Micellar Electrokinetic Capillary Chromatography - ACS Symposium

Chapter 6

Micellar Electrokinetic Capillary Chromatography 1

1

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M. J. Sepaniak , D. E. Burton , and M. P. Maskarinec 1

Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600 Analytical Chemistry Division, Oak Ridge National Laboratory, P.O. Box X, Oak Ridge, TN 37830

Downloaded by MONASH UNIV on December 14, 2015 | http://pubs.acs.org Publication Date: June 30, 1987 | doi: 10.1021/bk-1987-0342.ch006

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The incorporation of micelles in the mobile phase in capillary zone electroporesis permits the efficient separation of a variety of neutral compounds. Efficiencies in excess of 100,000 plates/m are rou tinely attained. The mass transport processes which are important in micellar electrokinetic capillary chromatography are described, along with the technique. The technique is particularly useful for biological separations. Preliminary data and discussion related to column selectivity and efficiency are presented. M i c e l l a r e l e c t r o k i n e t i c c a p i l l a r y chromatography, MECC, was f i r s t r e p o r t e d by T e r a b e , e t . a l . ( 1 ) . The t e c h n i q u e combines many o f t h e o p e r a t i o n a l p r i n c i p l e s and advantages o f m i c e l l a r l i q u i d chroma t o g r a p h y (2) and c a p i l l a r y zone e l e c t r o p h o r e s i s , CZE ( 3 ) . CZE i s performed u s i n g narrow-bore c a p i l l a r y columns (CA. 50 ym i . d . χ 100 cm) which a r e f i l l e d w i t h an aqueous b u f f e r s o l u t i o n . A l a r g e a p p l i e d e l e c t r i c f i e l d d r i v e s charged sample s o l u t e s , which a r e i n j e c t e d as a sharp p l u g , toward the d e t e c t i o n end o f t h e column. E f f i c i e n c i e s i n excess o f 100,000 p l a t e s / m a r e g e n e r a l l y o b s e r v e d . E l e c t r o o s m o t i c f l o w (4) p r o v i d e s a n o t h e r means o f t r a n s p o r t i n g s o l u t e s , i n c l u d i n g n e u t r a l s , t h r o u g h t h e column. In CZE, d i f f e r e n c e s i n t h e v i s c o u s d r a g o f n e u t r a l s o l u t e s , p r i m a r i l y as a r e s u l t o f s i z e d i f f e r e n c e s , can p r o v i d e f o r t h e i r s e p a r a t i o n ( 3 ) . However, t h e s e d i f f e r e n c e s a r e u s u a l l y v e r y s m a l l and, c o n s e q u e n t l y , t h e t e c h n i q u e i s not v e r y u s e f u l f o r s e p a r a t i n g n e u t r a l compounds. With t h e MECC t e c h n i q u e , a s u r f a c t a n t i s added t o the m o b i l e phase a t a c o n c e n t r a t i o n above i t s c r i t i c a l m i c e l l e concentration. The r e s u l t i n g m i c e l l e s p r o v i d e an e f f e c t i v e mecha nism f o r s e p a r a t i n g n e u t r a l compounds. N e u t r a l s o l u t e s a r e s e p a r a t e d based on t h e i r d i f f e r e n t i a l p a r t i t i o n i n g between an e l e c t r o o s m o t i c a l l y - p u m p e d m o b i l e phase and t h e h y d r o p h o b i c i n t e r i o r o f t h e

0097-6156/87/0342-0142$06.00/0 © 1987 American Chemical Society

In Ordered Media in Chemical Separations; Hinze, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


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Micellar Electrokinetic Capillary Chromatograohy

