Size Exclusion Chromatography

3 Pressure-Programmed Controlled-Flow Supercritical Fluid Chromatograph E. W. ALBAUGH and D. BORST Gulf Research & Development Company, Pittsburgh, PA 15230

Downloaded by CORNELL UNIV on June 14, 2017 | http://pubs.acs.org Publication Date: March 30, 1984 | doi: 10.1021/bk-1984-0245.ch003

P. TALARICO Waters Associates, Milford, MA 01757

Supercritical fluid chromatography is a form of chromatography in which the system is held near the critical temperature of the mobile phase and pressure utilized to effect solvency and hence migration. The advantages of this technique have been shown to be increased mass transfer and the migration of high molecular compounds. Most of the instruments designed for this technique have not attempted to control the flow as pressure is programmed. In this paper, an instrument is described in which the inlet liquid flow is held constant and the pressure regulated by a pneumatically activated flow control valve at the exit of the column. This approach permits the use of a wide pressure program with a controlled flow and the use of several conventional liquid chromatographic detectors. Separations of model systems including normal aliphatic hydrocarbons, polynuclear aromatics and polymers with molecular weights ranging up to one million are reported. S u p e r c r i t i c a l f l u i d chromatography i s a form of chromatography i n which the temperature i s h e l d near the c r i t i c a l temperature of the mobile phase and pressure u t i l i z e d to e f f e c t solvency and hence m i g r a t i o n . The advantages of t h i s technique have been shown to be increased m^s^ ^ r a n s f e r and the m i g r a t i o n of h i g h molecular weight compounds. ' ' ' Since t h i s work was r e p o r t e d , h i g h p e r f o r mance l i q u i d chromatography has made r a p i d advancement and over shadowed much of the e a r l y appeal of s u p e r c r i t i c a l f l u i d chroma tography. However, i n the area of wide molecular weight-range samples, s u p e r c r i t i c a l f l u i d chromatography w i t h p r e s s u r e program ming appears to have advantages. J e n t o f t has demonstrated the p o t e n t i a l of t h i s technjlaue and d e s c r i b e d the design of a pressure programmed instrument. I n t h i s instrument the system pressure 0097 6156 84 0245 0047$06.00/0 © 198#«^fiiCi^y^ciety

Society Library

1155 16th S i M, W. Washington, 0. C. 20G36 Provder; Size Exclusion Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

SIZE E X C L U S I O N C H R O M A T O G R A P H Y

48

was c o n t r o l l e d by programming the i n l e t pressure, but the f l o w was not c o n t r o l l e d . Bartman, i n an instrument designed f o r use w i t h carbon d i o x i d e , has used a flow meter and a motor d r i v e n ex pansion, v a l v e a t the column e x i t to r e g u l a t e pressure and gas flow. ! vThe c u r r e n t s t a t e of the. f i e l d has been reviewed by Randal, G e r e , and P e a d e n . In t h i s paper, an instrument i s d e s c r i b e d i n which the i n l e t l i q u i d flow r a t e i s h e l d constant and the pressure r e g u l a t e d by a pneumatically actuated flow c o n t r o l v a l v e a t the e x i t of the column. This approach permits the use of a wide-range pressure program w i t h a c o n t r o l l e d flow. A l s o , by s e l e c t i n g mobile phases that a r e l i q u i d s a t ambient l a b o r a t o r y c o n d i t i o n s , s e v e r a l types of c o n v e n t i o n a l l i q u i d chromatographic d e t e c t o r s may be u t i l i z e d . ft


