A New Family of Organic Polymer-Based High-Efficiency Gel

10 A New Family of Organic Polymer-Based High-Efficiency Gel Permeation Chromatography Columns

Size Exclusion Chromatography Downloaded from pubs.acs.org by YORK UNIV on 12/04/18. For personal use only.

HERMAN

S.

SCHULTZ,

PETER

G.

ALDEN,

a n d JURIS L .

EKMANIS

Waters Associates, M i l f o r d , MA 01757

The ULTRASTYRAGEL family of columns for separation by molecular size was studied using calibration curves and Probe Mixtures. The Probe Mixtures consisted of combinations of small molecules, polystyrene oligomers and high molecular weight polymer standards. Two experimental mixed pore size columns with very broad pore size distribution were also evaluated. Columns (30 cm long) were evaluated for their ability to resolve the Probe Mixtures using various combinations of one to four columns. The Probe Mixtures serve as qualitative but visually very apparent indicators of resolving power. Use of such Probe Mixtures can facilitate understanding of the interaction among amount of pores, distribution of pore sizes, number of plates, and resolving power. This in turn leads to optimum utilization of combinations of the columns. Shorter analysis times can then be attained utilizing sets of one or two columns of the proper pore size. In 1964 Moore and Hendrickson {1,2) introduced the technique o f "Gel Permeation Chromatography*' (GPC) f o r determining molecular weight d i s t r i b u t i o n s o f polymer samples. Moore's work introduced the use o f chromatographic column packings c o n s i s t i n g o f then considered s m a l l porous s p h e r i c a l o r g a n i c polymer p a r t i c l e s ( 3 7 - 7 5 u ) . These p a r t i c l e s were made from h i g h l y c r o s s l i n k e d copolymers o f s t y r e n e s and d i v i n y l benzenes. They became a v a i l a b l e as a f a m i l y o f columns under the name STYRAGEL. Subsequently, much more e f f i c i e n t f a m i l i e s (3,4) o f columns became a v a i l a b l e as p a r t i c l e s i z e s were reduced. The columns are g e n e r a l l y a p p r o p r i a t e f o r r e s o l u t i o n o f oligomers through v e r y h i g h molecular weight polymers t h a t are s o l u b l e i n o r g a n i c solvents. The mechanism o f s e p a r a t i o n was by molecular volume o r

0097-6156/84/0245-O145S07.25/0 © 1984 American Chemical Society

146

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

s i z e . This was equated w i t h molecular weight a f t e r c a l i b r a t i o n w i t h v e r y narrow molecular weight d i s t r i b u t i o n standards. These standards were d e f i n e d by primary molecular weight measurements such as l i g h t s c a t t e r i n g , u l t r a c e n t r i f u g a t i o n , osmometry, e t c . . H i s t o r i c a l l y , and even today, p o l y s t y r e n e standards a r e the most r e a d i l y a v a i l a b l e . They a r e d e f i n e d w i t h l e a s t ambiguity. The s u b j e c t packings e x h i b i t l i t t l e o r no a d s o r p t i o n (non-size) e f f e c t s when used t o r e s o l v e compounds and polymers i n a p p r o p r i a t e mobile phases. I t was noted e a r l y i n the h i s t o r y o f the s u b j e c t t h a t columns o f a p p r o p r i a t e pore s i z e c o u l d even be used t o r e s o l v e mixtures o f s m a l l o r g a n i c molecules (5,j>,2) · Packings based on s i l i c a s f o r s e p a r a t i o n by molecular s i z e are a l s o p r e s e n t l y a v a i l a b l e . However, the molecular s i z e range a v a i l a b l e i s more l i m i t e d and p o s s i b i l i t y o f encountering a d s o r p t i o n e f f e c t s i s more l i k e l y . Such columns must be throughly evaluated f o r each new type o f sample f o r which a s e p a r a t i o n and/or molecular weight d i s t r i b u t i o n i s d e s i r e d . One s u b j e c t o f t h i s p a p e r i s t h e d e s c r i p t i o n and i l l u s t r a t i o n o f t h e chromatographic c h a r a c t e r i s t i c s and c a p a b i l i t i e s o f a new f a m i l y o f s t y r e n e based GPC columns designated by the name ULTRASTYRAGEL (4,8,9,10). The p o s s i b i l i t i e s c r e a t e d by the c o n s i d e r a b l e i n c r e a s e i n e f f i c i e n c y l e a d s t o t h e need f o r r e a s s e s s m e n t o f how t o e v a l u a t e and u t i l i z e columns o f d i f f e r e n t pore s i z e ranges. T h i s i s r e l a t i v e to t h e extended banks o f columns c o n v e n t i o n a l l y used. Much higher speed w i t h g r e a t e r r e s o l u t i o n than h i t h e r t o p o s s i b l e can now be a t t a i n e d i n a g i v e n situation. Alternatively, e x t r a o r d i n a r y r e s o l u t i o n i s p o s s i b l e when time i s n o t an i s s u e and e x t e n d e d banks o f t h e s e columns c a n be u s e d . T h i s new f a m i l y o f columns i s made p o s s i b l e by new suspension p o l y m e r i z a t i o n processes f o r s m a l l p a r t i c l e s (11), coupled w i t h improved i n s i g h t s i n t o t h e r e l a t i o n s h i p s ôF~ p a r t i c l e s i z e d i s t r i b u t i o n s and the a r t o f column packing. C a r e f u l l y c o n s t r u c t e d Probe M i x t u r e s based on s m a l l molecules and p o l y s t y r e n e standards a r e used as standardized reference p o i n t s t o b e t t e r d e f i n e t h e f u n c t i o n a l c a p a b i l i t i e s o f i n d i v i d u a l columns and column combinations. The r e s u l t u s i n g the method o f Probe M i x t u r e s t o evaluate columns i s b e t t e r than can be a t t a i n e d from c a l i b r a t i o n curves alone and i s e s p e c i a l l y useful i n t h i s high resolution c a p a b i l i t y s i t u a t i o n . Experimental In most cases, c a l i b r a t i o n curves were determined a t ambient o r e l e v a t e d temperatures using the Waters 150C High Temperature G e l Permeation Chromatograph which i n c l u d e s a s e n s i t i v e r e f r a c t i v e index d e t e c t o r . Otherwise, a modular system c o n s i s t i n g o f a Waters Model M6000A Solvent D e l i v e r y System, a Waters Model U6K I n j e c t o r and a Waters Model 401 Refractometer, were used a t ambient temperature. The mobile phase a t room temperature was

