Size Exclusion Chromatography - American Chemical Society

13 Deuterium Oxide Used to Characterize Columns for Aqueous Size Exclusion Chromatography HOWARD G. BARTH Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 11, 2015 | http://pubs.acs.org Publication Date: March 30, 1984 | doi: 10.1021/bk-1984-0245.ch013

Research Center, Hercules Incorporated, Wilmington, DE 19899 FRED Ε. REGNIER Department of Biochemistry, Purdue University, West Lafayette, IN 47907

In order to characterize size-exclusion chroma tographic (SEC) columns, both the interstitial volume and the pore volume of a packed column must be determined. This information is required for the construction of a calibration curve as well as to obtain SEC distribution coefficients. In aqueous SEC, either glucose or deuterium oxide (D O) are commonly used to measure the total permeation volume of a column. Using LiChrospher silica packings with a glycerylpropyl silane bonded phase (SynChropak GPC), we found that the elution volume of D O was significantly greater than the results obtained for glucose. Controlled-pore glass packings which have narrower pore-size distributions did not exhibit this property. From these results, it appears that the silica packing contains a population of micropores which are accessible only to low molecular weight probes. 2

2

Size e x c l u s i o n chromatography (SEC) i s a s e p a r a t i o n process by which molecules are f r a c t i o n a t e d by s i z e on the b a s i s o f d i f f e r e n t i a l p e n e t r a t i o n i n t o porous p a r t i c u l a t e m a t r i c e s . Elution volume ( V ) o f any given molecular species r e l a t i v e t o another of d i f f e r e n t s i z e i s dependent on the pore diameter o f the m a t r i x , p o r e - s i z e d i s t r i b u t i o n , pore volume ( V | ) , i n t e r s t i t i a l volume ( V ) and column dimensions. Use o f SEC t o estimate molecular s i z e i s achieved by p l o t t i n g the l o g o f the molecular weight o f a s e r i e s o f c a l i b r a n t s against t h e i r e l u t i o n volume. Since V i s a f u n c t i o n of V and V|, i t s magnitude w i l l be dependent on the geometry o f a column. A more u s e f u l and fundamental parameter than e l u t i o n volume i s the dimensionless s i z e e x c l u s i o n d i s t r i b u t i o n c o e f f i c i e n t (K ) which i s r e l a t e d t o V| and V by the equation: e

0

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0097-6156/84/0245-0207S06.00/0 © 1984 American Chemical Society

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

SIZE E X C L U S I O N

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Use of K i n s t e a d of V i n the c a l i b r a t i o n of columns produces a c a l i b r a t i o n curve that i s independent of column dimen sions and pore volume. To o b t a i n Kp f o r any species r e q u i r e s the determination of V and V | i n a d d i t i o n to V . V is u s u a l l y taken as the e l u t i o n volume of an excluded polymer w h i l e V | i s equal t o V - V . The volume i s the t o t a l permeation volume of the column and i s measured w i t h a low molec u l a r weight compound t h a t t o t a l l y permeates p a r t i c l e m a t r i c e s . Deuterium oxide (D2O) has been used to determine V«j i n SEC columns because i t s low molecular weight assures high m a t r i x permeation and i t s high d i f f u s i o n c o e f f i c i e n t i s u s e f u l i n determining column e f f i c i e n c y (1-3). ( I t should be noted t h a t i n aqueous mobile phases, DHO would be present a f t e r i n j e c t i n g D2O i n t o a column because of hydrogen exchange.) I n a d d i t i o n to D2O, t r i t i a t e d water (THO) has been used as a low molecular weight probe of V i n SEC (1.4-7). Marsden (4.8)» however, cautions t h a t t r i t i u m exchange w i t h i n the c r o s s l i n k e d p o l y saccharide m a t r i x c o u l d r e s u l t i n e r r o r s when THO i s used t o determine V^. From measurements w i t h H2* 0, Marsden found t h a t K f o r THO was 1.09 ( 8 ) . The assumption has g e n e r a l l y been made i n SEC w i t h m a t r i c e s g r e a t e r than 100Â pore diameter t h a t there i s l i t t l e , i f any, s i z e d i s c r i m i n a t i o n of molecules l e s s than 500 d a l t o n s , i . e . , they would a l l e l u t e at Vj. During our s t u d i e s w i t h SynChropak, a high-performance SEC packing c o n s i s t i n g of LiChrospher s i l i c a w i t h a g l y c e r y l p r o p y l s i l a n e bonded phase, we found to our s u r p r i s e t h a t the e l u t i o n volume of D2O was s i g n i f i c a n t l y g r e a t e r than t h a t of glucose which we had p r e v i o u s l y used as a low molecular weight c a l i b r a n t (9-11). The problem of determining V i n SEC i s s i m i l a r t o t h a t of determining zero r e t e n t i o n time ( t ) i n other l i q u i d chroma tography columns. Recently, there have been s e v e r a l papers d e a l i n g w i t h the determination of r e t e n t i o n time of a r e t a i n e d peak i n HPLC (12-19). In high-performance reversed-phase chromatography, McCormick and Karger (15) and Berendsen, et a l . , (16) have employed D2O to measure t . Neidhart et a l . , (12.14) took a d i f f e r e n t approach by determining the r e t e n t i o n times of a s o l u t e as a f u n c t i o n of temperature. Since the enthalpy of a d s o r p t i o n of a s o l u t e onto a s t a t i o n a r y phase i s n e g a t i v e , the e l u t i o n time of a r e t a i n e d species should decrease w i t h i n c r e a s i n g temperature. However, none of these methods r i g o r o u s l y examines the p o s s i b i l i t y t h a t m i c r o p o r o s i t y may a l s o cause d i f f e r e n c e s i n t between s o l u t e s . This paper d e s c r i b e s the extent of r e t e n t i o n time d i f f e r e n c e s between D2O and glucose on bonded phase i n o r g a n i c supports. D

