Narrow-Bore, Microparticle-Packed Column High Performance Liquid

8 Narrow-Bore, Microparticle-Packed Column High Performance Liquid Chromatography Utilization and Constraints

Downloaded by PEPPERDINE UNIV on September 28, 2017 | http://pubs.acs.org Publication Date: April 26, 1984 | doi: 10.1021/bk-1984-0250.ch008

FRANK J. YANG Walnut Creek Division, Varian Instrument Group, Walnut Creek, CA 94598 An overview of microbore column technology is given. The potential and the constraints of micro -bore columns are discussed. The driving forces in microbore column HPLC are (1) the compatibility of the flow rates with mass spectrometer and flame -based detectors; (2) a total saving of up to 99.9% solvent consumption; (3) the high resolving power of the long fused-silica columns packed with small particles; and (4) the opportunities in new detector development and new applications. To utilize the available potential of microbore packed columns, the liquid chromatograph including injectors, pumps, and detectors must be improved to match the chromatographic requirements. Modifica tions of existing commercial liquid chromatograph for the practice of microbore column HPLC are also described. The d r i v i n g f o r c e s f o r t h e r a p i d d e v e l o p m e n t and g r o w t h i n microbore column HPLC a r e (1) s a v i n g s i n s o l v e n t c o n s u m p t i o n : a t o t a l s a v i n g o f up t o 99.9% can be a c h i e v e d when n a r r o w - b o r e m i c r o p a r t i c l e packed columns o r o p e n - t u b u l a r m i c r o - c a p i l l a r y columns a r e used; (2) the high separation power using long column and s m a l l p a r t i c l e s (e.g., 3 μπι); (3) the c o m p a t i b i l i t y o f t h e column eluent flow r a t e s w i t h a mass spectrometer and flame based d e t e c t o r s ; and (4) o p p o r t u n i t i e s i n new d e t e c t o r d e v e l o p m e n t , e.g., a l a s e r based s p e c t r o s c o p i c d e t e c t o r . M i c r o b o r e column HPLC, however, r e q u i r e s s u b s t a n t i a l improvement o f t h e conven t i o n a l l i q u i d chromatograph to meet the o p e r a t i o n a l requirements and chromatographic performance of microbore columns. I t i s the purpose of t h i s manuscript to compare four types of microbore columns reported i n the l i t e r a t u r e . The u t i l i z a t i o n of the narrow-bore packed column HPLC i n terms of instrumentation requirements f o r i n j e c t i o n valves, pumping systems, and detectors are a l s o discussed. 0097-6156/ 84/ 0250-0091 $06.00/ 0 © 1984 American Chemical Society

Ahuja; Ultrahigh Resolution Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

ULTRAHIGH RESOLUTION

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CHROMATOGRAPHY

Columns S i n c e t h e i n t r o d u c t i o n of o p e n - t u b u l a r columns f o r gas chromatography by Golay' ' i n 1958, developments i n open-tubular column gas c h r o m a t o g r a p h i c technology and techniques have made open-tubular column GC a very important t o o l f o r a c h i e v i n g high s e p a r a t i o n power and speed f o r complex sample a n a l y s i s * I n l i q u i d chromatography, attempts to a c h i e v e h i g h r e s o l u t i o n and high speed LC a n a l y s i s have been made by many researchers i n the d e v e l o p m e n t o f m i c r o b o r e column HPLC* T a b l e I shows t h e operating parameters of four types of microbore columns reported i n the l i t e r a t u r e * A b r i e f comparison of these four types o f microbore columns a r e given below* Downloaded by PEPPERDINE UNIV on September 28, 2017 | http://pubs.acs.org Publication Date: April 26, 1984 | doi: 10.1021/bk-1984-0250.ch008