10.

SCHULTZET AL.

New Organic Polymer-Based

GPC Columns

147

toluene and a t 140°C was 1,2,4-trichlorobenzene. Standard f l o w r a t e was 1 ml/minute. Model 401 Re f r a c tome t e r s e n s i t i v i t y was 4X o r 8X. P o l y s t y r e n e s t a n d a r d s were o b t a i n e d f r o m W a t e r s A s s o c i a t e s , M i l f o r d , MA, and Ibyo Soda Manufacturing Co., Japan. E s p e c i a l l y g r e a t care was taken i n h a n d l i n g standards above one m i l l i o n molecular weight t o minimize t h e p o s s i b i l i t y f o r shear degradation. These standards were used a t 0.02% c o n c e n t r a t i o n , prepared f r e s h d a i l y , and 50-100 u l o f s o l u t i o n was i n j e c t e d per column. P l a t e s were determined using ortho dichlorobenzene and corroborated w i t h d i c y c l o h e x y l p h t h a l a t e , r e s u l t i n g i n s i m i l a r values f o r a l l columns, except w i t h t h e 10 A columns where t h e d i c y c l o h e x y l p h t h a l a t e value was used. These markers were i n j e c t e d as 3-5% (w/v) s o l u t i o n s (10 ul)· Both the tangent and 5 sigma methods were used t o c a l i b r a t e p l a t e s (12) and both methods were used t o judge the q u a l i t y o f a column. S p e c i a l a t t e n t i o n was p a i d t o minimizing band spreading due to instrumentation since t h i s i s e s p e c i a l l y d e l e t e r i o u s t o e f f i c i e n c y when v e r y h i g h p l a t e columns a r e used. A measure o f band spreading was determined by measuring the volume o f t h e band width o f a 10 u l i n j e c t i o n o f 3% o r t h o dichlorobenzene w i t h no column i n the instrument and a minimal volume connector. The l e n g t h o f tubing i n the system was kept as s h o r t as p o s s i b l e and o n l y 0.009" I.D. t u b i n g was used between t h e i n j e c t o r and d e t e c t o r . Samples were i n j e c t e d immediately a f t e r l o a d i n g i n t o the i n j e c t o r t o minimize d i f f u s i o n o f the sample i n the sample loop. The volume o f t h e band w i d t h was c a l c u l a t e d b y t h e equation, System Band Spreading