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EXPERIMENTAL A schematic diagram of the instrument i s shown i n Figure 1. The l i q u i d mobile phase flows from the r e s e r v o i r , through a heated chamber f o r degassing, and a 10 μ f i l t e r t o a s y r i n g e pump (Ruska, Cat. No. 1441 w i t h a Boston R a t i o t r o l v a r i a b l e speed motor con t r o l ) . A s a f e t y r e l i e f l i n e leads from the pump through a 207 bar rupture d i s c i n the r e s e r v o i r . A l i n e from the pump a l s o runs to a pneumatic pressure t r a n s m i t t e r (Moore Model No. 1735) which provides the process (pressure) s i g n a l f o r a c o n t r o l l e r (pressure) (Moore, N u l l m a t i c C o n t r o l l e r , Model 50). From the pump the s o l vent flows to s h u t - o f f v a l v e s A and Β (High-Pressure Equipment Co. Model 15-12 AF1-316). With Β c l o s e d and A open, the s o l v e n t flows through the sample v a l v e (Chromatronix Model HPSV, w i t h 25 μΐ loop) and i n t o the column oven. The sample v a l v e i s enclosed i n an oven w i t h a maximum temperature of 200PC which i s maintained by a temperature c o n t r o l l e r (West Guardsman, J r . ) . When Β i s open and A i s c l o s e d , the s o l v e n t flows through the preheater and i n t o the oven. The preheater c o n s i s t s of 2 f t of 1/8 i n . s t a i n l e s s s t e e l tubing wound around a Chromaiox heater and coated w i t h 2 i n . of i n s u l a t i o n . The temperature i s maintained 10°C above the column oven temperature. Inside the column oven, the s o l v e n t flows through 0.75 m of 0.009 i n . I.D. c o n d i t i o n i n g c o i l , through a low dead-volume tee c o n t a i n i n g a thermocouple t o monitor s o l v e n t temperature, and then to the column. The column oven, w i t h a 425°C maximum temper a t u r e , i s heated by two 2 - k i l o w a t t wire wound heaters which are c o n t r o l l e d w i t h a Gulton Model 2GB C o n t r o l l e r which provides e i t h e r i s o t h e r m a l o r programmed temperature c o n t r o l . A f t e r l e a v i n g the oven, the mobile phase flows through 1 m of 0.009 i n . c a p i l l a r y tubing which i s immersed i n a heat ex changer to r e t u r n the s o l v e n t to ambient temperature. A c o n t r o l v a l v e , (Research C o n t r o l Valve, P r e c i s i o n Products, T u l s a , Okla homa, Type 78S w i t h a P-9 trim) w i t h a low dead-volume head as shown i n Figure 2, i s placed a f t e r the heat exchanger. T h i s v a l v e i s p o s i t i o n e d by the c o n t r o l l e r ( p r e s s u r e ) . The s e t p o i n t of the

Provder; Size Exclusion Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1984.



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CHROMATOGRAPHY


SIZE E X C L U S I O N

Figure 2 .

M o d i f i e d research v a l v e .



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c o n t r o l l e r (pressure) I s p o s i t i o n e d by a v a r i a b l e speed motor which i s r e g u l a t e d by a GKHT-2 motor c o n t r o l l e r (G. K. K e l l e r Corp.). For i s o b a r i c o p e r a t i o n , the set p o i n t i s d r i v e n t o the d e s i r e d p o s i t i o n and the d r i v e d e a c t i v a t e d . The c o n t r o l l e r then maintains the s e l e c t e d pressure. For pressure programming, the motor speed i s s e l e c t e d t o d r i v e the set p o i n t a t the d e s i r e d r a t e . I n o p e r a t i o n , the flow r a t e i s f i r s t set by the pump con t r o l and then the pressure a d j u s t e d . A siphon counter (Waters A s s o c i a t e s , Model C908) i s placed a t the end of the system to measure v o l u m e t r i c f l o w . The s o l v e n t system used here c o n s i s t e d of cyclohexane and 5% e t h a n o l . This mobile phase w i l l d i s s o l v e many petroleum f r a c t i o n s , produce a s t a b l e base l i n e , and i s compatible under ambient l a b o r a t o r y c o n d i t i o n s w i t h many common l i q u i d chromatographic de t e c t o r s . The system was operated a t 10°C above the c r i t i c a l tem perature of cyclohexane (280°C). The s t a t i o n a r y phase u t i l i z e d was 75-100 mesh P o r o s i l C packed i n t o f o u r , 4 - f t lengths of 1/4 i n . O.D. s t a i n l e s s s t e e l tubing f i t t e d w i t h 10 μ snubber, swagelock 1/4 i n . to 1/16 i n . unions. The minimum pressure i n t h i s system a t 1 ml/min. flow r a t e and 280°C i s 20.6 bar. The maximum o p e r a t i n g pressure of the instrument i s 206.8 bar. As the pressure i s i n c r e a s e d , the flow r a t e i s constant from 20.6 to 48.2 bar and the base l i n e i s ex c e l l e n t . I n the r e g i o n from approximately 48.2-55.2 bar the flow r a t e slows s l i g h t l y and the base l i n e r i s e s w i t h an u l t r a v i o l e t d e t e c t o r . This change i s r e p r o d u c i b l e and b e l i e v e d due to a phase change i n the s o l v e n t system. From approximately 55.2 t o 206.8 bar the flow r a t e i s again constant and the base l i n e ex c e l l e n t . Depending upon the pressure program r a t e , a short per iod of compression i s i n i t i a l l y r e q u i r e d , and then the flow s t a bilizes . RESULTS AND DISCUSSION The s e p a r a t i o n of a mixture of aromatic compounds (benzene, naph thalene, anthracene, chrysenes, and benz(a)pyrene) a t 31 bar i s shown i n Figure 3. This chromatogram was obtained w i t h a P e r k i n Elmer Model 250 u l t r a v i o l e t d e t e c t o r w i t h the high-pressure c e l l p l a c e d a f t e r the c o o l i n g heat exchanger and before the flow con t r o l v a l v e . A s i m i l a r chromatogram i s obtained w i t h an Isco Model UA4 w i t h a 10 mm micro c e l l placed a f t e r the flow c o n t r o l valve. The e f f e c t o f pressure (measured a t the pump) on t h i s separa t i o n can be seen i n Figure 3 and Table I . As the pressure i s i n creased, the r e t e n t i o n volume of benzene and naphthalene c o n t i n u a l l y i n c r e a s e , w h i l e the r e t e n t i o n volume o f anthracene, chrysene, and benz(a)pyrene f i r s t i n c r e a s e , go through a maximum, and then decrease. The maximum s e p a r a t i o n occurs a t 38.5 bar w h i l e the minimum s a t i s f a c t o r y s e p a r a t i o n volume ( s h o r t e s t a n a l y s i s