1

Open-Tubular Columns Open-tubular columns f o r l i q u i d chromatography were i n v e s t i g a t e d by I s h i i and a s s o c i a t e s / " ' Y a n g / * ^ and T i j s e n e t . a l / ' Columns r e p o r t e d a r e 10-300 jjm I.D. and 0.5-35 m i n l e n g t h * LC s t a t i o n a r y phases were e i t h e r p h y s i c a l l y c o a t e d o r c h e m i c a l l y bonded on t h e i n n e r w a l l o f t h e column. The t y p i c a l m o b i l e s o l v e n t f l o w r a t e s a r e n o r m a l l y ranged from 0.01 t o 1 ji£/min. The t h e o r e t i c a l performance p o t e n t i a l and p r a c t i c a l c o n s t r a i n t s of o p e n - t u b u l a r column LC have been d i s c u s s e d by Y a n g / ' T i l s s e n / ' P r e t o r i u s and S m u t s / ' G u i o c h o n / ' and H a l a s z e t . βίΛ ' T h e o r e t i c a l l y , open-tubular column could achieve s t a t e o f - t h e - a r t packed column p e r f o r m a n c e i n t e r m s o f speed o f a n a l y s i s , column e f f i c i e n c y and peak c a p a c i t y , i f t h e opent u b u l a r column I.D. c o u l d be reduced t o a p p r o a c h t h e p a r t i c l e diameter of the s t a t e - o f - t h e - a r t LC packed columns. For example, open- t u b u l a r columns w i t h I.D. l e s s t h a n 7*5 μπι have b e t t e r r e s o l v i n g power than that obtainable by using the s t a t e - o f - t h e a r t 5 Mm p a r t i c l e s packed columns i n a given a n a l y s i s time* Many r e p o r t s ' ^ have been g i v e n d e a l i n g w i t h t h e c o n s t r a i n t s of open-tubular column LC instrumentation* There i s no doubt t h a t o p e n - t u b u l a r column LC poses t h e most s t r i n g e n t r e q u i r e m e n t s on t h e pump (e.g., 0*01 ji£/min c o n t r o l l e d f l o w r a t e ) , I n t e r f a c e t u b i n g and c o n n e c t o r s (e.g., 1 μ £ ) i n t o narrowbore packed columns i s allowed i f a s o l u t e on-column f o c u s s i n g technique^ ' ' i s applied. I n p r i n c i p l e , a weak s o l v e n t m o d i f i e r can e i t h e r be p r e s e n t i n the sample s o l u t i o n o r be introduced j u s t before the i n j e c t i o n of the sample. An example of the on-column f o c u s s i n g e f f e c t on peak r e s o l u t i o n i s shown i n F i g u r e 3. F i g u r e 3-a shows a chromatogram of f o u r t h i o l compounds eluted from a 50 cm χ 250 μπι, 5 μπι C^g reversed phase column using 100% methanol as solvent f o r both mobile phase and sample s o l u t i o n . F i g u r e 3-b shows the improvement i n peak r e s o l u t i o n and d e t e c t i o n o b t a i n e d under the same c o n d i t i o n s except that the i n j e c t e d sample s o l u t i o n contains 13% water i n methanol. The e f f e c t of the weak solvent m o d i f i e r depends upon the r e l a t i v e s o l v e n t s t r e n g t h o f the s o l v e n t m o d i f i e r to the m o b i l e s o l v e n t and the amount r e l a t i v e to the column volume. In general, a weak


1 2

1 6

1 2

2 4



F i g u r e 2. The m o d i f i e d V a l c o 0.5 μ 1 i n t e r n a l l o o p s a m p l i n g v a l v e f o r s p l i t i n j e c t i o n i n n a r r o w - b o r e p a c k e d c o l u m n HPLC ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 12, c o p y r i g h t E l s e v i e r S c i . P u b l . Co.)


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Microbore Column HPLC

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a

I

0

I

10 t , min R

k 0

I

10 tu, min

F i g u r e 3 a . R e s o l u t i o n a n d peak shape o f ( 1 ) b e n z e n t h i o l ; ( 2 ) h e x a n e t h i o l ; ( 3 ) o c t a n e t h i o l and ( 4 ) d o d e c a n e t h i o l e l u t e d f r o m a 50 cm χ 250 μπι, 5 μπι C^g r e v e r s e p h a s e f u s e d - s i l i c a c o l u m n . M e t h a n o l was u s e d a s s o l v e n t f o r b o t h m o b i l e a n d sample s o l u t i o n . Sample volume was 0.6 μ 1. F i g u r e 3b. Improved peak r e s o l u t i o n a n d d e t e c t i o n by u s i n g 13% H2O i n t h e 0.6 μ 1 sample s o l u t i o n . A l l e x p e r i m e n t a l conditions were i d e n t i c a l t o t h a t f o r F i g u r e 3 a . ( R e p r o d u c e d w i t h permission f r o m R e f . 24, c o p y r i g h t P r e s t o n P u b l . I n c . )


100

ULTRAHIGH RESOLUTION CHROMATOGRAPHY

s o l v e n t m o d i f i e r volume w h i c h i s more than 1% o f t h e column volume c a n produce the e f f e c t o f on-column s o l u t e f o c u s s i n g i n narrow-bore packed column HPLC.


