Ordered Media in Chemical Separations - ACS Publications

Chapter 15

High-Performance Liquid Chromatography Using a β-Cyclodextrin-Bonded Silica Column: Effect of Temperature on Retention Haleem J. Issaq, Maureen L. Glennon, Donna E. Weiss, and Stephen D. Fox

Downloaded by FUDAN UNIV on December 16, 2016 | http://pubs.acs.org Publication Date: June 30, 1987 | doi: 10.1021/bk-1987-0342.ch015

Program Resources, Frederick Cancer Research Facility, National Cancer Institute, P.O. Box B, Frederick, MD 21701

The effect of temperature, and of temperature and pH on the retention of a selected group of compounds using a beta-cyclodextrin column was studied. The results indicated that a plot of lnk' vs. 1/T gave linear r e l a t i o n s h i p s f o r anthraquinone, methyl anthraquinone, ethyl anthraquinone, naphthalene and biphenyl using a mobile phase of methanol/water. However, a non linear relationship was observed for a selected group of dipeptides employing a mobile phase of methanol/ammonium acetate at the following pH's: 4, 5.5 and 7. The retention times decreased with an increase in the temperature of the column except that for certain dipeptides the retention times increased. The s e p a r a t i o n f a c t o r (a) values decreased by approximately 10% with increase in column temperature from 25°C to 77°C.

The u s e o f h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y (HPLC) f o r t h e s e p a r a t i o n o f v a r i o u s g r o u p s o f compounds, u s i n g a β-cyclodextrin bonded s i l i c a c o l u m n i s w e l l e s t a b l i s h e d ( 1 - 8 ) . The e f f e c t o f t h e v o l u m e o f o r g a n i c m o d i f i e r i n t h e e l u e n t was s t u d i e d ( 9 ) , n o q u a n t i t a t i v e d a t a has been p u b l i s h e d on the e f f e c t o f temperature on r e t e n t i o n u s i n g a b e t a - c y c l o d e x t r i n bonded s i l i c a . This study d e a l s w i t h t h e e f f e c t o f t e m p e r a t u r e on t h e c a p a c i t y f a c t o r o f ( a ) naphthalene and b i p h e n y l , (b) a n t h r a q u i n o n e , m e t h y l - and e t h y l a n t h r a q u i n o n e , and ( c ) p - n i t r o a n i 1 i n e . Also, theeffect of temperature and pH on t h e r e t e n t i o n a n d r e s o l u t i o n o f a s e l e c t e d group o f d i p e p t i d e s was i n v e s t i g a t e d . EXPERIMENTAL The HPLC s y s t e m c o n s i s t e d o f a H e w l e t t Packard (Avondale, PA, USA) HP1090 L i q u i d Chromatograph e q u i p p e d w i t h a h e a t e r , a p h o t o d i o d e This chapter not subject to U.S. copyright Published 1987 American Chemical Society

Hinze and Armstrong; Ordered Media in Chemical Separations ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Effect of Temperature on Retention in HPLC