(ul) = (W^) (F) (1000)/(CS)

where W i s peak width a t 4.4% peak h e i g h t (cm.), F i s f l o w r a t e (mi/minute), CS i s c h a r t speed (cm/minute). T y p i c a l band spreading w i t h i n t h e instrument should be 100 u l o r l e s s . Band spreading s i g n i f i c a n t l y g r e a t e r than 100 u l i n d i c a t e s an instrument problem t h a t must be c o r r e c t e d . F i g u r e 1 d e f i n e s t h e Probe M i x t u r e s based on s m a l l molecules through h i g h molecular weight p o l y s t y r e n e standards and t h e c o n c e n t r a t i o n s and volumes used per column. The ULTRASTYRAGEL f a m i l y a t ambient temperatures can be used w i t h o r g a n i c s o l v e n t s such a s t o l u e n e , t e t r a h y d r o f u r a n , methylene c h l o r i d e , chloroform, e t c . I t has a l s o been used a t e l e v a t e d temperatures w i t h a p p r o p r i a t e s o l v e n t s such a s c h l o r i n a t e d benzenes, c r e s o l s , and dimethyl formamide f o r p o l y o l e f i n s , p o l y e s t e r s and other polymers r e q u i r i n g e l e v a t e d temperatures. A l l f i g u r e s a r e based on ULTRASTYRAGEL columns.

American Chemical Society Library 1155 16th St f i W, Washington» D. C. 20038


MIX 1

Benzene, ortho xylene, 50/50 by volume

MIX 2

0 . 5 % Polystyrene O l i g o m e r Mix " 3 0 0 " — 10 identified c o m p o n e n t s M W 161-1098, peak at 370

MIX 3

2 . 0 % Bezene 0 . 1 6 % Polystyrene O l i g o m e r 0 . 1 0 % Polystyrene O l i g o m e r 0 . 0 3 % 2800 M W Polystyrene 0 . 0 3 % 6200 M W Polystyrene

MIX 4 ( 0 . 0 3 % of each)

MW 2,800 6,200 10,200 16,700 42,800 107,000 186,000 422,000 Injection V o l u m e :

Ratio Successive Components

Mix " 3 0 0 " Mix "1000" Standard Standard

MIX 5 ( 0 . 0 3 % of each)

MW

Ratio Successive Components

422,000 1,260,0005,480,000-

2.99X 4.35X

MIX 6 ( 0 . 0 3 % of each) 1,260,0008,420,000 ·

6.68X

Mix 1: 0.50 μΙ / c o l u m n , neat Mix 2: 30μΙ, 0 . 5 % solution/column All Other Mixtures: 50 μΙ/column

F i g u r e 1. D e f i n i t i o n o f s m a l l molecules and p o l y s t y r e n e probe mixtures 1 t o 6; i n j e c t i o n volumes and c o n c e n t r a t i o n s .

10.

SCHULTZ ET AL.


GPC

Columns

149

R e s u l t s and D i s c u s s i o n D e f i n i t i o n s o f Columns Using C a l i b r a t i o n Curves

