Survey of Chromatographic and Electromigration Separations

F. Svec, T.B. Tennikova and Z. Deyl (Editors) Monolithic Materials

Journal of Chromatography Library, Vol. 67 9 2003 Elsevier Science B.V. All rights reserved.

Chapter 24

Survey of Chromatographic and Electromigration Separations Ivan MIKSIK and Zden6k DEYL

Institute of Physiology, Academy of Sciences of the Czech Republic, Videnskb 1083, 142 20 Prague 4, Czech Republic

CONTENTS

24.1 24.2 24.3 24.4 24.5 24.6 24.7 24.8 24.9 24.10 24.11 24.12

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hy drocarbons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alcohols, phenols, oxo compounds, carbohydrates and organic a c i d s . . . Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amino acids, peptides and proteins . . . . . . . . . . . . . . . . . . . . Nucleic acids and their constituents . . . . . . . . . . . . . . . . . . . . Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluorescent dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

623 624 652 653 653 653 654 655 655 656 656 656

24.1 I N T R O D U C T I O N

As indicated in a number of places throughout this book, monolithic sorbents in their wide diversity have brought new dimensions to both chromatographic and (perhaps even more) to electromigration separations. While electromigration separations are used preferably on the analytical scale, various chromatographic operation modes (typically membrane separations) can be exploited also for preparative purposes, in particular if bioaffinity separation mechanisms are used. As with any developing

624

Chapter 24

technique the first applications are always directed towards complex artificial test samples, while the practical aspects and applications to naturally occurring mixtures will surely come at a later stage. Nevertheless, the separations of test mixtures show clearly the potential of this methodology and pave the way for its practical use in the future. As even detailed information about working procedures is unlikely to save the reader having to consult the original papers, we have decided to present an overview in a (rather extensive) tabular form (Table 24.1), and then to give some general summarizing comments. 24.2 HYDROCARBONS

Hydrocarbons, in particular the aromatic ones, are frequently used as test mixtures for monolithic packings: naphthalene, chlorobenzene and a number of polycyclic hydrocarbons were successfully separated by CEC on reversed-phases monolithic sorbents (C8, C18) [3-5], frequently sol-gel bonded, using acetonitrile and different proportions of aqueous phase (typically Tris or MOPS buffers, the proportion of which in the eluent usually does not exceed 10 %) as the mobile phase. For purely chromatographic separations (without the involvement of the electrodriven flow) C3 or C 18 monoliths with acetontrile-Tris buffer, acetonitrile-water or methanol-water mobile phases were used for the separation of alkylbenzenes (an extensive study on this subject can be found in ref. [6]). Tetraethoxysilane-n-octyltriethoxysilane hybrid gels also appear applicable for CEC separations of this category of compounds [10]. Further, aromatic hydrocarbons like toluene and its homologues can also be separated on methacrylate-based monoliths, typically methyl methacrylate or butyl methacrylate-ethylene dimethacrylate copolymers [ 16,17,20,21 ]. These separations are generally electrokinetic in rather long capillaries (up to 1 m), and exploited phosphate- or borate buffers of pH 7.0-9.2 containing specific proportions of acetonitrile as background electrolyte. In order to ensure adequate electroosmotic flow, 2-acrylamido-2methyl-1-propanesulfonic acid was incorporated in the monolithic packing. Reversed-phase-based separations can be effected not only with hydrophobized silica based monoliths but also with 2-hydroxyethyl methacrylate-piperazine diacrylamide copolymers possessing C 18 ligands, as reported in ref. [8]. Styrene-divinylbenzene copolymers are also applicable for HPLC and CEC separations with acetonitrile-water mobile phases [2,15].

Applications I c~

0.0 o

o 0

o

I

.,r,

.o

c~

o:8

c.)

o

Old)

~ .,.., o

r~

t~

.,.-~

r

(1.)

o

o

o

t..)

o~ [,c..,)

o~o o ~

~o

~~

~

~.~.~ ~.~..~

9- r

~

'~

~~

~'n

~

~~

~~g,

~

,co ~

~

~

.,., .~

~

~.~

~

c.)