a r r a y d e t e c t o r , an HP 85B s y s t e m c o n t r o l l e r , and an HP 3392A integrator. The m o b i l e p h a s e s o l v e n t s w e r e f i l t e r e d u s i n g a M i l l i p o r e f i l t e r h o l d e r ( M i l f o r d , MA, USA) w i t h M i l l i p o r e f i l t e r s h a v i n g a pore s i z e o f 0.5 urn o r g a n i c and 0.45 urn a q u e o u s . The pH meter used t o a d j u s t the pH o f the m o b i l e phases was a F i s h e r Accumet M o d e l 320 ( F a i r Lawn, ΝJ, USA). The s a m p l e s were d i s s o l v e d i n methanol o r 0.1 HC1 ( d i p e p t i d e s ) . S e p a r a t i o n s were c a r r r i e d out on a 250 mm χ 4.6 mm C y c l o b o n d I c o l u m n f r o m A d v a n c e d S e p a r a t i o n T e c h n o l o g i e s , I n c . ( W h i p p a n y , NJ, USA) which has b e t a - c y c l o d e x t r i n bonded t o 5u s p h e r i c a l s i l i c a p a r t i c l e s . MAIËLïAL S. A n t h r a q u i n o n e , methyl-* and e t h y l a n t h r a q u i n o n e , naphthalene and b i p h e n y l were p u r c h a s e d f r o m A l d r i c h C h e m i c a l Co., Inc. ( M i l w a u k e e , WI, USA). D i p e p t i d e s were o b t a i n e d from Sigma ( S t . L o u i s , M0, USA), Chemical Dynamics Corp. (South P l a i n f i e l d , NJ, USA), and U.S. B i o c h e m i c a l Corp. ( C l e v e l a n d , OH, USA). The f o l l o w i n g were p u r c h a s e d f r o m F i s h e r ( F a i r Lawn, N J , USA): h y d r o c h l o r i c a c i d , ammonium a c e t a t e and g l a c i a l a c e t i c a c i d . The ammonium a c e t a t e b u f f e r used was p r e p a r e d by weighing 0.77 grams o f ammonium a c e t a t e i n a p p r o x i m a t e l y 700 ml o f M i l l i - Q water. The pH was a d j u s t e d t o 4, 5.5 o r 7 u s i n g g l a c i a l a c e t i c a c i d and t h e v/v m e t h a n o l / b u f f e r a d j u s t e d a c c o r d i n g l y t o one l i t e r . A f t e r the v/v m e t h a n o l / b u f f e r was made t o one l i t e r , the m o b i l e phase was t h e n f i l t e r e d and d e g a s s e d b e f o r e use. The methanol, HPLC grade, was purchased from B u r d i c k and J a c k s o n Labs (Muskegon, MI, USA). The b u f f e r s were p r e p a r e d u s i n g water from a M i l l i p o r e ( M i l f o r d , MA, USA) M i l l i - Q water system. RESULTS AND

DISCUSSION 1

A p l o t o f t h e l o g a r i t h m o f t h e c a p a c i t y f a c t o r ( k ) a g a i n s t the i n v e r s e o f t h e a b s o l u t e temperature (T) f o r naphthalene and b i p h e n y l i s g i v e n i n F i g u r e 1 . The temperature range used was 25°C t o 65°C. At h i g h e r temperatures the r e t e n t i o n times o b t a i n e d were c l o s e t o the v o i d volume. F i g u r e 1 shows a l i n e a r r e l a t i o n s h i p between k* and 1/T. The s l o p e o f t h e l i n e g i v e s t h e e n t h a l p y v a l u e ( Δ Η ) f o r n a p h t h a l e n e (2.81 K C a l / m o l e ) and f o r b i p h e n y l (3.59 K C a l / m o l e ) . A s l i g h t d e c r e a s e was o b s e r v e d i n t h e s e p a r a t i o n f a c t o r α ( T a b l e I ) where α i s d e f i n e d as t / t ,„ where t . i s c o r r e c t e d r e t e n t i o n time r 2 r 1 r ( t . - t ) and t , t , amd t are r e t e n t i o n t i m e s o f u n r e t a i n e d Π ro ro r1 r2 peak, naphthalene and b i p h e n y l , r e s p e c t i v e l y . 0

t f

o

1

1

0

Table I

E f f e c t o f Temperature on α v a l u e s o f naphthalene and b i p h e n y l

Temperature °C

α

25 35 45 55 65

2.62 2.51 2.47 2.44 2.44


262

O R D E R E D M E D I A IN C H E M I C A L SEPARATIONS

The c o r r e c t e d r e t e n t i o n t i m e s f o r n a p h t h a l e n e and b i p h e n y l d e c r e a s e d by 83% e a c h , by changing t h e column temperature from 25°C t o 65°C. T h i s i n d i c a t e s t h a t ( a ) t h e i n t e r a c t i o n o f t h e s e t w o p o l y n u c l e a r a r o m a t i c hydrocarbons w i t h t h e b e t a - c y c l o d e x t r i n c a v i t y i s t h e same under s i m i l a r e x p e r i m e n t a l c o n d i t i o n s , i . e . , m o b i l e phase c o m p o s i t i o n and column t e m p e r a t u r e , and (b) t h a t t h e b e s t s e p a r a t i o n i s o b t a i n e d a t t h e l o w e s t temperature. F i g u r e 2 shows a p l o t o f I n k ' v s 1/T f o r a n t h r a q u i n o n e , methyl-and e t h y l a n t h r a q u i n o n e between 25°C and 55°C. At temperatures h i g h e r than 55°C t h e r e t e n t i o n t i m e s were t o o c l o s e t o t h e v o i d v o l u m e t o g i v e any m e a n i n g f u l d a t a . The ΔΗ values obtained f o r anthraquinone ( A ) , m e t h y l - (MA) and e t h y l a n t h r a q u i n o n e ( E A ) , w e r e 2 . 6 9 , 3.18 a n d 3.59 K C a l / m o l e r e s p e c t i v e l y . The s e p a r a t i o n f a c t o r v a l u e s f o r e a c h p a i r a r e g i v e n i n T a b l e I I . A g a i n α v a l u e s showed a s l i g h t d e c r e a s e w i t h temperature v a r i a t i o n s . These r e s u l t s a r e i n a g r e e m e n t w i t h t h o s e o b s e r v e d by C o l i n e t a l . ( 1 0 ) f o r r e v e r s e d p h a s e m a t e r i a l . The l a r g e r t h e temperature the s m a l l e r t h e α v a l u e .