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 11, 2015 | http://pubs.acs.org Publication Date: March 30, 1984 | doi: 10.1021/bk-1984-0245.ch013

CHROMATOGRAPHY

e

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Experimental


Apparatus. Pumping systems used i n these s t u d i e s f o r h i g h performance columns were a V a r i a n 8500 syringe pump and a V a r i a n 5000 i s o c r a t i c pump. An A l t e x 110A was employed f o r the cont r o l l e d - p o r e g l a s s (CP6) columns. Waters A s s o c i a t e s model 401 refractometers were used on a l l instruments. Stagnant mobile phase was kept i n the reference s i d e of the refractometer. Samples were i n j e c t e d w i t h a Eheodyne 70-10 i n j e c t i o n v a l v e using a 2 0 y l loop (lOOul f o r CPG columns). Columns. The packing m a t e r i a l s were lOum SynChropak and 37-74um c o n t r o l l e d - p o r e g l a s s w i t h g l y c e r y l s i l a n e bonded phase. SynChropak columns were purchased prepacked i n 25 cm χ 4.1 mm ID s t a i n l e s s s t e e l columns from SynChrom (Linden, IN). Nominal pore s i z e s were 100, 300, 1000 and 4000Â. CPG was dry packed i n t o s t a i n l e s s s t e e l columns using the t a p - f i l l procedure (20). Column dimensions were 100 cm χ 4.6 mm ID f o r the 1000, 1400, 2000 and 3000Â m a t e r i a l and 50 cm χ 4.6 mm ID f o r the 75Â packing. A d e s c r i p t i o n of these packings i s given i n Table I . Values l i s t e d i n the t a b l e were obtained from the manufacturer ( E l e c t r o n u c l e o n i c s I n c . ) .

TABLE I .

GLYCERYL-CPG COLUMN PACKING MATERIAL (200/400 mesh)

Nominal Mean Pore Pore S i z e Pore Pore S i z e . A Diameter. ft D i s t r i b u t i o n . +% Volume, cc/κ 75 1000 1400 2000 3000

75 1038 1489 1902 3125

6.0 7.3 6.4 10 10

Chemicals. Urea (99+%), glucose and D2O from A l d r i c h Chemical Co. (Gold L a b e l ) .