~

,,. 0

_~~

t--

r--

~o~ ~

9 ~ ~o

~~

,

~.~

r

.o

.<~

~

~l

,3=

~o~

625

~3

~3

626

0

"0

r

u~

<

0

o

~ ' ~

~

~

0 0

oo

9. ~ ,

dH

,.~

o

~

-~

.,.-4

"~

~=oxg

oo

oo~ ._~ ~.~

~

= ~ ~ x

x x ~.~

~--~~

oo

~_~

-~

Chapter 24

~o ~

~.

v~.~

< o ~ -~ ~

9

0

~

~ . -

~ 0

~+

o

~+

<~

0

<~

~,..~ o ,-~

~

Applications

oo

f~

o t~

t~

o ,..~ n~

0

o ,..~

0

o ,..~ n~

o t~

o

o ,..~

o.0

o t~

0

o

0

rll

r

oo

.o~

~~~

.,~:.?

,--i

,..,__o

~

~

r

~.-~

0

~,=

~ <

=

~2

~

~ ,..~ ~

_~ ~

o ~

<

~

o~ <

0

~~~~.-

= tq

0

0

<~~

"~,1::I

_~

0

N

References pp. 656-658

-?

::t.

o ,..~

~

o

o

~

-

o.

~

~

627

~o

.~

~o

628

0 o

(",1

0 ' ~o

0

0

.,4

N

~.~ 9

0 0

~

~,-~

.g

~

~)