0

Table I I E f f e c t o f temperature on α v a l u e s o f a n t h r a q u i n o n e s .

Temp °C 25 35 45 55

α (MA/A)

α (EA/MA)

1.53 1.48 1.45 1.44

1.51 1.50 1.48 1.47

The e f f e c t o f change i n column temperature on t h e r e t e n t i o n o f a n t h r a q u i n o n e , m e t h y l - and e t h y l a n t h r a q u i n o n e s between 25°C and 55°C was a decrease o f 75% 76% and 77% r e s p e c t i v e l y . F i g u r e 3 shows t h e e f f e c t o f temperature on t h e c a p a c i t y f a c t o r o f p - n i t r o a n i l i n e , from 0°C t o 7 7 ° C A m o b i l e phase c o n s i s t i n g o f 1 0 % methanol/water was employed. The r e t e n t i o n a t 0°C was 2 3 . 6 2 min. w h i l e a t 77°C was 2 . 2 8 , a t e n f o l d d e c r e a s e . This decrease i n r e t e n t i o n may be a t t r i b u t e d t o many f a c t o r s s u c h as i n c r e a s e d s o l u b i l i t y of the p - n i t r o a n i l i n e with increase i n temperature, which r e s u l t s i n l e s s s o l u t e - s t a t i o n a r y phase, and an i n c r e a s e i n s o l u t e m o b i l e phase i n t e r a c t i o n s ; i n c r e a s e i n mass t r a n s f e r , and d e c r e a s e i n the p r e s s u r e . A l s o t h e b i n d i n g c o n s t a n t o f any s o l u t e w i t h c y c l o d e x t r i n goes t o z e r o a t 80°C ( 1 1 ) . t

E F F E C T OF METHANOL I N THE ELUENT ON RETENTION. The e f f e c t o f i n c r e a s i n g t h e volume o f t h e o r g a n i c m o d i f i e r , m e t h a n o l , i n t h e m o b i l e phase on t h e r e t e n t i o n o f methyl a n t h r a q u i n o n e and naphthalene i s g i v e n i n F i g u r e 4. M e t h y l a n t h r a q u i n o n e and n a p t h a l e n e a r e more s o l u b l e i n m e t h a n o l than i n water so i n c r e a s i n g t h e volume o f methanol i n t h e m o b i l e phase s h o u l d r e s u l t i n t h e i n c r e a s e i n t h e s o l u b i l i t y o f b o t h compounds, a n d as a r e s u l t , a d e c r e a s e i n t h e r e t e n t i o n t i m e . A l s o , the presence o f methanol i n t h e m o b i l e p h a s e a f f e c t s r e t e n t i o n when c y c l o d e x t r i n bonded c o l u m n s a r e u s e d . Methanol i s much more t i g h t l y bound i n t h e c y c l o d e x t r i n c a v i t y t h a n


15.

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263



3.00 Γ

2.70 2.80

2.90

3.00

3.10

3.20

3.30

3.40

3.50

1/T χ 10- K" 3

F i g u r e 1.

1

E f f e c t o f temperature on t h e c a p a c i t y f a c t o r o f naphthalene (-•-) and b i p h e n y l (-0-) u s i n g a βc y c l o d e x t r i n column, 4.6 χ 100 mm, and a m o b i l e phase o f 45$ methanol/water a t a f l o w r a t e o f 1 ml/min.

3.00

3.10

3.20

3.30

3.40

3.50

1/T χ ίο- κ3

F i g u r e 2.