0.47 1.22 1.16 0.80 1.25

Surface Area. m /g 2

140 28 17.6 10 7.9

(99.8%) were obtained

Mobile Phase P r e p a r a t i o n . D i s t i l l e d water and 6M urea were f i l t e r e d under vacuum using a 0.22um membrane f i l t e r (Type GS, Millipore). Sample P r e p a r a t i o n i n 6M Urea. S o l u t i o n s of glucose were prepared d i r e c t l y i n 6M urea. D2O s o l u t i o n s were prepared by d i l u t i n g equal volumes of D 0 and 12M urea and the r e s u l t i n g s o l u t i o n was then d i l u t e d 1:1 w i t h 6M urea. 2


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E l e v a t e d Temperature S t u d i e s , The V a r i a n 5000 l i q u i d chromatograph and a Waters A s s o c i a t e s 401 d i f f e r e n t i a l r e f r a c t o m e t e r were employed. The column was heated w i t h a V a r i a n u n i v e r s a l heater b l o c k a t an estimated accuracy o f + 0.5 C. About 15-30 minutes were allowed f o r column e q u i l i b r a t i o n f o r a given temperature. The r e c o r d e r employed was a V a r i a n 9176. A 25 cm χ 4.6 mm ID long 300ft SynChropak column was used to evaluate temperature e f f e c t s . I n j e c t i o n s were made w i t h 5% D2O and 1.3 mg/ml glucose s o l u t i o n s . D2O gave a negative r e f r a c t i v e index response. Because of some peak t a i l i n g , the number o f t h e o r e t i c a l p l a t e s was based on peak width at one-half peak h e i g h t : N=5.54 (t /wi/2> . The pooled standard d e v i a t i o n ( a l l temperatures) of r e t e n t i o n time measurements (df=34) was + 0.007 minutes.


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P h y s i c a l Measurements on Supports. Pore diameter and volume were determined by mercury porosimetry. Micropores were estimated by the BET and t-curve methods (21, 2 2 ) . R e s u l t s and D i s c u s s i o n E l u t i o n Volume o f DgO and Glucose on C o n t r o l l e d - P o r e Glass and SynChropak Columns. The e l u t i o n volumes o f D2O and glucose on 100, 300 and 4000Â p o r e - s i z e SynChropak columns are given i n Table I I . As i n d i c a t e d , the e l u t i o n volume of D2O was g r e a t e r than t h a t of glucose i n a l l cases. Because o f the s m a l l e r hydrodynamic volume of D2O, as compared t o glucose, t h i s t r e n d was expected. However, the s i z a b l e e l u t i o n volume d i f f e r e n c e between D2O and glucose e x h i b i t e d by the 100 and 300Â columns i s s u r p r i s i n g . On the b a s i s o f t o t a l pore volume, V|, the percentage o f micropore volume t h a t was a v a i l a b l e t o D2O and not glucose was h i g h : 17.4 + 1.7% and 8.4 + 1.5%, r e s p e c t i v e l y , f o r the 100 and 300ft packings. The r e s u l t obtained w i t h the 4000ft column was w i t h i n experimental e r r o r . Glucose and D2O were a l s o t e s t e d on f i v e g l y c e r y l p r o p y l CPG packings o f 75, 1000, 1400, 2000 and 3000ft and the r e s u l t s are presented i n Table I I I . The percentage o f micropore volume t h a t was a v a i l a b l e t o D2O and not glucose was c l o s e t o o r w i t h i n the experimental e r r o r of V determination f o r a l l columns. e


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BARTH A N D REGNIER

211

TABLE I I . ELUTION CHARACTERISTICS OF D 0 AND GLUCOSE ON SYNCHROPAK COLUMNS* 2

Pore Diameter D 0, V (ml) Glucose, V (ml) Δ, ml V | , ml** Micropore volume, %*** 2

ÎOOA 2.58 2.34 +0.24 1.38 17.4+1.7

r


r

300& 2.82 2.68 +0.14 1.66 8.4+1.5

*

4000A 2.62 2.60 +0.02 1.47 1.4+1.7

Chromatographic c o n d i t i o n s : Mobile phase: H 0; Flow: 0.5 ml/min; Chart Speed: 1 in/min; Volume i n j e c t e d : 2 0 u l ; Sample c o n c e n t r a t i o n s : 1 mg/ml glucose and 5% D 0; Columns: 25 cm χ 4.1 mm ID; RI d e t e c t o r s e n s i t i v i t y : X4. ** i = V - V where V i s the e l u t i o n volume of D 0. For 4000Â columns, V = 0.35 (*»r L). For 100 and 300Â columns, V was obtained from 2 χ 1 0 d a l t o n dextran (1.20 and 1.16 m l , r e s p e c t i v e l y ) . *** Propagated e r r o r assuming flow r a t e p r e c i s i o n of + 1%. 2