t~

o~

oo

0

~>

o
r~,o

. ,..~

o~

~

0

0

0

r

O~

.o

~..~

~

,

o

~ ~.o

r~~

~ ~

~o

~~~'~ '~-~

~~o

o

~~o

0

.~.~

~

_

"~o .,4

0

~ o

0

Q,)

~-~

o

~9 ~ ~

Q,)

~

0

:L

"~,.~ ~o . - ~

0

~

0

o

o~ .~

f2~ r~

~ o

t~

0

Chapter 24

~

0

0

m.. o 0.'~

~

~

0

N

0

~

";~

0

~

::t.

.~ tr

Q,)

~

o

0

,-M tr t",l

~ > ~

,-.~

~=~ tr

9

~ ~

"~ ," 0

0

.

x=> ~

~

o

,'~

~c~?

~'~

~n

~

o"<,

0 o

-~o

r

~

-.-,

0

""

0

""

0

o

N

~o

~ ~ ~ , ~-

Applications

o

~

9~

0

o~,

c..)

o~

~

~~,~~

References pp. 656-658

o c'q

~0 o

o

0

o

~

~, 0

0

.~ ~

,.-2,

0,.~ ~ m

0

r.g3

~

~

.,.~

0o

o

00

o

,...,

"~ ~,

m

~

"~ o~

~

~176 ~-.N

ID

o

I~

.~

o

~ c',l

00

629

630

(D

*fi 0

,<

o

C

E

r

C

o

+~

o'~

9

t~

0

azeqeq

,.o

g~

o~

S=Z

o

~,

o

o,,.~

,-~,

<~ zl.

~s (D

o~

az~

~az

o

0

r

,~!-

0

-~o 0

0

Chapter 24

9 ,..~

Applications

t'q t'q

9~

o=~

r

-~-a

;>. ~

m

t",l X

~

o"~

0

~....q r~

,~ r~ .,.=

.~

~,

~ ~ ~..~ ~ .,,~ 0 > r.,r

=

=

0

r.~

t"q i_,.,.i

,-6

x

<=

0

0

exO

0

?-s

0

~o*

References pp. 656-658

~

t"q

0

r,~

~h

r,.)

,.t=

"~

.,..4

os

"~~

t"" ,---~ s

0

0

~'q , . 0

=o

0

z,

t'q

~

~

~

<

=~ . ,...~

o ~ "'~

oo ~

o ~

0

t"'-.

0a

o .~ ~

"-~ 9 9

0

0

=?.,

0

~ o

~

~

9

~

~

~=

.~ "~

,-,-, _

9

=~

0

o~

~~'~-~ ' ~ ~ ,~~ o~.""~

t"4

I::~ s

-~.~~.= ~ ~ ~ ~.~~ ~,

631

r~

632

n~

.=. 0 0

UJ

C',l

.<

= 0 0

0 0

0

.o

O~

o o

rj

O~

r

0

O0

0

n::l

~

n:~ r

~, ~x>

;;;,,

,-~

,t,~ t'~

0

r

='~

o

0

g~~.o 0

0

.,..~ " ~ o 0

,~

C',l

•

r 0

r

Ob

o.=

,,~

.~ ~oo~

.~ ~,~

i~

o

.~,~,

0

~ 0

o0 0

o~

0

< ~b

0 N

<

~'~

0

0

0

r~ ~ 0

C',l

0

0

n~

O~

~

0

Chapter 24

..Q

0

o0~

0

~o o 4~~

Applications

cq

t'xl

o

cq

t'xl

on

oo t"q

C 0

r~

"=o.~

o

o

re3

~.

tr

0

0

I

,,

n~

0

~ 2~

,,

~

,

~

.-.

"~..

~

,,

~~,

~.~

~

~

o

~~ ~

~

~

o

.~

.

~~o ~~~ ~~ ~.~~

~

C

~ ~ .~

.~

~

~~. ~ ~ . ~ "~ ~ ~ ~, ~

~c,l

~

~.~.~ . . . .

~

O,t~

~_

0

References pp. 6 5 6 - 6 5 8

6 33

~o r~

634

o o

<

o

(.,1

o

r~

o

f2~ r~

o

r

~ ~i~ o,.

"~

0"~

o

o

r~

o~,

~,

q

o

o,..i

6

r..)

A

9 ,..~

~o

("4 m

A

~

o

o

(',4

o

ot--~ o

o

o o . ..,,~

o

o

o~ o

o

0

. ,...~

0

<

r

r~

o

o

o o

6~ rae~

~o <

o

>.

~oeq

r~

0

. ,,.,.~

o

~z

Chapter 24

Applications

=~.B 0

o~

o ""

x

~

r

~ ,

0

~

f~ oo

~"~-.

o

0

~d

.o N

9

9

9

O'l

.~ =

~

0 n~

r

"u oed~

~

656-658

,.~

9-"~ -

< ~

ON,. ~

o ~ ~"

.

o--

"~

0

~

9.u o

o

tth

pp.

z~ References

N

0

.~

r 0"3

~9

.g ol) r

tlj

~.

m

~.~

,.wl

" ,-~

tch t',l t",.

0

011 0 ~

oo

~'~

""

r

0

o

N ?~ ~ Xo 4

o,...i

i

o

r162

o

o ,.,~

r

.,-i

~ 4.,-i

0

. ,..~

0

~1

tg3

I'r

o c',l

" ~ -o

O0

~ .~ es

,-~ 9

~'~

~

o

rn

o,..i 0

,x,

~2 z

~

o

.,-i

~d 0

i

,,,.,i

o,.-i

<'~

9,~,

N

n~ o ,.~

.o

0

i

ba~

zz

o o,1

r

635

C

636

o o

<

o

=x

~.~> ~,,-,

-~ ~ ~

t'-.. 06

~

~

~~ o~x

~_~,

=.~-,=

...~

~

q-~

'

' r-

o

o o

-.. ~

~ ~ 2.

.,.~

o

r ~

,.c:l o

~l " ~

~_

o

.,-,

,-

~

r

~

o

.-=

'...,

t:~

E~

_

Oq

o

r

r

I= o

"''

~

~ :.= ,

.,,_,

.,.~

-'a

rj

I o

C~

9o~ ~

~x 0

,.-1

o

o

0

o

o,.~

~

o

~=

o

~o

r

r

c'q

Chapter 24

f~

o cq

"=~~

x

Applications

o

o

~~

~

- e >o1~ cq

o

<

0

~

o

9 ,'-,

0 .,..~

t~

9~ , . ,

~:~

99,0

obq

oo

r~

~ ~

e~

""

tD

References pp. 656-658

oo

= 0.0

0

oo

~ "-'

"-~

-6d

0

~

I..~

9. ~

~

o~

;>

r--.i

o r,r

x

~

,.~.~

,,..~

~

:=

r,.)

,---,

.,..~

.4=

0

r

o ,.,.4 'x:;I

x

c',l

m

O

. ,...~

2 r./3

6

r

~.)

.4

O

637

638

"a

0 0

C',I

<

~ ,...~

x

o ,..~

i

.~ ~

x

~

~ o

~ .~

~9

~,

i

,

~D

0

X

[.o

o

0

0

-~ o..

~

o

~ ~.~

0

x

0

~

0

t~ i

~o x

~ ~

L~

r

.

~

0

ue

.~

n~ oo o

~

~,0

e~ ~

~

0

G

o

0

~2-

"~

~

~

~

Chapter 24

~ '~

, ~

•

t'xl

0

nT~

r

<

Applications

<

U2 U~

0

0 ~,..~

~

0

t~

~> ~,..~

r~

0

dr-


t~

0

~

0

~

~~

o~

~

0

m

-~

6.~

~.~

o

o G

0

r~

0

~ f~

oo

O,.Q

.~

0

<

.= ~ "'~

r.r

~ "'~

z.~

0

_~

o

~.~

r~

H ~ ,..4

07J ~"~

r/)

ol) r o,.Q

639

640

O

,..1

<

~

9 "~

~

• E

~,,,~ r~

m

O ~

~

.~2 ~

O ~

o,..i

~.<;>

< ~.~

r...)~

M

~ ~ :< ~ ~ "~ ~ ~

O

o ,,.~ O f2~ O

~o ~ ~ ' ~

,,=

rl=

0

~g

0

~

~

~

tr

O

~ ,~

~,..~

x

o,...i

o ,,,~

~D

I O

9

o, ~

!

zz

...2

Chapter 24

i ~

r

o

Applications

o

o~

-9

X

~o6Z

o

C',l

o

~ r

0

-~=

0

.-

~

~

.

~.~ -,~ ~

~

<

O

,.Q

rll

0

n~

Z

9

c~

.,--4

X tr

n~ 0 O

0

.,..4

0 0 01~ Ol)

o ,-~ ,sn

x~

n~

o

~: ~.~>

" ~ -~.~

~~-~~~~ ~ . . ~ ~o ~ ~ @,~

~.~ ,~.~

~

~~

~'~

I 0

9

References pp. 656-658

D~

641

642

0 o

0

Z r~ 0 o,-i

0

0

o

0 ~,~

n~ n~

0

o

0

n~

0

o

"~ ~.~0

o

.,.~

~

,

~

.~ ~ ~,

~

o'~ X 0 0

~o

~i o

.,.~

i o

~

?

~o~ .d

txl r X 0

IDD

~ ~,

?-~

i

~ i "~ ~

9 ,.~

'~- ~

o~-~ ,.. ~0

o,,.i

-

~

0

. ,..~

r 0 . X 0

0

,.~

-

~

?

.~

m

Chapter 24

oo

~~

~.. ~ o

r . ~ ' , . ~~

09

Applications

~0 t~

0

-o .,.~.

c~ X 0 0

~

0'-

~.~ ~

~~"~!" ~ t/h ~C',l

!

X

~~ |

eo

r~

< Z References pp. 656-658

t~ t~ 9,

0/}

,.o

0

n~

t~ t"q

r

~ .=

o

"~

L) = o

~

r

t~

< Z

o

oo

~

0

o

~

X

~

o"

""

0

~,~~~

o

~

~

~"

9,.,.,i

o

9 .-,

~ o~

643

644

o o

r~

o

r..)

o

o o

o

o

~d

o ,,.~

r~

o

n~

o

r..)

tr

,d o ~..~

Cxl 0 X 0

Tm

~.~

r~

d~

n~

n~

oF

:~ .. ~ . ~

o#

0

~

o

.,.:,

0 X 0 tt~

~

0

,.C~

"~e~ ''~

o o

ez ez

i ~

C h a p t e r 24

Applications

~ ~-'~

9