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m i c e l l e s , which a r e charged and moving a t a v e l o c i t y d i f f e r e n t from t h a t o f t h e m o b i l e phase due t o e l e c t r o p h o r e t i c e f f e c t s . The mass t r a n s p o r t phenomena o p e r a t i v e i n MECC a r e d e p i c t e d i n the expanded view o f a c a p i l l a r y s e c t i o n shown i n F i g u r e 1. The column c o n t a i n s t h e s u r f a c t a n t / b u f f e r s o l u t i o n . N e g a t i v e l y charged ( a n i o n i c ) s u r f a c t a n t i s c o n s i d e r e d i n t h e f i g u r e . P r i m a r y and s e c o n d a r y s o r b e d i o n s g e n e r a t e an e l e c t r i c double l a y e r p o t e n t i a l ( z e t a p o t e n t i a l ) a t t h e i n s i d e s u r f a c e o f t h e c a p i l l a r y . The l a r g e r the zeta p o t e n t i a l the greater the d i s p a r i t y i n the o v e r a l l m o b i l i t i e s o f p o s i t i v e and n e g a t i v e i o n s . Because o f t h i s d i s p a r i t y a n e t f l o w o f s o l v e n t ( e l e c t r o o s m o t i c f l o w ) r e s u l t s when an e l e c t r i c f i e l d i s a p p l i e d and s o l v a t e d i o n s move toward the e l e c trode of opposite s i g n . I n o u r work w i t h both a n i o n i c ( e . g . , sodium d o d e c y l s u l f a t e , SDS), and c a t i o n i c ( e . g . , c e t y l t r i m e t h y l ammonium c h l o r i d e , CTAC) s u r f a c t a n t s , t h e e l e c t r o o s m o t i c f l o w v e l o c i t y (Veo) opposes t h e e l e c t r o p h o r e t i c v e l o c i t y o f t h e m i c e l l e s (Vm,e) and i s o f a g r e a t e r magnitude. C o n s e q u e n t l y , two d i s t i n c t phases, the m o b i l e phase and t h e m i c e l l a r phase, e x i s t w i t h i n t h e column and m i g r a t e a t d i f f e r e n t v e l o c i t i e s toward t h e e l e c t r o d e w i t h t h e same charge as t h e m i c e l l e s . Veo i s p r o p o r t i o n a l t o t h e magnitude o f the z e t a p o t e n t i a l and t h e a p p l i e d p o t e n t i a l , w h i l e Vm,e i s p r o p o r t i o n a l t o t h e m i c e l l a r e l e c t r o p h o r e t i c m o b i l i t y and the a p p l i e d p o t e n t i a l . A s o l u t e ( S ) which p a r t i t i o n s between t h e m o b i l e and m i c e l l a r phases w i l l have a band v e l o c i t y ( V s ) t h a t i s i n t e r m e d i a t e between Vm and Veo. Chromatograms i n MECC d i f f e r from those o b s e r v e d i n c o n v e n t i o n a l e l u t i o n chromatography i n t h a t t h e r e i s g e n e r a l l y a l i m i t e d e l u t i o n r a n g e . R e t e n t i o n t i m e s , t , i n MECC a r e g i v e n by equat i o n 1 where t i s the r e t e n t i o n time o f a s o l u t e which i s n o t R

n

tp =

(1) R

+

(t /t )(l-R) 0

m

s o l u b i l i z e d by t h e m i c e l l e s , t i s t h e r e t e n t i o n time o f a s o l u t e which i s c o m p l e t e l y s o l u b i l i z e d , and R i s the f r a c t i o n o f s o l u t e not s o l u b i l i z e d ( i . e . , t h e MECC a n a l o g o f the r e t e n t i o n r a t i o i n conventional LC). The l i m i t e d e l u t i o n range o f MECC t r a n s l a t e s i n t o r e l a t i v e l y s h o r t s e p a r a t i o n t i m e s , but a d v e r s e l y , l i m i t s t h e peak c a p a c i t y o f the t e c h n i q u e . C e r t a i n fundamental c h a r a c t e r i s t i c s o f MECC t h a t i n f l u e n c e r e t e n t i o n have been i n v e s t i g a t e d ( 5 ) . The t e c h n i q u e has been used i n t h e a n a l y s i s o f a v a r i e t y o f samples i n c l u d i n g p h e n o l i c compounds ( 1 ) , p h e n y l t h i o h y d a n t o i n - a m i n o a c i d s ( 6 ) , and m e t a b o l i t e s o f v i t a m i n Bg ( 7 ) . In r e l a t e d e l e c t r o k i n e t i c s e p a r a t i o n techniques, s u b s t i t u t e d benzene compounds have been s e p a r a t e d based on t h e f o r m a t i o n o f i n c l u s i o n complexes w i t h an i o n i c c y c l o d e x t r i n d e r i v a t i v e i n t h e m o b i l e phase ( 8 ) and p o l y a r o m a t i c h y d r o c a r b o n s have been s e p a r a t e d based on s o l v o p h o b i c i n t e r a c t i o n s w i t h a t e t r a a k y l ammonium i o n i n t h e m o b i l e phase ( 9 ) . The e f f e c t s o f i n j e c t i o n p r o cedures on e f f i c i e n c y have a l s o been s t u d i e d ( 1 0 ) . m