H y d r a u l i c Systems f o r I s o c r a t i c and Gradient Microbore Column HPLC The d e s i g n o f h y d r a u l i c s y s t e m s f o r n a r r o w - b o r e packed column HPLC i s p a r t i c u l a r l y c h a l l e n g i n g because o f t h e low f l o w r a t e used i n narrow-bore packed columns. The p o t e n t i a l performance of narrow-bore packed columns i s g r e a t l y reduced by the c o n s t r a i n t of o p e r a b l e c o n s t a n t low f l o w r a t e s and t h e o p e r a b l e p r e s s u r e limit. A h i g h p r e s s u r e s y r i n g e pump o p e r a t e d under c o n s t a n t p r e s s u r e mode i s o f t e n used f o r low f l o w r a t e o p e r a t i o n i n narrow-bore packed column HPLC. Flow r a t e s as low as 0.05 jz£/min can be obtained. The drawback of constant pressure operation i s the p o t e n t i a l flow r a t e v a r i a t i o n due to a flow r e s i s t a n c e change i n the column. In gradient e l u t i o n narrow-bore packed column LC, both step g r a d i e n t " » ) and continuous g r a d i e n t ^ > > ) systems were reported. In step gradient operation, a strong solvent stored i n an i n j e c t i o n l o o p i s i n t r o d u c e d i n t o t h e m i c r o b o r e column a t a selected time using an i n l i n e i n j e c t o r switching valve. A m u l t i port v a l v e a l l o w s the a p p l i c a t i o n of m u l t i p l e step g r a d i e n t elution. The advantages of the step gradient technique a r e that o n l y a s i n g l e pump i s needed t o a c h i e v e the e f f e c t o f g r a d i e n t e l u t i o n and the gradient delay time i s minimum. A l i n e a r gradient has advantages i n terms of automation and ease i n p r a c t i c e . T a k e u c h i and I s h i i ' ' also described a continuous gradient system u t i l i z i n g a syringe pump and a syringe type m i x i n g v e s s e l . A s i n g l e r e c i p r o c a t i n g h y d r a u l i c pump modified f o r l i n e a r gradient e l u t i o n i n micropore column HPLC has r e c e n t l y been r e p o r t e d by v a n der Wal and Y a n g ' ' and i s shown i n F i g u r e 4. The u s e o f such a g r a d i e n t s y s t e m i n c o n j u n c t i o n with a narrow-bore f u s e d - s i l i c a m i c r o p a r t i c l e packed column a t a flow r a t e between 3 and 5 μΐ/min showed a r e t e n t i o n time and peak area r e p r o d u c i b i l i t y comparable to that obtained by a conventional HPLC system. 2

3 7

1 6 , 2 0

3 8

3 9

3 9

Detector Because of the s m a l l e l u t i o n peak volume obtained from narrowbore packed columns, c o n v e n t i o n a l c o n c e n t r a t i o n dependent d e t e c t o r s such as U V - v i s i b l e a b s o r b a n c e , f l u o r o m e t r i c , and e l e c t r o c h e m i c a l detectors must be purged with makeup solvent or m i n i a t u r i z e d to a l l o w minimum extra-column c o n t r i b u t i o n to peak spreading. A c e l l volume 1 ji£/min. Gradient e l u t i o n i n narrowbore packed column HPLC r e q u i r e s a s i g n i f i c a n t r e d u c t i o n i n the g r a d i e n t d e l a y t i m e o f c u r r e n t s y s t e m s and improvement i n g r a d i e n t l i n e a r i t y and r e p r o d u c i b i l i t y f o r t h e whole g r a d i e n t range (0 to 100%)· A m i c r o v o l u m e h i g h p r e s s u r e (up to a few thousand b a r ) d i r e c t i n j e c t i o n v a l v e i s d e s i r a b l e f o r s m a l l sample i n t r o d u c t i o n to the narrow-bore packed columns* The oncolumn s o l u t e f o c u s s i n g t e c h n i q u e s h o u l d be a p p l i e d i n l a r g e sample i n j e c t i o n s * Narrow-bore packed column HPLC today can be p r a c t i c e d w i t h some m o d i f i c a t i o n s of the conventional LC system* The u t i l i t y of the narrow-bore packed column HPLC, however, now must be demonstrated i n the a p p l i c a t i o n s of " r e a l - l i f e " samples.


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November 3, 1983


Narrow-Bore, Microparticle-Packed Column High Performance Liquid

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