1

E f f e c t o f temperature on t h e c a p a c i t y f a c t o r o f a n t h r a q u i n o n e (..Δ..), methyl a n t h r a q u i n o n e (-0-) and e t h y l a n t h r a q u i n o n e (-0-). E x p e r i m e n t a l c o n d i t i o n s are t h e same as i n F i g u r e 1.


264

ORDERED MEDIA IN CHEMICAL SEPARATIONS


£

1.50

.301 ι ι ι ι ι ι ι 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50

ι 1 3.60 3.70

1/T χ 10- K" 3

F i g u r e 3.

Same as i n F i g u r e 1, b u t p - n i t r o a n i l i n e and a m o b i l e phase o f 10% methanol/water.

o.oo

F i g u r e 4.

1

I 35

ι 40

1

1

1

45 % Methanol

50

55

E f f e c t o f volume o f methanol i n t h e e l u e n t on t h e r e t e n t i o n times o f methyl a n t h r a q u i n o n e (-0-) and n a p h t h a l e n e (-0-) u s i n g a β-cyclodextrin column, 4.6 χ 100 mm, and a f l o w r a t e o f 1 ml/min.


15.

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265

w a t e r , b u t l e s s t i g h t l y t h a n most h y d r o p h o b i c s o l u t e s . Since methanol i s p r e s e n t at h i g h c o n c e n t r a t i o n s compared t o t h e s o l u t e s , methanol can d i s p l a c e a s o l u t e from the c y c l o d e x t r i n c a v i t y ( 1 1 ) . EFFECT OF TEMPERATURE AND pH ON RETENTION. I t was o b s e r v e d i n a p r e v i o u s study (12) t h a t an i n c r e a s e i n the t e m p e r a t u r e r e s u l t e d i n a d e c r e a s e of r e t e n t i o n o f a s e l e c t e d group o f d i p e p t i d e s , w h i c h i s an expected behavior. H o w e v e r , when t h e pH o f t h e m o b i l e phase was i n c r e a s e d from 4.0 ( T a b l e I I I ) t o 5.5 ( T a b l e IV) t h e r e t e n t i o n o f some o f the d i p e p t i d e s i n c r e a s e d w i t h an i n c r e a s e i n t e m p e r a t u r e .


T a b l e III

E f f e c t o f column t e m p e r a t u r e on the r e t e n t i o n o f d i p e p t i d e s on a β - c y c l o d e x t r i n column u s i n g a m o b i l e o f 10$ MeOH/0.1 M ammonium a c e t a t e , pH 4

Dipeptide

27°C

Phe-Gly Phe-Ala Phe-Val Phe-Met Phe-Pro Phe-Ile Phe-Leu Phe-Tyr Phe-Phe Phe-Asp Phe-Glu

3.8 3.6 3.9 4.5 7.1 4.7 5.0 6.9 7.3 8.3 7.9

R E T 37°C 3.7 3.5 3.8 4.2 6.1 4.4 4.7 5.7 6.4 7.8 7.5

Ε Ν Τ I ON Τ 47°C 57°C 3.6 3.5 3.7 4.1 5.4

I[MIN.) I Μ Ε 67°C 77°C

3.5 3.4 3.7 3.9 4.5 4.0 4.2 4.5 5.2 7.2 7.0

3.5 3.5 3.7 4.0 4.8 4.1 4.4 4.8 5.5 7.5 7.2

4.3 4.5 5.2 5.9 7.7 7.4

$

phase

change

rll r21

t

3.5 3.4 3.6 3.9 4.2 4.0 4.1 4.3 5.0 7.1 6.9

/t

-14 - 8 -10 -20 -50 -22 -25 -47 -39 -17 -16

F o r e x a m p l e , t h e r e t e n t i o n t i m e s o f P h e - A l a d e c r e a s e d by 8$ by c h a n g i n g the t e m p e r a t u r e o f s e p a r a t i o n f r o m 2 7 ° C t o 7 7 ° C , a t pH 4, but i n c r e a s e d by 12$ at pH 5.5, and d e c r e a s e d by 9$ at pH 7, T a b l e V, u s i n g the same e x p e r i m e n t a l c o n d i t i o n s . The same was t r u e f o r P h e V a l and P h e - I l e . At pH 4.0 the r e t e n t i o n times o f Phe-Asp d e c r e a s e d by 17$, at pH 5.5 i t d e c r e a s e d by 15$ b u t i n c r e a s e d by 6$ a t pH 7. In g e n e r a l , h o w e v e r , r e t e n t i o n times d e c r e a s e d f o r t h e o t h e r d i p e p t i d e s , T a b l e s I I I - V , w i t h an i n c r e a s e i n t e m p e r a t u r e , b u t t h e amount o f d e c r e a s e was v a r i e d f o r d i f f e r e n t d i p e p t i d e s . Those w i t h the h i g h e s t r e t e n t i o n times at a c e r t a i n pH d i d not d e c r e a s e t h e most by c h a n g i n g t h e s e p a r a t i o n t e m p e r a t u r e f r o m 2 7 ° C t o 7 7 ° C . For example, P h e - P r o , has a t of 7.1 min. at pH 4 and 2 7 ° C , but a t ^ o f 4.2