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

Experimental Apparatus. The e x p e r i m e n t a l c o n f i g u r a t i o n used i n t h i s work i s shown i n F i g u r e 2. Columns were 25-100 \im i . d . f u s e d s i l i c a c a p i l l a r i e s which were p r o t e c t e d by a p o l i m i d e c o a t i n g . The column l e n g t h was g e n e r a l l y 50 - 100 cm. A 1 - 2 mm s e c t i o n of the p r o t e c t i v e c o a t i n g was removed near one end o f the column to p r o v i d e an o p t i c a l window f o r l a s e r - b a s e d f l u o r e s c e n c e d e t e c t i o n (10) u s i n g the 488 nm l i n e of a C y o n i c s Model 2001 A r laser for excitation, or UV absorbance d e t e c t i o n u s i n g a J a s c o UVIDEC-100-III d e t e c t o r w i t h a m o d i f i e d f l o w c e l l compartment ( 1 0 ) . H i g h v o l t a g e (0 - 40 kV) was s u p p l i e d by H i p o t r o n i c s r e g u l a t e d DC power s u p p l i e s (Models R30B and R40B). E l e c t r o d e s were e i t h e r p l a t i n u m w i r e or g r a p h i t e rods. Procedures. The c a p i l l a r y columns were r i n s e d w i t h 0.1 M HC1 p r i o r t o f i l l i n g w i t h m o b i l e phase. T y p i c a l m o b i l e phases were 0.01 M SDS and 0.01 M ^ 2 ^ 0 ^ , i n the case o f n e g a t i v e l y charged m i c e l l e s , and 0.02 M CTAC, 0.01 M Na HP0i , and 0.01 M ^ B ^ O y , i n the case o f p o s i t i v e l y charged m i c e l l e s . However, the s u r f a c t a n t c o n c e n t r a t i o n s were v a r i e d i n a column e f f i c i e n c y study and b u f f e r c o n c e n t r a t i o n s were o f t e n a d j u s t e d t o keep c u r r e n t s at l e v e l s t h a t m i n i mize h e a t i n g . E l e c t r o i n j e c t i o n was used t o i n t r o d u c e sample i n t o the column ( 1 0 ) . With t h i s t e c h n i q u e the s u r f a c t a n t / b u f f e r r e s e r v o i r a t the i n l e t o f the column i s r e p l a c e d by the sample s o l u t i o n . High v o l t a g e ( 2 - 5 kV) i s a p p l i e d f o r 5 - 3 0 seconds. The s u r f a c t a n t / b u f f e r r e s e r v o i r i s then r e t u r n e d and the h i g h v o l t a g e r e a p p l i e d to e f f e c t the s e p a r a t i o n . Chemicals. The s u r f a c t a n t s used i n t h i s work were o b t a i n e d from Sigma C h e m i c a l Co. and m o b i l e phases were p r e p a r e d u s i n g d i s t i l l e d and d e i o n i z e d water. The p u r i n e s were o b t a i n e d from B-L B i o c h e m i c a l s and Merck C h e m i c a l s . The b u f f e r s and the o t h e r t e s t s o l u t e s were o b t a i n e d from F i s h e r S c i e n t i f i c . The amines were d e r i v a t i z e d with 7-chloro-4-nitrobenzo-2,1,3-oxadizaole (NBD-C1) from R e g i s Chemicals u s i n g a p r o c e d u r e s u p p l i e d by the manufacturer.