52

CHROMATOGRAPHY



F i g u r e 3.

Separation o f benzene, naphthalene, anthracene,

chrysene, and benz(o:)pyrene at 50 and 31 b a r .


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Supercritical Fluid

Chromatograph

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time) occurs at 50 bar. At 51.7 bar, the h i g h e r molecular weight m a t e r i a l s are not r e s o l v e d .


Table I .

EFFECT OF PRESSURE ON SEPARATION OF POLYNUCLEAR AROMATIC COMPOUNDS

Compound P r e s s u r e , bar Benzene Naphthalene Anthracene Chrysene Benz(a)Pyrene

31 2. 5 4. 0 8. 5 18. 0 27. 5

40 5 8 13.5 26.0 35.5

Elution 44 .1 6 .0 8 .5 15 .0 24 .5 32 .0

Volume, ml 50 46 .5 8.0 6 .5 10.0 9 .0 13.0 13 .0 17 16 .5 18 .0 18.5

51 .7 8 .0 10 .0 10- 13 10- 13 10- 13

The s e p a r a t i o n of a somewhat h i g h e r molecular weight m a t e r i a l i s shown i n Figure 4. Here, a p o l y s t y r e n e w i t h an average molecular weight of 600 has been separated i n t o eleven components a t 50 bar. The e f f e c t of pressure programming i s a l s o shown. The program was s t a r t e d at 46 bar at a r a t e of 0.34 bar per min. As the p r e s sure i n c r e a s e s , the e l u t i o n volumes of the v a r i o u s compounds de crease and the peak widths become more narrow i n a manner s i m i l a r to that f o r temperature programming i n gas chromatography and s o l vent programming i n l i q u i d chromatography. At 65.5 bar the sample e l u t e s e s s e n t i a l l y as one peak. To demonstrate the behavior of h i g h molecular weight com pounds i n t h i s system, a s e r i e s of p o l y s t y r e n e standards were analyzed. The h i g h e s t molecular weight m a t e r i a l (average molecu l a r weight of 1,800,000) i s shown i n Figure 5. A pressure p r o gram r a t e of 0.69 bar per minute was used. A s m a l l amount of m a t e r i a l e l u t e s a t approximately 75.8 b a r , but the major p o r t i o n of the sample e l u t e s between 89.6 bar and 134.4 bar. A sample was taken from the 117.2 bar r e g i o n and analyzed by c o n v e n t i o n a l e x c l u s i o n chromatography. I t was found to have a molecular weight i n the range of 1,000,000. Thus, these high molecular compounds s u r v i v e the column and are r e s o l v e d a t pressures below 2000 p s i . The other lower molecular weight p o l y s t y r e n e standards e l u t e d a t correspondingly lower p r e s s u r e s . A s e r i e s of normal hydrocarbons were analyzed u s i n g the same chromatographic system and the Pye LCM I I flame i o n i z a t i o n detec t o r . I n Figure 6 i s shown the s e p a r a t i o n of a mixture of C 22' 4 o * A4 l hydrocarbons. With the h i g h molecular weight c a p a b i l i t y shown f o r the p o l y s t y r e n e s , t h i s system should a l s o handle the higher molecular weight s a t u r a t e d hydrocarbons that are beyond the range of gas chromatography. S e v e r a l p o l y n u c l e a r compounds c o n t a i n i n g both aromatic and a l k y l f u n c t i o n s were chromatographed. The higher the molecular weight of the compound, the g r e a t e r was the e l u t i o n volume, i n d i c a t i n g t h a t s e p a r a t i o n was not o c c u r r i n g according to the number of aromatic r i n g s . C