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TABLE I I I . ELUTION CHARACTERISTICS OF D 0 AND GLUCOSE ON GYCERYL ·- CPG COLUMNS* 2

Pore Diameter D 0, V ( m l ) Glucose, V (ml) Δ, ml V|, ml** Micropore volume, %*** 2

lOOOA 14.25 14.15

21a 5.75 5.65

r

1400Â 14.20 14.18

2000Â 13.70 13.65

3000Â 13.38 13.30

r

0.10 2.15 4.6+2,,7

0.02 8.38 0.2+1. 7

0.10 8.43 1.2+1.7

0.05 7.88 0.6+1..8

*

0.08 7.56 1+1.8

Chromatographic c o n d i t i o n s : Mobile phase: 0.5 M NaOAc; Flow: 0.5 ml/min; Chart Speed: 0.5 cm/min; Volume i n j e c t e d : ΙΟΟμΙ; Sample c o n c e n t r a t i o n s : 2 mg/ml glucose (X4) and 5% D 0 (X8); Columns : 100 cm χ 4.6 mm ID (50 cm χ 4.6 cm ID f o r 75Â); Pump: A l t e x 110A. ** V| a V - V where V i s the e l u t i o n volume of D 0. For 1000, 1400, 2000 and 3000Â columns, V » 0.35 (tTT «L). For 75Â columns, V was obtained from 2 χ 1 0 d a l t o n dextran. *** Propagated e r r o r assuming flow r a t e p r e c i s i o n of + 1%. 2

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SIZE EXCLUSION CHROMATOGRAPHY

Mercury porosimetry data o f these packings are given i n Table IV. I t i s o f i n t e r e s t t o note t h a t the p o r e - s i z e d i s t r i b u t i o n o f CPG i s s i g n i f i c a n t l y more narrow than t h a t of Syn Chropak, a s u r f a c e - m o d i f i e d porous s i l i c a ( L i C h r o s p h e r ) . These d i f f e r e n t p h y s i c a l c h a r a c t e r i s t i c s may help t o e x p l a i n the e x i s tence o f micropores i n SynChropak. Because of the wide p o r e - s i z e d i s t r i b u t i o n o f t h i s packing, i t seems reasonable t h a t t h i s m a t e r i a l a l s o contains a p o p u l a t i o n of micropores which are only a c c e s s i b l e t o D2O. I n mercury porosimetry measurements, the lower pore s i z e l i m i t i s about 30*.

TABLE IV. PHYSICAL CHARACTERISTICS OF SEC PACKINGS FROM MERCURY POROSIMETRY

Support Pore SynChropak (lOym diam.) Glyceryl-CPG (37~74um diam. ) 1000* 3000* Diameter 100* 75* 1000* 4000* Pore-size d i s tribution,μπι Dead-end volume, cc/g V|, cc/g* V , cc/g** Surface area, m /g 0

0.00440.06 1.66

0.020.30 1.55

0.140.9 0.84

0.0060.009 0.125

0.090.18 0

0.250.35 0

0.92 1.10 294

0.96 1.10 48.4

0.82 1.25 12.0

0.33 0.90 181

1.35 1.65 50

0.89 1.4 9.5

z

* **

Pore volume I n t e r s t i t i a l volume (measured t o 100 p s i )

Comparison of surface areas as determined by the BET and t-curve methods (21) i s another measure o f m i c r o p o r o s i t y s i n c e the l a t t e r technique w i l l estimate the surface area o f pores under 15* i n diameter. A SynChropak GPC-100 sample gave 201 m /g by the BET method and 216 m /g by the t-curve method. The 15 m /g d i f f e r e n c e i s a t t r i b u t e d t o micropores l e s s than 15*. I n c o n t r a s t , 75* pore diameter Glycophase CPG was found t o have 137 m /g o f surface area by both the BET and t-curve methods i n d i c a t i n g the absence o f micropores. Dead-end volume i s estimated from mercury porosimetry by measuring the amount o f mercury l i b e r a t e d from the packing when the a p p l i e d pressure i s r e l e a s e d . This measurement approximates the volume occupied by b l i n d channels o r pockets w i t h i n the i n t e r s t i t i a l and pore volumes. Assuming t h a t the i n t e r s t i t i a l volume o f the bed c o n s i s t s t o t a l l y o f b l i n d channels, then the minimum percentage o f dead-end volume w i t h i n the pores of the packing i s 61 and 47%, r e s p e c t i v e l y , f o r the 100 and 1000* 2