+

2

t

R e s u l t s and D i s c u s s i o n MECC s e p a r a t i o n s are g e n e r a l l y l i m i t e d to compounds which a r e r e a s o n a b l y s o l u b l e i n the m o b i l e phase. In the case of normal m i c e l l e s the sample components t o be s e p a r a t e d must have some s o l u b i l i t y i n the aqueous phase. I t i s i n t e r e s t i n g t o note t h a t good sample s o l u b i l i t y i n the a q u e o u s - m i c e l l e m i x t u r e does not a s s u r e an effective separation. The a d d i t i o n of m i c e l l e s to an aqueous s o l u t i o n can g r e a t l y i n c r e a s e the s o l u b i l i t y of h y d r o p h o b i c compounds. For example, the s o l u b i l i t y o f pyrene i n water i s enhanced by 10 when the water i s made 0.07 M i n SDS ( 2 ) . However, a l l of the h y d r o p h o b i c compounds i n a m i x t u r e tend to be n e a r l y c o m p l e t e l y s o l u b i l i z e d by the m i c e l l e s and e l u t e from a MECC column p o o r l y r e s o l v e d , w i t h r e t e n t i o n times near t . N e v e r t h e l e s s , we have u t i l i z e d the MECC t e c h n i q u e f o r e f f i c i e n t s e p a r a t i o n s o f a v a r i e t y of samples. For example, F i g u r e 3 i s the chromatogram of a s e p a r a t i o n o f a m i x t u r e o f benzene compounds s u b s t i t u t e d w i t h groups which d i f f e r g r e a t l y i n t h e i r 5

m


Micellar Electrokinetic Capillary Chromatography


SEPANIAK E T A L .

H

©

r

Θ

C^— | C ®*

Θ

(-)

3

©

£ A

Θ

(+)

Veo

Θ

Θ

Θ

e

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0 © 0

F i g u r e 1. Expanded view o f a MECC c a p i l l a r y column dynamics.

\

s e c t i o n showing

COLUMN

F i g u r e 2.

Diagram o f a p p a r a t u s .




F i g u r e 3. MECC chromatogram o f a n i l i n e ( A ) , p h e n o l ( C ) , and t o l u e n e ( D ) .

nitrobenzene(B),


6.


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chemical p r o p e r t i e s . The s e p a r a t i o n was performed w i t h a 60 ym i . d . χ 100 cm column c o n t a i n i n g 0.05 M CTAC, 0.01 M ^ 2 ^ 0 ^ and 6 χ 10"" Μ Νβ2Β^07· UV d e t e c t i o n a t 220 nm was employed. As e x p e c t e d the most h y d r o p h o b i c component, t o l u e n e i s e l u t e d l a s t . The weakly b a s i c a n i l i n e i s e l u t e d f i r s t u s i n g t h i s c a t i o n i c m i c e l l e system, w h i l e the weakly a c i d i c p h e n o l i s w e l l - r e t a i n e d and somewhat broadened r e l a t i v e t o the o t h e r components, both e f f e c t s presumably due t o e l e c t r o s t a t i c i n t e r a c t i o n s w i t h t h e p o s i t i v e l y charged m i c e l l e s . B i o l o g i c a l samples a r e p a r t i c u l a r l y amenable t o MECC s e p a r a t i o n ( 7 ) . F i g u r e 4 i s t h e chromatogram o f a s e p a r a t i o n o f a m i x t u r e o f p u r i n e s on a 75 ym i . d . χ 100 cm column c o n t a i n i n g 0.01 M SDS and 0.01 M Na HP0i . D e t e c t i o n was a t 280 nm u s i n g t h e absorbance d e t e c t o r . The p u r i n e s a r e e f f i c i e n t l y s e p a r a t e d i n l e s s t h a n 20 minute u s i n g an a p p l i e d p o t e n t i a l o f 20 kV. A h i g h e r p o t e n t i a l c o u l d be employed t o reduce t h e s e p a r a t i o n time but some l o s s i n e f f i c i e n c y would o c c u r ( s e e d i s c u s s i o n b e l o w ) . The o b j e c t i n any s e p a r a t i o n i s t h e r e s o l u t i o n o f t h e com ponents o f a sample. R e s o l u t i o n , Rg, i n MECC i s g i v e n by Equa t i o n 2 ( 5 ) where Ν i s the number o f t h e o r e t i c a l p l a t e s f o r t h e 3


2

N

l/2

Rs = — 4

f

α - 1

(

k»

) ( α

1 - t /t 0

1 + k''

m

°—J5

) ( 1 +

)

(2)