ULTRASTYRAGEIgcolumns (Figure 2) are a v a i l a b l e i n s i x pore s i z e s (100A t o 10 A d e s i g n a t i o n ) and are the same w i t h respect t o pore s i z e d i s t r i b u t i o n as l a r g e r p a r t i c l e s i z e STYRAGEL and uSTYRAGEL columns. The t o t a l molecular weight range i s from approximately 50 (small molecules) to over ten m i l l i o n molecular weight based on p o l y s t y r e n e standards. T h i s i s the approximate upper l i m i t f o r v a l i d use o f such standards (13). The second column i n F i g u r e 2 t a b u l a t e s a c o n s e r v a t i v e estimate o f the optimum molecular w i g h t range f o r each column based on i n t e r p r e t a t i o n o f c a l i b r a t i o n curves developed using s m a l l molecules and p o l y s t y r e n e standards. The t h i r d column i n F i g u r e 2 i n d i c a t e s the o f t e n broader u t i l i t y range as shown by the use of standard Probe M i x t u r e s . The f o u r t h column i n F i g u r e 2 l i s t s minimum column e f f i c i e n c i e s i n terms o f p l a t e s . Most columns s i g n i f i c a n t l y exceed these minimum v a l u e s . The l o g molecular weight v s . e l u t i o n volume c a l i b r a t i o n curves o f the s i x i n d i v i d u a l 30 cm long columns i s presented i n Figure 3. The h i g h e s t molecular weight p o l y s t y r e n e standard used was 8 . 4 m i l l i o n . For the 10 A° column, i t i s apparent t h a t t h e e x c l u s i o n l i m i t has n o t y e t been r e a c h e d . Figure 4 i l l u s t r a t e s the same, f o r banks o f t h r e e columns- c o n s i s t i n g o f 10 A°, 10^A° and 10 A°, o r 100A, 500A° and 10 A°. Figure 5 presents c a l i b r a t i o n curves f o r a four column bank c o n s i s t i n g o f 10 A° through 10~A and, f o r comparison, two s e t s o f two columns c o n s i s t i n g of 10 A° p l u s 10 A° and 10 A p l u s 10 A . The middle curve f o r the bank of four columns, p l o t t e d using a h a l f s c a l e f o r comparison purposes, i s e s s e n t i a l l y " l i n e a r " f o r most o f i t s length. T h i s h i s t o r i c a l l y has been considered d e s i r a b l e f o r c a l i b r a t i o n purposes although moderately s l o p i n g curves today can be handled r e a d i l y by the use o f computer based methodology. With the s u b j e c t columns, the augmented r e s o l v i n g power, due t o h i g h p l a t e s , o f a r e l a t i v e l y s m a l l e r amount o f pores i n a g i v e n pore s i z e range becomes u s e f u l f o r . c a l i b r a t i o n rjurposes i n n o n - l i n e a r p o r t i o n s o f curves. The 10 A p l u s 10 A column c o m b i n a t i o n i n F i g u r e 5 i s a good example o f t h i s . It is r e l a t i v e l y d e f i c i e n t i n p o r e amount a t t h e l o w e r m o l e c u l a r weight çnd but has g r e a t e r c a p a b i l i t y than the comparable 10 A p l u s 10 A° combination a t the h i g h molecular weight end t o an undetermined degree beyond the h i g h e s t molecular weight standard. T h i s i s i n d i c a t e d by the use o f Probe M i x t u r e s t o be d i s c u s s e d and confirmed by merqury porosimetry measurements o f pore s i z e . The 10 A° p l u s 10 A combination i n F i g u r e 5 i s c l o s e t o , but not q u i t e , l i n e a r . However, i t has been used t o o b t a i n approximate molecular weight d i s t r i b u t i o n s . b

J

150

SIZE E X C L U S I O N

COLUMN 100 À 500 À 103À 10* À 10* À

CHROMATOGRAPHY

OPTIMUM MW RANGE (Judged from calibration curves)

FUNCTIONAL MW RANGE (Judged from probe mixtures)

MINIMUM COLUMN EFFICIENCIES, N

50-1,500 100 · 10,000 200 - 30,000 5,000 · 600,000 50,000 · 4,000,000 200,000 · * 10,000,000

50-1,500 100-15,000 200 - 40,000 3,000-1,000,000 30,000 - 8,000,000 200,000 · % 10,000,000

10,000 14,000 14,000 14,000 14,000 14,000

1,000 · 4,000,000

500 · 8,000,000

14,000 ppf

t a n

ppf ppf ppf ppf ppf ppf

MIXED P O R E MP-35

("D" Type, s e e text) F i g u r e 2. U l t r a s t y r a g e l GPC column s p e c i f i c a t i o n s based on p o l y s t y r e n e s t a n d a r d s , t o l u e n e as m o b i l e phase ( l m l / m i n ) .

10,000,000

Ί

COLUMNS: SAMPLES:

As Indicated Polystyrene Standards and n-Hydrocarbons

INJECTION VOLUME: FLOW RATE: MOBILE PHASE: DETECTOR: TEMPERATURE:

50 μΙ per column 1 ml/min Toluene RI, 4X Ambient

Elution Volume Figure 3 . curves.

I n d i v i d u a l U l t r a s t y r a g e l column

calibration

δ*

9

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10.

SCHULTZETAL.