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SEC


SynChropak m a t e r i a l s . The minimum percentage of dead-end pores w i t h i n the 4000Â SynChropak i s 0%. Because of the much l a r g e r p a r t i c l e diameter of the CP6 packings, one would expect that b l i n d channels w i t h i n the packed bed would be n e g l i g i b l e . I n view of t h i s , the 75Â CPG packing would have a maximum of 38% of dead-end volume. The 1000 and 3000Â CPG packings have no dead-end pores. The i m p l i c a t i o n of these f i n d i n g s i n terms of column e f f i c i e n c y w i l l be presented i n a f u t u r e paper (23). E f f e c t of Flow Rate on E l u t i o n Volume of D2O and Glucose. I n order t o r u l e out the p o s s i b i l i t y that the increased r e t e n t i o n volume of D 0 was caused by deuterium exchange on e i t h e r r e s i d u a l s i l a n o l groups on the packing or h y d r o x y l groups on the g l y c e r y l p r o p y l s i l y l s t a t i o n a r y phase, the e l u t i o n volume of DHO was determined as a f u n c t i o n of flow r a t e . As shown i n F i g u r e 1, there was no s i g n i f i c a n t d i f f e r e n c e i n e l u t i o n volume when the f l o w r a t e was v a r i e d from 0.10 t o 2.0 ml/min (23.4 t o 1.2 minute residence time, r e s p e c t i v e l y ) . For a c o n t r o l , the e l u t i o n volume of glucose i s a l s o given. I t should be emphasized that even i f deuterium exchange were o c c u r r i n g , the r e s u l t i n g H 0 molecules would not be detected. Furthermore, DHO peaks were symmetrical; the absence of a t a i l e d peak i s f u r t h e r c o n f i r m a t i o n t h a t secondary e q u i l i b r i u m was not o c c u r r i n g . 2

2

E f f e c t of D 0 Concentration on E l u t i o n Volume. I f deuterium exchange were o c c u r r i n g , one would a l s o expect t h a t the exchange e q u i l i b r i u m would be dependent on D 0 c o n c e n t r a t i o n . In view of t h i s , 0.625 t o 10% D 0 was i n j e c t e d and the r e s u l t i n g r e t e n t i o n times and peak heights are shown i n Table V. The r e s u l t s c l e a r l y demonstrate that there was no D 0 concen t r a t i o n dependency of e i t h e r r e t e n t i o n volume or peak h e i g h t . 2

2

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TABLE V. EFFECT OF INJECTION CONCENTRATION ON PEAK HEIGHT AND RETENTION VOLUME OF D 0* 2

D0 2

Concentration. % 10 5 2.5 1.25 0.625

* **

V r . ml** 2.55 2.52 2.54 2.54 2.54

Height, cm**

DRI A t t e n u a t i o n

14.1 14.2 14.3 14.1 14.2

Chromatographic c o n d i t i o n s : See Table I I , 100Â Average of t r i p l i c a t e 20ul i n j e c t i o n s

16 8 4 2 1 column


214


6M Urea as the Mobile Phase. The only p o s s i b l e p a r t i t i o n i n g mechanism that c o u l d be r e s p o n s i b l e f o r D 0 r e t e n t i o n i s hydrogen bonding to the g l y c e r y l p r o p y l s i l y l s t a t i o n a r y phase which i s h i g h l y u n l i k e l y because of c o m p e t i t i o n between D 0 and the H 0 mobile phase. However, to r u l e t h i s out, D 0 and glucose were chromatographed i n a 6M urea mobile phase u s i n g a 100Â column. The r e s u l t s , given i n Table V I , are s i m i l a r t o the data obtained u s i n g water as the mobile phase (Table I I ) , i n d i c a t i n g t h a t the urea mobile phase had no s i g n i f i c a n t e f f e c t on e l u t i o n volume of D 0. I t i s of importance t o note that i t was d i f f i c u l t to prepare a 5% D 0 s o l u t i o n i n 6M urea so that the c o n c e n t r a t i o n of urea would be i d e n t i c a l t o that of the mobile phase. Because of the high urea content, a r e l a t i v e l y s m a l l d i f f e r e n c e between the urea c o n c e n t r a t i o n i n the i n j e c t e d s o l u t i o n and i n the mobile phase, produced a urea peak. In view of t h i s , the urea content of the i n j e c t e d s o l u t i o n was adjusted to minimize i n t e r f e r e n c e . 2

2


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TABLE V I .