(t /t )k'' 0

m

f

column, k i s the c a p a c i t y f a c t o r ( i . e . , moles o f s o l u t e i n m i c e l l a r phase d i v i d e d by moles i n the m o b i l e p h a s e ) , and α i s t h e s e l e c t i v i t y f a c t o r ( k ^ / k ' j ) f o r a d j a c e n t peaks i n a chromatogram. R e s o l u t i o n i n MECC depends on f o u r f a c t o r s , e f f i c i e n c y ( N ) , s e l e c t i v i t y ( a ) , c a p a c i t y ( k ) , and e l u t i o n range ( a s r e f l e c t e d i n t /t ). The l a s t f a c t o r i l l u s t r a t e s an i m p o r t a n t d i f f e r e n c e between MECC and c o n v e n t i o n a l e l u t i o n chromatography which employs a t r u e s t a t i o n a r y phase. The optimum k* i n MECC depends on t /t b u t i s g e n e r a l l y i n t h e range o f 2 - 5. As t / t approaches z e r o t h e l a s t term i n E q u a t i o n 2 drops o u t and the MECC t e c h n i q u e resembles c o n v e n t i o n a l e l u t i o n chromatography. We a r e c u r r e n t l y s t u d y i n g e x p e r i m e n t a l f a c t o r s which i n f l u e n c e s e l e c t i v i t y and e l u t i o n r a n g e . The importance o f t h e s e f a c t o r s i s demonstrated i n F i g u r e 5 which i s t h e chromatogram o f s e v e r a l n i t r a t e d p o l y a r o m a t i c h y d r o c a r b o n s t h a t were s e p a r a t e d u s i n g t h e same c o n d i t i o n s as i n F i g u r e 4 e x c e p t t h a t the d e t e c t i o n wavelength was 230 nm. Chromatographic e f f i c i e n c y f o r t h i s s e p a r a t i o n i s e x c e l l e n t b u t r e t e n t i o n s e l e c t i v i t y i s l a c k i n g . The same s o l u t e s can e a s i l y be r e s o l v e d by c o n v e n t i o n a l r e v e r s e d phase LC, even though t h a t t e c h n i q u e e x h i b i t s much lower column e f f i c i e c y . The poor s e l e c t i v i t y u s i n g MECC f o r t h i s sample c o u l d be a g e n e r a l r e s u l t o f t h e l a r g e k' v a l u e s f o r t h e s o l u t e s , o r i t c o u l d i n d i c a t e poor r e t e n t i o n d i s c r i m i n a t i o n among t h e s o l u t e s u s i n g t h i s p a r t i c u l a r a n i o n i c m i c e l l a r system. As w i t h o t h e r forms o f chromatography, m a x i m i z i n g column e f f i c i e n c y i s c r i t i c a l t o t h e o v e r a l l development o f the t e c h nique. The m a j o r i t y o f o u r r e s e a r c h i n MECC has f o c u s e d on s t u d i e s o f e x p e r i m e n t a l f a c t o r s which i n f l u e n c e column e f f i c i e n c y ( 1 1 ) . 1

Q

Q

m

m

Q

m

A m e r i c a n Chemical Society Library 1155 16th St., N.W. Washington, D.C. 20036 In Ordered Media in Chemical Separations; Hinze, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.



A

F i g u r e 4· MECC chromatogram o f the p u r i n e s a d e n i n e ( A ) , 6 - m e t h y l p u r i n e ( B ) , 2 h y d r o x y p u r i n e ( C ) , 6,6-dimethylaminop u r i n e ( D ) , and x a n t h i n e ( E ) .

Β

F i g u r e 5. MECC chromatogram o f 1 , 4 - d i n i t r o n a p h t h a l e n e ( A ) , l - n i t r o n a p h t h a l e n e ( B ) , 9 - n i t r o a n t h r a c e n e ( C ) , and 1 - n i t r o pyrene(D).


6.

SEPANIAK E T A L .