GPC

Columns

153

F i g u r e 6 shows c a l i b r a t i o n c u r v e s f o r t h r e e o t h e r two column combinations, each r e p r e s e n t i n g 30,000 t o 40,000 p l a t e s per s e t . Ihe 10 A° p l u s 10 A curve can be i n t e r p r e t e d to show a d e f i c i e n c y i n r e l a t i v e pore p o p u l a t i o n i n the range e q u i v a l e n t to about 50,000 t o 600,000 molecular weight. The other two, p r o p e r l y c a l i b r a t e d , can c o n c e i v a b l y be used f o r d e t e r m i n a t i o n o f molecular weight d i s t r i b u t i o n s . However, u t i l i t y for r e s o l u t i o n o f s p e c i f i c polymodal mixtures i s too d i f f i c u l t t o assess from c a l i b r a t i o n curve alone. How much curvature o f a c a l i b r a t i o n curve t r a n s l a t e s i n t o u t i l i t y o r non-utility? C a l i b r a t i o n curves i n d i c a t i n g pore s i z e p o p u l a t i o n s a l l have the same shape f o r g i v e n column combinations whether the p l a t e count l e v e l i s 5000 p l a t e s o r 20,000 p l a t e s o r 80,000 p l a t e s . C a l i b r a t i o n curves o f two column banks each c o n s i s t i n g o f two experimental mixed pore columns are presented i n F i g u r e 7. Ihe calibration curves were determined at 140 C with t r i c h l o r o b e n z e n e as mobile phase. Each i n d i v i d u a l column i n a set has e x a c t l y the same pore s i z e d i s t r i b u t i o n so t h a t each can be used i n d i v i d u a l l y i f the r e s o l v i n g c a p a b i l i t y i s s u f f i c i e n t for a s p e c i f i c s i t u a t i o n . The c a l i b r a t i o n curves f o r the "NW" type and the "D" type column, banks should be -compared r e s p e c t i v e l y t o the 10 A° p l u s 10 A° and 10 A° p l u s 10 A° banks i n F i g u r e 5. The comparisons i n d i c a t e t h a t i t i s p o s s i b l e to a t t a i n a v e r y wide range of c a p a b i l i t i e s f o r screening and many q u a l i t y c o n t r o l purposes w i t h a s i n g l e 30cm column. The s i n g l e column i s operated a t 1 t o 1.5 ml/minute, and between e x c l u s i o n times o f 4-6 minutes and t o t a l permeation time o f 8-12 minutes. I t should be remembered t h a t a l l events take p l a c e w i t h i n one volume o f pores o f a column o r bank o f columns. The "D" Type (or MP-35) mixed pore column i s l i n e a r f o r a major p o r t i o n o f i t s l e n g t h as shown i n F i g u r e 7. The a v a i l a b i l i t y o f c a l i b r a t i o n c u r v e s f o r i n d i v i d u a l columns and v a r i o u s combination banks o f columns a f f o r d s no more than a g e n e r a l i n s i g h t , based on pore d i s t r i b u t i o n , i n t o the performance o f the v e r y h i g h r e s o l v i n g power columns. The use o f c a r e f u l l y c o n s t r u c t e d standard Probe M i x t u r e s w i l l now be d i s c u s s e d t o evaluate i n more d e t a i l the c a p a b i l i t y o f d i f f e r e n t column combinations. 0

Use of Probe Mixtures t o Define Columns and T h e i r Performance

Probe M i x t u r e s serve as v i s u a l l y v e r y apparent i n d i c a t o r s o f r e s o l v i n g power. The mixtures were c o n s t r u c t e d and standardized to cover the molecular weight range from s m a l l o r g a n i c molecules through h i g h molecular weight polymodal m i x t u r e s . Figure 1 d e f i n e s Probe M i x t u r e s 1 t o 6. The components o f a mix were chosen so t h a t there would not l i k e l y be b a s e l i n e r e s o l u t i o n unless there was a v e r y f a v o r a b l e i n t e r p l a y between a h i g h p l a t e value and the amount of pores i n a g i v e n s i z e range, r e s u l t i n g