min.

at

77°C,

a

decrease

min at pH 4 and 2 7 ° C , and Also,

a t^

the one w i t h the l o w e s t

o f 50$, w h i l e Phe-Asp of

t^

7.1

at

(Phe-Ala,

77°C,

a

has a t ^ o f

decrease

of

pH 4) d i d not d e c r e a s e

8.3 17$. the

most, except at pH 5.5 ( P h e - G l y ) . T h e r e f o r e , i t c o u l d be c o n c l u d e d t h a t the degree o f d e c r e a s e i n r e t e n t i o n w i t h an i n c r e a s e i n t e m p e r a t u r e , i s not a f u n c t i o n o f t ^ a t 2 7 ° C , b u t o f t h e s t r u c t u r e and p h y s i c a l p r o p e r t i e s o f t h e m o l e c u l e i n q u e s t i o n , and the p r o p e r t i e s o f the m o b i l e phase, aqueous, pH, b u f f e r , i o n i c strength


266



T a b l e IV

E f f e c t o f column temperature on t h e r e t e n t i o n o f d i p e p t i d e s on a β- c y c l o d e x t r i n column u s i n g a m o b i l e phase o f 10$ Me0H/0.1 M ammonium a c e t a t e , pH 5.5

Dipeptide

27°C

R E T E N T I O N T I M E (MIN.) 37°C 47°C 57°C 67°C 77°C


4.2 4.2 4.7 5.4 7.8 6.0 6.3 8.0 9.2 8.3 8.7

4.2 4.2 4.8 5.3 6.9 6.0 6.2 7.8 8.4 7.9 8.3

Table V

4.1 4.2 4.9 5.2 6.1 5.9 6.1 7.2 7.7 7.7 8.1

4.1 4.3 5.2 5.3 5.3 6.1 6.1 6.5 7.4 7.4 7.8

4.1 4.2 5.0 5.2 5.6 6.0 6.1 6.8 7.6 7.5 7.9

$ change t / t 7

7

0 + 12 + 17 - 1 -43 + 2 - 5 -35 -25 -15 -12

4.2 4.5 5.3 5.3 5.0 6.1 6.0 6.3 7.2 7.2 7.8

E f f e c t o f column temperature on t h e r e t e n t i o n o f d i p e p t i d e s on a β-cyclodextrin column u s i n g a m o b i l e phase o f 10$ Me0H/0.1 M ammonium a c e t a t e , pH 7 R Ε Τ Ε Ν Τ I

0

Ν

Τ I M Ε

$ change

(MIN.)