The " p l u g - l i k e " v e l o c i t y f l o w p r o f i l e f o r e l e c t r o k i n e t i c a l l y pumped c a p i l l a r y columns ( s e e F i g u r e 1) i s i m p o r t a n t i n m i n i m i z i n g r e s i s t a n c e t o mass t r a n s f e r w i t h i n t h e mobile phase ( 4 ) . Hydros t a t i c a l l y - p u m p e d c a p i l l a r i e s , have p a r a b o l i c f l o w p r o f i l e s which tend t o s e v e r e l y d i s p e r s e s o l u t e bands u n l e s s extreme narrow-bore ( i . d , s l e s s than 10 ym) c a p i l l a r i e s a r e employed ( 1 2 ) . Fortun a t e l y , l a r g e r c a p i l l a r i e s , w i t h l e s s s t r i n g e n t d e t e c t o r volume r e q u i r e m e n t s , can be e f f i c i e n t l y used i n MECC. Under the p r o p e r c o n d i t i o n s , column e f f i c i e n c y i n MECC i s outstanding. I n one s e p a r a t i o n o f p u r i n e compounds we o b t a i n e d o v e r 600,000 p l a t e s / m f o r t h e o p h y l l i n e . However, t h e e f f e c t s o f parameters such as column d i m e n s i o n s , a p p l i e d v o l t a g e , and t h e c o n c e n t r a t i o n o f the b u f f e r and s u r f a c t a n t on e f f i c i e n c y can be very dramatic. A b r i e f d i s c u s s i o n o f how these parameters influence efficiency follows. Narrow-bore c a p i l l a r y tubes d i s s i p a t e t h e heat g e n e r a t e d i n the e l e c t r o p h o r e t i c p r o c e s s v e r y e f f i c i e n t l y . N e v e r t h e l e s s , we have observed h e a t i n g e f f e c t s when power d i s s i p a t i o n s exceed about 2 watts/m. T h i s o c c u r s f o r 75 ym i . d . χ 100 cm columns, c o n t a i n i n g 10~ M b u f f e r , when a p p l i e d v o l t a g e s exceed about 30 kV. The h e a t g e n e r a t e d produces a t r a n s v e r s e temperature g r a d i e n t w i t h i n t h e column. Since e l e c t r o p h o r e t i c m o b i l i t y i n c r e a s e s with temperature the " p l u g - l i k e " f l o w p r o f i l e shown i n F i g u r e 1 i s d i s t o r t e d and column e f f i c i e n c y i s degraded. MECC s e p a r a t i o n s a r e conducted i n open c a p i l l a r i e s , hence eddy d i f f u s i o n i s not p r o b l e m a t i c . However, t h e columns behave i n many ways l i k e packed columns, w i t h t h e m i c e l l e s f u n c t i o n i n g as u n i f o r m l y s i z e d and e v e n l y d i s p e r s e d p a c k i n g p a r t i c l e s . I n packed columns, r e s i s t a n c e t o mass t r a n s f e r i n t h e mobile phase i s reduced ( i . e . , e f f i c i e n c y improved) when s m a l l e r p a r t i c l e s a r e used because the " d i f f u s i o n d i s t a n c e " between p a r t i c l e s i s d e c r e a s e d . Average " i n t e r - m i c e l l a r " d i s t a n c e i s t h e analogous parameter i n MECC. This d i s t a n c e can be d e c r e a s e d by i n c r e a s i n g s u r f a c t a n t c o n c e n t r a t i o n . In p r e l i m i n a r y e x p e r i m e n t s , i n c r e a s i n g SDS c o n c e n t r a t i o n s from j u s t above i t s c r i t i c a l m i c e l l e c o n c e n t r a t i o n (about 7 χ 1 0 ~ M) t o 5 χ 10" M r e s u l t e d i n 5 - 10 f o l d d e c r e a s e s i n p l a t e h e i g h t s f o r t e s t solutes. 2

3

D e s p i t e t h e improved mass t r a n s f e r c h a r a c t e r i s t i c s o f t h e " p l u g - l i k e " f l o w p r o f i l e s observed i n MECC, " i n t r a - c o l u m n " r e s i s t ance t o mass t r a n s f e r i s s i g n i f i c a n t a t h i g h e r f l o w v e l o c i t i e s ( i . e . , a t h i g h a p p l i e d v o l t a g e s ) . A l t h o u g h n o t as d r a m a t i c as i n our work w i t h h y d r o s t a t i c a l l y - p u m p e d open c a p i l l a r y LC, we have o b s e r v e d improvements i n e f f i c i e n c y w i t h t h e MECC t e c h n i q u e when column diameter i s r e d u c e d . T h i s i s i l l u s t r a t e d i n F i g u r e 6. Peaks A and Β c o r r e s p o n d t o t h e NBD-C1 d e r i v a t i v e s o f e t h y l a m i n e and c y c l o h e x y l a m i n e s e p a r a t e d on a 75 ym i . d . column and d e t e c t e d by l a s e r f l u o r o m e t r y . Peak C i s NBD-cyclohexylamine from a 25 ym i . d . column o f t h e same l e n g t h . E f f i c i e n c y i s about a f a c t o r o f 4 b e t t e r w i t h t h e s m a l l e r d i a m e t e r column. The a p p l i e d v o l t a g e used t o o b t a i n F i g u r e 6 was 20 kV. The e f f e c t i s even more d r a m a t i c a t h i g h e r v o l t a g e s which r e s u l t i n g r e a t e r f l o w r a t e s . Optimum v o l t a g e s f o r t h e columns used i n t h i s