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D

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Ο

73

δ π

Q

m m Χ

Ν

S S

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1

&

7

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I

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m Η

Ν

§

χ

η

ρ

156

SIZE EXCLUSION C H R O M A T O G R A P H Y

i n the needed r e s o l v i n g power. In F i g u r e 1, the t a b u l a t i o n s f o r Mixes 4 , 5 and β i n d i c a t e the s m a l l v a l u e s chosen f o r the r a t i o s o f molecular weights o f the adjacent standards. The c r i t i c a l o p e r a t i o n a l assumption t h a t makes i t p o s s i b l e to draw c o n c l u s i o n s i n a g i v e n comparison s i t u a t i o n about the e f f e c t of p l a t e and pore amount i s t h a t a constant volume and a constant absolute amount o f s o l u t e was i n j e c t e d per column t o normalize comparisons. I f pore amount per column i s c o n s t a n t , then i n c r e a s e i n r e s o l u t i o n w i t h s e v e r a l columns o f the same k i n d i n s e r i e s i s due o n l y to the increased amount of p l a t e s . C o n v e r s e l y , i f p l a t e s o f a column bank a r e t h e same then d i f f e r e n c e s i n r e s o l u t i o n are due t o d i f f e r e n c e s i n the amount of pores o f a p p r o p r i a t e s i z e . A l s o , a l l the other a p p r o p r i a t e o p e r a t i n g parameters are constant f o r each comparison. The f o l l o w i n g group o f comparisons w i l l i l l u s t r a t e d i f f e r e n t i s s u e s i n v o l v i n g the i n t e r p l a y o f pores, p l a t e s , and r e s o l v i n g power. The times on the f i g u r e s are maximum v a l u e s f o r t o t a l permeation volumes a t a f l o w r a t e o f 1 ml/miη. Small molecule Mix 1 (MW=78, 106) i s used i n Figure 8 w i t h s e t s o f t h r e e , two, or one 10 A columns. This f i g u r e i l l u s t r a t e s t h a t a l a r g e amount o f p l a t e s makes up f o r i n s u f f i c i e n t pores to a t t a i n r e s o l u t i o n . The r e s o l u t i o n i s f u n c t i o n a l evidence f o r the e x i s t e n c e o f s u f f i c i e n t pores o f appropriate s i z e . The p r e v i o u s g e n e r a t i o n o f lower e f f i c i e n c y (i.e. 5000 p l a t e s per column) 10 A° columns were never considered t o have r e s o l v i n g power i n t h i s molecular weight range. The same probe i s used i n F i g u r e 9. TWo 100A° and two 500A column banks are compared w i t h the 500A° columns having t w i c e as many p l a t e s . The 100A° bank having more pores i n the a p p r o p r i a t e range, r e s u l t e d i n approximately the same degree o f resolution. The same p o i n t s are i l l u s t r a t e d i n Figure 10 using Mix 2, a p o l y s t y r e n e oligomer mix w i t h components i n the 161 t o 1,098 range and h i g h e s t p o p u l a t i o n peak a t 370. The bottom row a c r o s s the figure i s a comparison o f the same amount o f p l a t e s . The 100A s e t g i v e s the best r e s u l t s a t the same p l a t e l e v e l . In the top row where a l l s e t s c o n t a i n t h r e e columns, the 500A bank having twice as many p l a t e s - i s comparable to the 100A bank. I t should be noted t h a t the 10 A° bank s t i l l has u s e f u l n e s s i n t h i s range f o r screening purposes due to h i g h p l a t e s . The remainder of t h i s paper i l l u s t r a t e s t h a t f a m i l i e s o f reference chromatograms can be developed t o determine i f a g i v e n column combination i s the b e s t one t o be used w i t h an unknown polymer o r polymer m i x t u r e , the f a m i l i e s o f chroma tog rams being b a s e d on t h e s t a n d a r d Probe M i x t u r e s , one t o f o u r c o l u m n combinations and d i f f e r e n t p l a t e l e v e l s . Extended banks o f lower e f f i c i e n c y columns would be r e q u i r e d t o a t t a i n the same degree o f r e s o l u t i o n o f even one o f these ULTRASTYRAGEL columns. Figure^11 i n d i c a t e s the performance o f t h r e e and one column banks of 10 A , 10 A° or 10 A columns using polymodal Probe Mix f

10.


SCHIJLTZ E T A L .

Three W Â Columns

Two 1 0 A Columns 3

One 1 0 A Column 3

GPC Columns

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65,000 Plates 36 Minutes



F i g u r e 8. Comparison 3, 2, 1 10 A° column sets i l l u s t r a t i o n l a r g e r amount o f p l a t e s makes up f o r i n s u f f i c i e n t pore amount ; Probe M i x 1, benzene and ortho xylene.

Two 100Â Columns

Two 500À Columns



F i g u r e 9. Comparison 100A° and 5 0 0 A ° two column sets ; p l a t e s versus pore amount; Probe Mix 1.