57°C

67°C

77°C

4.5

4.5

4.4

4.2

4.8

4.7

4.5

4.4

5.6

5.5

5.3

5.1

4.9

-13 - 9 -16

5.8

5.6

5.3

5.0

4.8

-30

8.1

7.0

6.3

5.7

5.2

-48

7.1

6.6

6.3

5.9

5.7

4.9 5.4

7.3 10.6

6.7

6.3

5.9

5.7

5.4

-33

8.4

7.3

6.5

6.1

5.5

-55

11.9

10.0

8.9

8.2

7.7

6.9

-48

4.3

4.3 4.6

4.4

4.1

4.3

4.5

•

4.6

4.4

4.7

4.8

-11

Dipeptide

27°C

37°C

47°C


4.6

4.6

4.7

4.8

5.5 6.3

4.5

t

r77

/

t

r27

-30

6

and o r g a n i c m o d i f i e r . I t s h o u l d be emphasized t h a t t h e m o b i l e phase used i n t h i s s t u d y was 5$ methanol i n 95$ 0.005 M ammonium a c e t a t e , pH 4, 5.5 o r 7. H o r v a t h and coworkers ( 1 3 ) i n s t u d y i n g t h e r o l e o f a c i d i c amine p h o s p h a t e b u f f e r s as e l u e n t s o b s e r v e d t h a t " b e s i d e s t h e i r c l a s s i c a l s t a t i c r o l e t o m a i n t a i n t h e pH o f a s o l u t i o n c o n s t a n t , b u f f e r s may p l a y a v a r i e t y o f o t h e r r o l e s a n d a f f e c t s i g n i f i c a n t l y t h e p r o p e r t i e s and e f f i c i e n c y o f a chromatographic system". One o f t h e s e e f f e c t s i s t h e masking o f t h e s i l i n o l g r o u p s a t t h e s t a t i o n a r y p h a s e s u r f a c e by t h e weak amine component o f t h e buffer.


15.

ISSAQ ET AL.

267


TEMPERATURE AS A SEPARATION PARAMETER. W h i l e temperature may be used t o improve the s e p a r a t i o n f a c t o r , a q u i c k g l a n c e a t T a b l e s I I I - V , r e v e a l t h a t i n most c a s e s t h e above s t a t e m e n t i s not t r u e , but i n c e r t a i n cases i t h o l d s . Phe-Phe and Phe-Glu had r e t e n t i o n t i m e s o f 7.3 and 7.9 a t 27°C r e s p e c t i v e l y , w h i l e the r e t e n t i o n times a t 77°C are 5.0 and 6.9 which i s an i n c r e a s e i n t - t , f r o m 0.6 t o 1.9 rà rι min. O t h e r examples can be found i n T a b l e s I I I - V . Such temperature e f f e c t s are not u n u s u a l . C h e m i e l o w i e c and S a w a t z k y ( 1 4 ) u s e d t e m p e r a t u r e as a s e p a r a t i o n parameter f o r a group of p o l y n u c l e a r a r o m a t i c h y d r o c a r b o n s (PAH) on C „ r e v e r s e d phase column u s i n g a 0

Q


l ο

m o b i l e p h a s e o f a c e t o n i t r i l e / w a t e r . They observed t h a t the e l u t i o n o r d e r o f some PAH a r e r e v e r s e d by temperature v a r i a t i o n s , w h i c h a r e "entropy dominated s e p a r a t i o n s " . Snyder (15) i n commenting on the above s t u d y (14) and the e f f e c t o f temperature on r e t e n t i o n c o n c l u d e d t h a t i n a c h r o m a t o g r a p h i c system the dependence o f the c a p a c i t y f a c t o r k on temperature i s determined by the e n t h a l p y (ΔΗ°): d ( l o g k ) / d ( 1 / T ) - ΔΗ°/4.57 (1) 1