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CHROMATOGRAPHY



F i g u r e k. Separation o f low molecular p o l y s t y r e n e "by pressure programming.


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PRESSURE BAR MIRROR IMAGE F i g u r e 5. Pressure s e p a r a t i o n o f 1,800,000 molecular weight p o l y s t y r e n e "by pressure programming.

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F i g u r e 6. Separation o f C g, C ^ , C , C^ , and hydrocarbons a t U8.3 b a r m i r r o r . 2

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SIZE EXCLUSION

CHROMATOGRAPHY

In the area of p o l a r compounds, phenol, r e s o r c i n o l , and ben z o i c a c i d were chromatographed. Phenol gives a n e a r l y symmetri c a l peak w i t h e s s e n t i a l l y no t a i l i n g . R e s o r c i n o l i s separated from phenol but shows some t a i l i n g . Benzoic a c i d f a l l s i n the same e l u t i o n r e g i o n as r e s o r c i n o l , but t a i l s to a much g r e a t e r extent. In one a p p l i c a t i o n , styrene s t i l l bottoms were chromato graphed. Here styrene and the i n d i v i d u a l lower molecular weight oligomers were separated, and as the pressure was i n c r e a s e d , the higher molecular weight p o l y s t y r e n e e l u t e d . The instrument described here has been found to be essen t i a l l y t r o u b l e - f r e e . Pressure s e t t i n g s and c o n t r o l are reproduc i b l e , r e q u i r i n g only the p o s i t i o n i n g of a s w i t c h . One of the a t t r a c t i v e a t t r i b u t e s of s u p e r c r i t i c a l f l u i d chromatography i s the s h o r t time r e q u i r e d f o r the column to reach e q u i l i b r i u m when con d i t i o n s are changed. A f t e r o p e r a t i n g a t 172.4 b a r , f o r example, the instrument can be r a p i d l y depressurized to 20.7 b a r and the system, i n c l u d i n g the columns, e q u i l i b r i a t e d w i t h i n a few minutes. The column c o n d i t i o n i n g problems o f t e n found i n high-pressure l i q u i d chromatography-solvent programming were not experienced here. The r e p r o d u c i b i l i t y of e l u t i o n volumes i s comparable t o i s o c r a t i c h i g h pressure l i q u i d chromatography. LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Giddings, J . C . , Myers, Μ. Ν . , and King, J . W., J. Chroma togr. S c i . , 7, 276(1969). Giddings, J . C . , Science, 162, 7(1968). Su, S. T . , Rynders, G. W. Α., Anal. Chem. Act., 38 31(1967). Gouw, T. H . , Jentoft, R. E., J. Chromatogr., 68, 303(1972). Doran, T . , Soc. Analyt. Chem., 117, May 1974. Jentoft, R. E., Gouw, T. H . , J. Chromatogr. S c i . , 8, 138(1970). Bartman, D., Berichte der Bunsen-Gesellschaft Bd. 76, NY 3/4, 336(1972). Randal, L. G . , Separation Science and Technology, 17(1) 1(1982). Gere, D. R., Board, R., McManigill, Anal. Chem., 54, 736(1982). Peaden, P. Α., Lee, M. L., J. Liq. Chromatogr. 5(2), 179(1982).

RECEIVED October 13, 1983


Size Exclusion Chromatography

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