0. 10 0. 05 0. 03 0. 03 0. 03 0. 01 0. 01 0. 26 0. 05 0. 02 0. 01 0. 18 0. 45 0. 02 0. 05

0 .19 0 .11 0 .22 0 .13 0 .03 0 .22 0 .04 0 .05

7.89 4. 36 0. 04 0. 21 0. 09 5. 47 0. 08 4.,15

0. 20 0. 13 0. 15 0. 16 0. 08 0. 32 0. 05 0. 06

16.59 13.15 0. 99 0. 64 0. 32 14. 33 0. 38 11.86

2.05 1.03 0. 68 0. 34 0. 09 1. 34 0. 22 0. 84

Saccharides: L~(+)-Arabinose D-Cellibiose 2-Deoxy-D-Ribose Dulcitol Fructose L-(-)-Fucose D~(+)-Galactose D-Glucose Inositol Lactose D-(+)-Maltose Maltotriose D-Mannitol D-(+)-Mannose a-D-Melibiose D—Raffinose I>~(-)-Ribose Sorbitol Sucrose D-(+)-Xylose Nucleosides: Adenosine Cytidine 2-Deoxyadenosine 2-Deoxycytidine 2-Deoxyuridine Guanine Thymidine Uridine




S. Methanol 1. D-(-)-Ribose 2. D-Mannitol. 3. S o r b i t o l .

SA2

WITH RESIDUAL AMINES (PHASE B)

WITHOUT RESIDUAL AMINES (PHASE E)

0

10 20 Time (min.)

10

20 30 Time (min.)

F i g u r e 4. The S e p a r a t i o n s of a Mixture Phenylboronic Acid Stationary Phases. Mobile Phase = water. Flowrate 1.0 ml./min. U l t r a v i o l e t Detection at 195 nm.

of Saccharides on

β



12.



Complexation

219

Table I I I . Percentage Increase i n Normalized Capacity Factors f o r Strongly Interacting Saccharides and Nucleosides on Phenylboronic Acid Stationary Phases % Increase i n Normalized k* Solute

Phases D & Β

Phases D & £

Phases Β

Adenosine

319

1287

231

Cytidine

501

2082

263

Dulcitol

139

2072

810

Fructose

168

2653

926

Guanine

421

1736

252

D-Mannitol

241

2556

679

I>-(-)-Ribose

175

2065

687

Sorbitol

244

2622

692

Uridine

469

2331

327



220


Table IV. Capacity Factors of Saccharides and Nucleosides on Phase D .·, T«25°C. Solute

Water

pH 6.0

0.42 0.05 0.16 3.34 4.14 0.14 0.12 0.05 0.08 0.12 0.05 0.04 3.99 0.21 0.09 0.07 2.22 7.15 0.05 0.08

0.12 0.06 0.07 0.66 0.50 0.11 0.10 0.06 0.07 0.08 0.04 0.05 1.07 0.11 0.08 0.07 0.41 2.13 0.09 0.08

0.05 0.00 0.01 0.49 0.15 0.07 0.04 0.01 0.01 0.06 0.00 0.00 0.41 0.06 0.02 0.00 0.15 0.78 0.01 0.02

0.04 0.00 0.01 0.15 0.13 0.06 0.03 0.03 0.02 0.04 0.01 0.00 0.14 0.03 0.02 0.01 0.12 0.25 0.02 0.05

16.59 13.15 0.99 0.64 0.32 14.33 0.38 11.86

3.65 2.55 0.89 0.29 0.22 2.81 0.25 1.69

2.23 0.95 0.81 0.23 0.15 2.08 0.20 1.67

0.91 0.34 0.52 0.12 0.10 0.58 0.15 0.31

pH 5.0

pH

4.0

Saccharides : L-(+)-Arabinose D-Cellibiose 2-Deoxy-D-Ribose Dulcitol Fructose L-(-)-Fucose D~(+)-Galactose D-Glucose Inositol Lactose D-(+)-Maltose Maltotriose D-Mannitol D-(+)-Mannose a-D-Melibiose D-Raffinose D-(-)-Ribose Sorbitol Sucrose D-(+)-Xylose Nucleosides : Adeno s ine Cytidine 2 *-Deoxyadenosine 2'-Deoxycytidine 2'Deoxyuridine Guanine Thymidine uridine


12.