f

0

Where Τ i s t h e a b s o l u t e temperature and ΔΗ i s i n c a l o r i e s . A c c o r d i n g t o S n y d e r ( 1 5 ) , " i f the l i n e a r dependence o f ΔΗ on l o g k* i s e x a c t , then s o l u t e r e t e n t i o n o r d e r w i l l be u n c h a n g e d as s e p a r a t i o n temperature i s v a r i e d " . T h i s i s r e f e r r e d t o as " r e g u l a r " t e m p e r a t u r e b e h a v i o r . He c o n c l u d e s t h a t " t e m p e r a t u r e i s n o t an e f f e c t i v e parameter f o r a l t e r i n g α ( t h e s e p a r a t i o n f a c t o r ) v a l u e s and maximizing r e s o l u t i o n i n " r e g u l a r " s y s t e m s . However, f a i l u r e o f r e l a t i o n s h i p s s u c h as e q u a t i o n 1 may be due t o : (1) r e t e n t i o n o f s o l u t e m o l e c u l e s by more than one mechanism or (2) marked d i f f e r e n c e i n t h e m o l e c u l a r s h a p e s o f two s o l u t e s w i t h s i m i l a r r e t e n t i o n i n a p a r t i c u l a r LC system. I t i s worth w h i l e t o e x a m i n e t h e r e s u l t s i n l i g h t o f H o r v a t h s ( 1 3 ) and S n y d e r s ( 1 5 ) o b s e r v a t i o n s , remembering t h a t we a r e d e a l i n g w i t h a b u f f e r e d m o b i l e p h a s e a n d a b e t a c y c l o d e x t r i n bonded s i l i c a column. A p l o t o f Ink* vs 1/T at each pH s h o u l d r e v e a l t h e e f f e c t o f pH and Τ on the p r o p e r t i e s and e f f i c i e n c y o f t h e c h r o m a t o g r a p h i c system. F i g u r e 5 shows a p l o t o f Ink' vs 1/T a t each o f the m o b i l e phase pH's studied. I t i s c l e a r t h a t a l i n e a r r e l a t i o n s h i p e x i s t s a t pH 4 and pH 5.5 w h i c h has s i m i l a r s l o p e s ( Δ Η ) . The l i n e a r r e l a t i o n s h i p i n d i c a t e s t h a t t h e same m e c h a n i s m o f s e p a r a t i o n i s t a k i n g p l a c e throughout the experiment 27°C - 77°C. However, a t pH 7 i t i s c l e a r t h a t t h e r e e x i s t s two d i f f e r e n t mechanisms o f s e p a r a t i o n . The same i s t r u e f o r Phe-Asp, F i g u r e 6. The n o t i c e a b l e d i f f e r e n c e b e t w e e n F i g u r e s 5 and 6 i s t h e e f f e c t o f pH on r e t e n t i o n a t pH 4 and 5.5. P l o t s o f I n k vs 1/T f o r Phe-Gly ( F i g u r e 7) and P h e - I l e ( F i g u r e 8) show t h a t one m e c h a n i s m i s i n v o l v e d a t pH 7 w h i l e two d i f f e r e n t mechanisms a r e i n v o l v e d a t pH 4 and 5.5. For Phe-Tyr, the l i n e a r r e l a t i o n s h i p ( o n e m e c h a n i s m ) i s o b s e r v e d a t pH 5.5 w h i l e two mechanisms o f s e p a r a t i o n a r e each observed at pH 4 and pH 7, F i g u r e 9. D i f f e r e n t r e s u l t s from the above ( F i g u r e s 4-8) were observed f o r p l o t o f I n k v s 1/T f o r P h e - V a l . At pH 5.5 t h e v a l u e o f I n K d e c r e a s e d , F i g u r e 10, w h i l e i n t h e o t h e r c a s e s ( F i g u r e s 4-8) i t i n c r e a s e d . A l s o , note t h a t a l t h o u g h two d i f f e r e n t m e c h a n i s m s a r e 0

0

1

1


1


268

1.80

60' 2.80

1

2.90

L 3.00

1

1

3.10

3.20


1/T χ

F i g u r e 5.

10-

ι 3.30

ι 3.40

1

K"

3

E f f e c t o f t e m p e r a t u r e on t h e c a p a c i t y f a c t o r o f PheG l u u s i n g a 3 - c y c l o d e x t r i n column and a m o b i l e phase o f 5% methanol/0,005 M ammonium a c e t a t e a t a pH o f 4 (-0-), 5.5 (-0-) and 7 (·.Δ..) a t a f l o w r a t e o f 1 ml/min.

601

2.80

Δ 1

1

1

1

2.90

3.00

3.10

3.20

1

3.30

J

3.40

1/T χ ίο- κ3

F i g u r e 6.

1

Same c o n d i t i o n s as i n F i g u r e 5, b u t Phe-Asp.

1.00 r

.301 2.80

1 2.90

1 3.00

1 3.20

1

3.10

1/T χ ΙΟ"

3

F i g u r e 7.

κ-

« 3.30

1 3.40

1

Same c o n d i t i o n s as i n F i g u r e 5, b u t Phe-Gly.


15.

ISSAQ ET A L .


1.50 r

.50' 2.80

1

2.90

1

3.00

ι 3.10

ι 3.20 3

ι 3.30

ι 3.40

1


1/T χ 10- Κ"

F i g u r e 8.

Same c o n d i t i o n s as i n F i g u r e 5, but P h e - I l e .

2.80

2.90

3.00

3.10

3.20

3.30

3.40

1/T χ ΙΟ" Κ" 3

F i g u r e 9.

1

Same c o n d i t i o n s as i n F i g u r e 5, b u t Phe-Tyr.

1.20

r

401

'

2.80

1

1

1

1

1

1

2.90

3.00

3.10

3.20

3.30

3.40

3

1

1/T χ ίο- κ-

F i g u r e 10. Same c o n d i t i o n s as i n F i g u r e 5, b u t P h e - V a l .