ELUTION OF D 0 2

D 0, V (ml) Glucose, V (ml) Â, ml V|, ml Micropore volume, % 2

r

r

IN 6M UREA*

2.59 2.32 0.27 1.38 19.3+1.7

Chromatographic c o n d i t i o n s : Flow: 1.0 ml/min; Chart speed: 2.5 in/min; 100Â Synchropak column. See Table I I f o r other c o n d i t i o n s .

E f f e c t of Temperature on E l u t i o n Volume. The heat of s o l u t i o n of a s o l u t e (ΔΗ) (heat l o s s when 1 mole of s o l u t e i s t r a n s f e r r e d from the mobile phase to the s t a t i o n a r y phase) i s r e l a t e d to the p a r t i t i o n c o e f f i c i e n t (K) as f o l l o w s : Log Κ « &

0

I*" + C 2.30 RT

(2)

Since K=k* V / V where k* i s the c a p a c i t y f a c t o r [ k ' = ( t - t ) / t ] , t and t are the e l u t i o n times of a r e t a i n e d and unretained peak, r e s p e c t i v e l y , V i s the volume of mobile phase, V i s the volume of s t a t i o n a r y phase and C i s a constant, then M

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215

f

Thus, ΔΗ can be r e a d i l y determined by p l o t t i n g l o g k versus 1/T. I f ΔΗ i s z e r o , there are no s o l u t e - p a c k i n g i n t e r a c t i o n s other than an entropie c o n t r i b u t i o n ( s i z e s e p a r a t i o n ) . S i n c e , by d e f i n i t i o n , k* > 1, the r e t e n t i o n time of glucose was used f o r t and the r e t e n t i o n time of D 0 was used f o r t . The r e t e n t i o n times of glucose and D 0 as a f u n c t i o n of column temperature using a 300Â SynChropak column are i n Table V I I . As i n d i c a t e d , the percent d i f f e r e n c e i n r e t e n t i o n time between D 0 and glucose was about 4.5% f o r a l l temperatures. These r e s u l t s were c l o s e to the 5.2% d i f f e r e n c e obtained from Table I I . The s m a l l e r value obtained i n t h i s study was probably caused by d i f f e r e n c e s i n the two l o t s of s i l i c a used i n the colums. 0

2

r

2


2

TABLE V I I . TIME OF D 0 2

EFFECT OF COLUMN TEMPERATURE ON THE ELUTION AND GLUCOSE USING A 300* SYNCHROPAK COLUMN*

Column Temp. *C P r e s s u r e , p s i Glucose 29 420 6.512 39 348 6.496 49 290 6.468 60 246 6.436 70 218 6.422

t r . min E^O D i f f e r e n c e . % k*. D 0 6.802 4.4 0.0445 6.776 4.3 0.0431 6.752 4.4 0.0439 6.732 4.6 0.0459 6.712 4.5 0.0451 2

Chromatographic c o n d i t i o n s : Mobile phase: H 0; Flow: 0.5 ml/min; Chart Speed: 5 cm/mi η ; Volume i n j e c t e d : 2 0 u l ; Sample c o n c e n t r a t i o n s : 1.3 mg/ml glucose and 5% D 0; Detector: RI X8; Column: 25cm χ 4.6mm ID SynChropak 300*. 2