221

Complexation


8T

CM—Τ

Τ

4.0

— — I

5.0 pH

OF

60 MOBILE

f7.0

PHASE

F i g u r e 5. The Dependence of S a c c h a r i d e C a p a c i t y on a Phenylboronic Acid Stationary Phase (Phase D) on Mobile-Phase pH. Flowrate = 1.0 ml./min. Refractive Index Detection.




222

Even though the r e s i d u a l amine: phenylboronic a c i d r a t i o (from Table I) i s higher for Phase Β than for Phase D, the average normalized k's for interacting solutes on Phase D are l a r g e r than on Phase B. There are s e v e r a l p o s s i b l e explanations for this observed dependence of the capacity on the silane backbone s t r u c ture: 1. The polymeric backbone (Phases A and B) i s composed of some amine moieties which remain i n a c c e s s i b l e both to r e a c t i o n w i t h a - b r o m o - 4 - t o l y l - b o r o n i c acid and to solvent modifications. Therefore, the r a t i o i s an i n f l a t e d estimate of the a c t u a l amine moieties available for interaction with the other moieties or with solvent. 2. The polymeric backbone could c o n s i s t of t e r m i n a l amines having more freedom of movement than i n the monomeric phase. These surface-bound moieties would t h e r e f o r e be expected to be more a c c e s s i b l e to solvent modification which would weaken the interactions between neighboring bound molecules. 3. Surface p o l y m e r i z a t i o n could a l s o increase the average pore diameter of the bounded phase (by plugging smaller pores) reducing the number of s i t e - s i t e i n t e r a c t i o n s and thus r a i s i n g the pKa of the phase (2). The r e l a t i v e l y slow k i n e t i c s of complexation has been pro posed as a cause of the g e n e r a l l y low e f f i c i e n c i e s of boronate s t a t i o n a r y phase systems (19). The plate numbers of interacting s o l u t e s i n these studies are about 90% of otherwise observed and assymetry i s unchanged. The l a t t e r i s unusual since previous work showed t a i l i n g ascribed to the presence of a "non-linear isotherm" (20). Perhaps the combination of s u r f a c e b u f f e r i n g and mixed bonded phase e f f e c t creates a more favorable, more uniform sorp tion environment. CONCLUSION The observed d i f f e r e n c e s i n normalized d i o l capacity factors i n dicate that residual surface amines do lower the apparent pKa of phenylboronic s t a t i o n a r y phases by either s i t e - s i t e interactions or by the buffering of the surface environment. Incorporating the b u f f e r i n t o the surface structure instead of i n the mobile phase i s advantageous i n lowering the mobile-phase pH r e q u i r e d to achieve r e t e n t i o n on weakly basic stationary phases, for c o l l e c ting fractions i n preparative chromatography and i n extending the l i f e t i m e of chromatographic equipment (especially columns with s i l i c a substrates). Although most of the s o l u t e c a p a c i t y i n a chromatographic system depends on the mobile-phase composition and the major stationary-phase moiety, some capacity may r e s u l t from residual surface species. Even i f these residual functions do not interact d i r e c t l y with the solutes, they can modify the s u r f a c e environment r e s u l t i n g i n a change i n the o v e r a l l performance of the stationary phase. Similar surface buffering effects should be of use i n other types of i o n i c e q u i l i b r i a where i t i s desirable to a l t e r the apparent i o n i z a t i o n constant. A more complete under standing of these stationary-phase s u r f a c e i n t e r a c t i o n s should lead to the further refinement of separation strategies. Acknowledgments

T h i s work was supported, i n p a r t , by a grant (to CHL) from the National Science Foundation, CHE-85-000658.


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