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4 c o n s i s t i n g o f e i g h t p o l y s t y r e n e standards. The d i a g n o s t i c p a t t e r n s f o r optimum use range o f each column type can be seen as a f u n c t i o n o f -JPgre s i z e and d i s t r i b u t i o n a t comparable p l a t e levels. The 10 h columns r e s o l v e w e l l i n the 2,800-42,000 standard range. The 10 A° columns $re optimum i n 16,700 through 422,000 s t a n d a r d r a n g e . The 10 A° columns f u n c t i o n t o a s l i g h t l y l e s s degree-in the optimum range f o r the 10 A columns but poox^v i n the 10 A° range. However, any a c t i v i t y a t a l l i n the 10 A range would Jse unexpected w i t h p r e v i o u s l y a v a i l a b l e lower p l a t e l e v e l 10 A columns. The optimum combination f o r Mix 4 would c o n s i s t o f 10 A° and 10 A° columns r a t h e r than the standard mixed banks used c o n v e n t i o n a l l y . A s i m i l a r comparison i s shown i n Figure 12 using Mix 5 i n the 422,000 t o 5.48 m i l l i o n range. I t can be i n t e r p r e t e d i n a s i m i l a r manner t o draw c o n c l u s i o n s about e f f e c t i v e n e s s versus pore s i z e and increased r e s o l u t i o n due to increased p l a t e s , e v e r y t h i n g e l s e being e q u a l . Ihe 16 minute magker f o r expected e x c l u s i o n volume o f the t h r e e column 10 A chromatogram i n d i c a t e s c o n s i d e r a b l e amount o f p o r e s i z e volume t h a t i s a v a i l a b | e f o r components g r e a t e r than 5.48 m i l l i o n standard. The 10 A° and 10 A° columns have b e t t e r performance than p r e v i o u s l y expected. Four banks of columns are used i n F i g u r e 13 t o determine the optimum t h r e e column combination f o r Mix 5. Each bank has the same l e v e l o f t o t a l p l a t e s (approximately 50,000). The best r e s u l t i s w i t h the 10 A° bank. P a t t e r n s o f c a p a b i l i t i e s a r e d e v e l o p e d i n F i g u r e s 14 through 18 using s e v e r a l Probe M i x t u r e s f o r two,, column combinations and the f u l l 10 A°, 10 A°, 10 A° and 10 A° bank c a l i b r a t e d i n F i g u r e s 5 and 6. Comparisons r e v e a l the power o f the two column combinations and b e t t e r d e f i n e the p r e l i m i n a r y r a n g e s o f use o b t a i n e d f r o m t h e c a l i b r a t i o n c u r v e s . The c a p a b i l i t i e s o f a number o f two column combinations are i l l u s t r a t e d i n Figure 14 r e l a t i v e t o a four column c o n v e n t i o n a l bank i n the molecular weight range of Probe Mixture 4. For e x a m p l e , t h e 10 A° a l u s 10 A° s e t shows c o n s i d e r a b l e 10 A a c t i v i t y and t h e 10 A° p l u s 10 A° s e t shows l e s s . I t can t h e r e f o r e be concluded t h a t the 10 Ao column i n the f i r s t s e t i s c o n t r i b u t i n g 10 A°activity. In choosing between a 500A° p l u s 10 A s e t and a 10 A° p l u s 10 A° s e t f o r use i n the range o f the Probe M i x t u r e , the second s e t has more a c t i v i t y (and e f f e c t i v e pores) i n the approximately 100,000 t o 190,000 molecular weight range. Therefore, i t would be the column s e t o f c h o i c e . F i g u r e s 15 t h r o u g h 18 s h o u l d be examined as a g r o u p . P a t t e r n s o f performance and comparisons are developed f o r Pgo^e Mixtures 3, 4, 5 and 6 using two column s e t s 10 A p l u s 10 A , 10 A p l u s 10 A , Experimental "D" Type Mixed Pore and Experimental "NW" Type Mixed Pore. They a l l have a c t i v i t y down t o a s u r p r i s i n g l y low molecular weight range. Each f i g u r e shows a comparison using a s i n g l e Probe M i x t u r e . The Probe M i x t u r e s Q

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6

CHROMATOGRAPHY

Experimental " D " ULTRASTVRAGEL™ MP-35 MIXED PORE 32,800 PLATES

A

Experimental "NW" DEVELOPMENT "NW" MIXED PORE 31,400 PLATES

F i g u r e 15. P a t t e r n s o f c a p a b i l i t i e s ; two s e l e c t e d or mixed pore column s e t s ; Probe Mix 3.

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F i g u r e I T . P a t t e r n s o f c a p a b i l i t i e s ; two s e l e c t e d i n d i v i d u a l or mixed pore column s e t s p l u s reference f o u r column s e t ; Probe M i x 5.