2

The decrease i n s o l u t e r e t e n t i o n time w i t h column tempera t u r e was caused i n p a r t by the expansion of mobile phase as i t entered the heated column. For example, there was a 1.3-1.4% increase i n f l o w r a t e when the temperature was increased from 29 to 70 C. The p r e d i c t e d value based on the expansion c o e f f i c i e n t of water i s 0.8%. As shown i n Table V I I there appears to be no s i g n i f i c a n t change of k w i t h respect to temperature. These data were p l o t t e d using Equation 3 and from l i n e a r r e g r e s s i o n a n a l y s i s , the heat of s o l u t i o n was +0.18 Kcal/mole. Since ΔΗ should be negative, t h i s low value i s o b v i o u s l y caused by experimental e r r o r . Furthermore, the ΔΗ c a l c u l a t e d from the standard e r r o r of the estimate (+1 standard d e v i a t i o n u n i t s ) of the l i n e a r r e g r e s s i o n l i n e i s ±0.17 Kcal/mole. Since ΔΗ i s zero or i s very c l o s e to z e r o , Equation 3 reduces to e

f

log k

f

- C

f


(4)


216

and the f r e e energy change when DHO i s t r a n s f e r r e d from the mobile phase to the s t a t i o n a r y phase i s of the form G^TâS. Thus the r e t e n t i o n time of D 0 i s caused by e n t r o p i e r a t h e r than e n t h a l p i c i n t e r a c t i o n s w i t h the packing. These r e s u l t s c o n f i r m that the e x i s t e n c e of micropores must be r e s p o n s i b l e f o r the d i f f e r e n c e i n e l u t i o n volume between glucose and D 0. The e f f e c t of temperature on column e f f i c i e n c y i s a l s o shown i n F i g u r e 2. As expected, the number of t h e o r e t i c a l p l a t e s generated by D 0 was s i g n i f i c a n t l y g r e a t e r than f o r glucose because of i t s higher d i f f u s i o n c o e f f i c i e n t . The temperature dependency of glucose appears to be s i g n i f i c a n t l y g r e a t e r than f o r D 0. For example, a column temperature change from 29 t o 70*C, r e s u l t s i n a 50% i n c r e a s e i n e f f i c i e n c y f o r glucose as compared to only 10% f o r D 0. Since the r e l a t i o n s h i p between temperature and d i f f u s i o n c o e f f i c i e n t i s l i n e a r as p r e d i c t e d by the WiIke-Chang equation, one would expect a much higher p l a t e count f o r D 0. A p o s s i b l e e x p l a n a t i o n f o r these r e l a t i v e l y low values f o r D 0 c o u l d be d i s r u p t i o n of the packed column bed at e l e v a t e d temperatures which would a f f e c t the narrower D 0 peak more than the glucose peak. 2

2


2

2

2

2

2

2

Conclusions From these s t u d i e s w i t h SynChropak SEC packings and c o n t r o l l e d p o r o s i t y g l a s s , i t i s concluded that the s i l i c a packing c o n t a i n s a p o p u l a t i o n of micropores which are d i f f e r e n t i a l l y a c c e s s i b l e t o low molecular weight probes of t o t a l permeation volume. I t i s not known, however, i f the m i c r o p o r o s i t y i n the 100 and 300Â SynChropak SEC packings i s the r e s u l t of the r a t h e r wide pores i z e d i s t r i b u t i o n and whether a l l s i l i c a s c o n t a i n micropores. The e x i s t e n c e of micropores i n a SEC packing and the f r a c t i o n a t i o n of low molecular weight probes presents a dilemma as to what should be used as V i n c a l c u l a t i n g K of high molecular weight s p e c i e s . I t i s recommended t h a t the c o r r e s ponding monomer (except i n the case of p r o t e i n s ) be used when c o n s t r u c t i n g a c a l i b r a t i o n curve f o r a given polymer. For example, i n the case of c e l l u l o s i c s , glucose would be the low molecular weight c a l i b r a n t of c h o i c e . D 0 i s best used t o determine column e f f i c i e n c y because of i t s s e n s i t i v i t y toward chromatographic peak broadening and extracolumn e f f e c t s ( 2 3 ) . However D 0 may s t i l l be used to estimate V? i n some cases. In view of Freeman*s s t u d i e s on the use of normal alkanes and p o l y s t y r e n e s to probe the macroporosity of porous m a t e r i a l s ( 2 4 ) , the r e s u l t s presented here would suggest t h a t low molecu l a r weight species ranging from twenty (deuterium oxide) t o s e v e r a l thousand d a l t o n s may be used to d e f i n e m i c r o p o r o s i t y of a SEC support. The ease w i t h which t h i s i s achieved may a l l o w r o u t i n e examination of m i c r o p o r o s i t y i n new support m a t e r i a l s and a more exact d e f i n i t i o n of t o t a l permeation volume i n SEC. T