269

270


i n v o l v e d a t e a c h , pH 4 and pH 7, t h e s l o p e s a r e d i f f e r e n t and o p p o s i t e o f each o t h e r . The above e x a m p l e s , F i g u r e s 5-10, c l e a r l y i n d i c a t e the r o l e a b u f f e r may p l a y and how i t e f f e c t s the p r o p e r t i e s and e f f i c i e n c y o f t h e c h r o m a t o g r a p h i c s y s t e m . These results, t h e r e f o r e , a g r e e w i t h what was o b s e r v e d e a r l i e r by H o r v a t h et a l . (13) and o t h e r s ( 1 4 , 1 5 ) . A n o t h e r e x p l a n a t i o n f o r t h e non l i n e a r r e l a t i o n s h i p between Ink* vs 1/T t h a t may be p o s t u l a t e d here i s t h a t each i n c l u s i o n complex w i l l d i s s o c i a t e a t a d i s t i n c t t e m p e r a t u r e . I t i s p o s s i b l e t h a t t h e b r e a k s i n the c u r v e s i n d i c a t e t h i s t e m p e r a t u r e (11).


ACKNOWLEDGMENTS The a u t h o r s w o u l d l i k e t o t h a n k Dr. D. A r m s t r o n g ( T e x a s T e c h . U n i v e r s i t y ) f o r h e l p f u l d i s c u s s i o n and c o n s t r u c t i v e comments. By a c c e p t a n c e o f t h i s a r t i c l e , the p u b l i s h e r o r r e c i p i e n t a c k n o w l e d g e s t h e r i g h t o f t h e U.S. Government t o r e t a i n a n o n e x c l u s i v e , r o y a l t y f r e e l i c e n s e and t o any c o p y r i g h t c o v e r i n g the a r t i c l e . This project has b e e n f u n d e d a t l e a s t i n p a r t w i t h F e d e r a l funds from the Department o f H e a l t h and Human S e r v i c e s , under c o n t r a c t number N01C0-23910 w i t h Program R e s o u r c e s , I n c . The c o n t e n t s o f t h i s p u b l i c a t i o n do not n e c e s s a r i l y r e f l e c t the views o f the Department o f H e a l t h a n d Human S e r v i c e s , n o r d o e s m e n t i o n o f t r a d e names, commercial p r o d u c t s , or o r g a n i z a t i o n s imply e n d o r s e m e n t by t h e U.S. Government.

LITERATURE CITED 1. Armstrong, D.W., Alak, Α., DeMond, W., Hinze, W.L. and Riehl, T.E., J. Liquid Chromatogr. 1985, 18, 261-269 and references there in. 2.

Armstrong, D.W. and DeMond, W., J. Chromatogr. Sc. 1984, 22, 411-415.

3. Armstrong, D.W., 57, 481-484.

DeMond, W., and Czech, B.P., Anal. Chem. 1985,

4. Ward, T.J. and Armstrong, D.W., J. Liq. Chromatogr. 1986, 9, 407-423 and references there i n . 5.

Abidi, S.L., J. Chromatogr. 1986, 362, 33-46.

6.

Issaq, H.J., Weiss, D.E., Ridlon, C., Fox, S.D. and Muschik, G.M., J. Liq. Chromatogr. 1986, 9, 1791-1801.

7.

Issaq, H.J., McConnell, J.H., Weiss, D.E., Williams, D.G. and Saavedra, J.E., J. Liq. Chromatogr. 1986, 9, 1783-1790.

8.

Issaq, H.J., J. Liq. Chromatogr. 1986, 9

229-233.


15. ISSAQETAL. Effect of Temperature on Retention in HPLC 271

9.

Hinze, W.L., Riehl, T.E., Armstrong, D.W., Ward, T., Anal. Chem. 1985, 57, 237-242.

DeMond, W., Alak, Α.,

10. Colin, H., Diez-Masa, J.C., Guiochon, G., Czajkowska, T. and Miedziak, I., J. Chromatogr. 1978, 167. 41-65. 11. Armstrong, D.W.,

Private communication.


12. Ridlon, C. and Issaq, H.J., J. Liq. Chromatogr., in press. 13. Melander, W.R., Stoveren, J. and Horvath, Cs., J. Chromatogr. 1979, 185, 111-127. 14. Chemielowiec, J. and Sawatzky, H., J. Chromatogr. Sci. 1979, 17, 245-252. 15. Snyder, L.R., J. Chromatogr. 1979, 179, 167-172. RECEIVED May

11,

1987


Ordered Media in Chemical Separations - ACS Publications

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