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CHROMATOGRAPHY

have o v e r l a p p i n g molecular weight ranges. Tbgether they c o n s t i t u t e a chromatographic f i n g e r p r i n t f o r the performance o f each s e t . O v e r a l l , the Experimental "D" Type and the 10 A p l u s 10 A° s e t s have a broader molecular weight u t i l i t y range than the Experimental "NW" Type and 10 A p l u s 10 A° s e t s . However, the l a t t e r have somewhat more c a p a b i l i t y a t the h i g h e r molecular weight pore s i z e end. The 10 A p l u s 10 A° s e t i s d i f f i c u l t t o d i s t i n g u i s h from the Experimental "D" Type s e t on the b a s i s o f Probe M i x t u r e s a l o n e . A b e n e f i t o f t h e Mixed Pore columns i s t h a t they combine the range o f a c t i v i t y i n t o one 30 centimeter v e r y h i g h p l a t e count column and can be used t o a t t a i n maximum speed i n s i t u a t i o n s where t h e amount o f r e s o l u t i o n i s s u f f i c i e n t . Another b e n e f i t o f the "D" Type column i s t h a t i t i s " l i n e a r " over a major p o r t i o n o f the molecular weight range, simplifying i t s use f o r molecular weight distribution c a l c u l a t i o n purposes. 0

Conclusions I t i s important t o understand the i n t e r p l a y o f pore amount and pore s i z e d i s t r i b u t i o n versus p l a t e s on column r e s o l v i n g power. T h i s i s necessary t o f u l l y u t i l i z e the performance c a p a b i l i t i e s of the new ULTRASTYRAGEL f a m i l y o f columns t o o b t a i n the optimum h i g h r e s o l u t i o n and speed a p p r o p r i a t e f o r a s p e c i f i c use situation. C a l i b r a t i o n curves a r e u s e f u l t o p u t one i n t o t h e r i g h t s e p a r a t i o n range b u t t h e use o f c a r e f u l l y c o n s t r u c t e d standard Probe M i x t u r e s d e f i n e more s p e c i f i c a l l y the performance molecular weight range o f the s u b j e c t columns. Stated i n o t h e r terms, i f one has a l a r g e amount o f p l a t e s , the l e s s pores i n a g i v e n range are r e q u i r e d f o r r e s o l u t i o n , e v e r y t h i n g e l s e being e q u a l . " S u f f i c i e n t pore amount" i s d e f i n e d f u n c t i o n a l l y by t h e a b i l i t y to resolve i n a s p e c i f i c s t i u a t i o n . "Sufficiency" l e v e l o f p o r e amount i s l e s s w i t h i n c r e a s i n g p l a t e s . Very h i g h e f f i c i e n c y columns o f f e r the c a p a b i l i t y t o r e s o l v e many m i x t u r e s of s m a l l o r g a n i c molecules and polymodal polymer products without t h e methods development needed when s e p a r a t i o n s a r e attempted by o t h e r mechanisms.

Literature Cited 1. Moore, J.C., J. Polym. Sci., A2 835 (1964) 2. Moore, J.C., Hendrickson, J.G., J. Polym. Sci., Part C. No. 8, 233 (1965) 3. Limpert, R.J., Cotter, R.L., Dark, W.A., Amer. Lab. 6 #5, 63 (1974) 4. Schultz, H.S., Ekmanis, J.L., Tisdale, V.R., Baptiste, A.J., Crossman, L.W., paper presented at Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, N.J., March 8-13, 1982. Abstract No. 392.

10.

SCHULTZ ET AL.

New Organic Polymer-Based GPC Columns

5. Smith, W.B., Kollmansberger, A., J. Phys. Chem. 69 4157 (1965) 6. Hendrickson, J.G., Moore, J.C., J. Polym. Sci., A4 167 (1966) 7. Gazes, J., Gaskill, D.R., Separation Sci., 2 421 (1967) 8. Schultz, H.S., Alden, P.G., Ekmanis, J.L., paper presented at Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, N.J.,March8-13, 1982. Abstract No. 393. 9. Schultz, H.S., Alden, P.G., paper presented at Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, N.J., March 7-12, 1983. Abstract No. 955. 10. Schultz, H.S., Alden, P.G., Ekmanis, J.L., paper presented at 185th ACS National Meeting, Seattle, WA, March 20-25, 1983. Abstract No.ORPL200;Organic Coatings and Applied Science Proceedings, 48 945 (1983) 11. Schultz, H.S., unpublished work. 12. Yau, W.W., Kirkland, J.J., Bly, D.D., "Modern Size Exclusion Chromatography" Wiley-Interscience, New York, 1979. 13. Slagowski, E.L., Fetters, L.J., McIntrye, D.,Macromolecules 7 394 (1974) RECEIVED

October 13, 1983

A New Family of Organic Polymer-Based High-Efficiency Gel

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