D

2

2


BARTH A N D REGNIER

Deuterium Oxide for Aqueous SEC

217

3.0 r

D 0 2

»GLUCOSE 2.0 -


1.0 -

0.2

0.4

0.6

0.8

1.0

1.2

1.4

FLOW R A T E ,

1.6

1.8

2.0

ml/min

Figure 1. Influence o f flow r a t e on e l u t i o n volume o f D 0 and glucose. The column was a SynChropak 100* column. See Table I I f o r c o n d i t i o n s . 2

11,000

9,000 CO LU

* GLUCOSE

t—

< _J

^

7,000

£

5,000

ο LU DC

I— 3,000

1,000

10

20

30

40

50

60

70

COLUMN TEMPERATURE, °C

Figure 2. Influence o f temperature on column e f f i c i e n c y using a SynChropak 300* column. See Table V I I f o r conditions.



218

Acknowledgments The h e l p f u l d i s c u s s i o n s w i t h Walter J . Freeman and the e x c e l l e n t t e c h n i c a l a s s i s t a n c e o f David A l l e n Smith are appreciated. We a l s o thank James F. Carre f o r p r o v i d i n g and i n t e r p r e t i n g the porosimetry and BET data.


Literature Cited 1. Bio-Rad Laboratories "A Laboratory Manual on Gel Chromatography"; Richmond, CA, 1971. 2. Karch, K.; Sebestion, I.; Halasz, I.; Engelhardt, H. J. Chromatogr. 1976, 122, 171. 3. Rochas, C.; Domard, A.; Rinaudo, M. Eur. Polym. J. 1980, 16, 135. 4. Marsden, N.V.B. Ann. Ν. Y. Acad. Sci. 1965, 125. 428. 5. Yoza, N.; Ohashi, S. J. Chromatogr. 1969, 41, 429. 6. Ohashi, S.; Yoza, N. J. Chromatogr. 1966, 24, 300. 7. Obrink, B.; Laurent, T.C.; Rigler, R. J. Chromatogr. 1967. 31, 48. 8. Marsden, N.V.B. J. Chromatogr. 1971, 58, 304. 9. Barth, H.G.; Regnier, F.Ε. J. Chromatogr. 1980, 192, 275. 10. Barth, H.G. J. Liq. Chromatogr. 1980, 3, 1481. 11. Barth, H.G.; Smith, D.A. J. Chromatogr. 1981, 206. 410. 12. Neidhart, B.; Kringe, K.P.; Brockmann, W. J. Liq. Chromatogr. 1981, 4, 1875. 13. Grushka, E.; Colin, H.; Guiochon, G. J. Liq. Chromatogr. 1982, 5, 1391. 14. Neidhart, B.; Kringe, K.P.; Brockmann, W. J. Liq. Chromatogr. 1982, 5, 1395. 15. McCormick, R.M.; Karger, B.L. Anal. Chem. 1980, 52, 2249. 16. Berendsen, G.E.; Schoenmakers, P.J.; Galen L.D.; Vigh, G.; Puchory, Z.V.; Inczecly, J. J. Liq. Chromatogr. 1980, 3, 1669. 17. Slaats, E.H.; Markovski, W.; Fekete, J.; Poppe, H. J. Chromatogr. 1981, 207, 299. 18. Kristulovic, A.M.; Colin, H.; Guichon, G. Anal. Chem. 1982, 54. 2438. 19. Billet, H.A.H.; van Dalen, J.P.J.; Schoenmakers, Ρ.J.; Galan, L.D. Anal. Chem. 1983, 55, 847. 20. Snyder, L.R.; Kirkland, J.J. "Introduction to Modern Liquid Chromatography"; J. Wiley and Sons: New York, 1979; p. 207. 21. Lippens, B.C.; Linsen, B.G.; de Boer, J.H. J. Catalysts 1964, 3, 32. 22. Unger, K.K. "Porous Silica"; Elsevier Scientific Publishing Co.: Amsterdam, 1979. 23. Barth, H.G., results to be published. 24. Freeman, D.H.; Poinescu, I.C. Anal. Chem. 1977, 49, 1183. RECEIVED

December 20, 1983


Size Exclusion Chromatography - American Chemical Society

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