149



TIME ( m l n . )

F i g u r e 6. MECC chromatogram o f NBD-ethylamine(A) and N B D - c y c l o h e x y l a m i n e ( B ) , u s i n g a 75 ym i . d . column and NBD-cyclohexylamine u s i n g a 25 ym i . d . column. The m o b i l e phase c o n t a i n e d 0.01 M SDS and 0.003 Μ N a H P 0 i f 2


6.

SEPANIAK E T A L .


151


work were about 10 kV, which corresponded t o an e l e c t r o o s m o t i c f l o w v e l o c i t y o f about 0.1 cm/s. H i g h e r v o l t a g e s r e s u l t i n more r a p i d s e p a r a t i o n s but e f f i c i e n c y i s degraded due t o r e s i s t a n c e t o mass t r a n s f e r w i t h i n t h e m o b i l e phase ( " i n t e r - m i c e l l a r " and " i n t r a column") and e v e n t u a l l y due t o the f o r m a t i o n o f temperature gradients. Lower v o l t a g e s r e s u l t i n l o n g a n a l y s i s times and a r e d u c t i o n i n e f f i c i e n c y due t o e x c e s s i v e a x i a l d i f f u s i o n . In summary t h e MECC t e c h n i q u e shows promise f o r t h e h i g h e f f i c i e n c y s e p a r a t i o n o f a v a r i e t y o f samples which a r e a t l e a s t s p a r i n g l y s o l u b l e i n water. Fundamental s t u d i e s o f column s e l e c t i v i t y are i n progress i n our laboatory. The r e s u l t s o f these s t u d i e s should provide i n s i g h t s f o r choosing s u i t a b l e m i c e l l e / b u f f e r s o l u t i o n s f o r s e p a r a t i n g sample components which d i f f e r i n s p e c i f i c molecular p r o p e r t i e s . Acknowledgment T h i s r e s e a r c h was sponsored j o i n t l y by t h e D i v i s i o n o f C h e m i c a l S c i e n c e s , O f f i c e o f B a s i c Energy S c i e n c e s , U. S. Department o f E n e r g y , under c o n t r a c t DE-AS0-83ER13127 w i t h t h e U n i v e r s i t y o f Tennessee ( K n o x v i l l e ) , t h e O f f i c e o f H e a l t h and E n v i r o n m e n t a l R e s e a r c h , U. S. Department o f Energy, under c o n t r a c t DE-ACO-84OR21400 w i t h t h e M a r t i n M a r i e t t a Energy Systems, I n c . , Oak R i d g e , Tennessee, and under appointment t o t h e L a b o r a t o r y P a r t i c i p a t i o n Program a d m i n i s t e r e d by Oak Ridge A s s o c i a t e d U n i v e r s i t i e s f o r t h e U.S. Department o f Energy.

Literature Cited 1. Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, Α.; Ando, T. Anal. Chem. 1984, 56, 111-113. 2. Armstrong, D. W. Separation and Purification Methods 1985, 14(2), 213-304. 3. Jorgenson, J.W.;Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. 4. Martin, M.; Guiochon, G.; Walbroehl, Y.; Jorgenson, J. W. Anal. Chem. 1985, 57, 559-661. 5. Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, 834-841. 6. Otsuka, K.; Terabe, S.; Ando, T. J. Chromatogr. 1985, 332, 219-226. 7. Burton, D. E.; Sepaniak, M. J.; Maskarinec, M. P. J. Chromatogr. Sci., 1986, 24, 347-351. 8. Terabe, S.; Ozaki,H.;_Otsuka,K.;Ando, T. J. Chromatogr. 1985, 332, 211-217. 9. Walbroehl, Y.; Jorgenson, J. W. Anal. Chem. 1986, 58, 479-481. 10. Burton, D. E.; Sepaniak, M. J.; Maskarinec, M. P. Chromatographia, in press. 11. Sepaniak, M. J.; Cole, R. Anal. Chem. submitted for publication. 12. Guiochon, G. Anal. Chem. 1981, 53, 1318-1325. RECEIVED

December 19, 1986


Micellar Electrokinetic Capillary Chromatography - ACS Symposium

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