-

~~~~

~o".= tr~ t"q

.

~ ..= .~ ~ ,::; ~ ~ ~ ~

0 r~ t"q

f~

References pp. 656-658

.=i o,..~

x t~

~ o-~

~

~ ~.

~ ~ ~ ~

~

<

C~

<

F~

0

0 ~ 9

c~

0

0 o 0 0

o ~ ~ ~

~ ~ ~ ~

< Z

r~

E

. ,..~

0

o

645

646

. ,.~ 0 0

C',l

U~

<

0

n~

0 0 r

0

.o

0

0

k~

C',l

o

_=

-.~

r

o

N N cl, r

n~ o,-~

0

0

0

9

".~

d

.

"~

o

~0

.o

X

o,..~ .,..~

"*

,~" ~q

~~

O

o~

d~

0

,fi

0 90,..~ CD

~

~ ~

.=~

Chapter 24

~ "~o

.o

o

Applications

~9

0 t',l 0

o0~

0

o

~

-~

~

ora~ (",1

fi~

~ ~4 i4

0

o

N

~D 0 0

< References pp. 656-658

~

X

0

;>

0

. ,..~

~x

o

v'3 t~

0

0

~

~

,.-2

0 0 ~,1 , . 0

0 0

~

.~

0

=

~

V'3

o ~ 0 0,.~

< ~

~2 0 f2~

f2~

0

0

o t~

~

o

~'~

eq

0

q..~ 0

0

~

0 ~D

0 0

O

~

i i

c5 & "~

0

oe.:.

~o

~D

~eq ~c5

647

.5

c

648

. ,-~

o o

<

9

-~

"

~

o o ~

0

o o

o

X

o

r~ ~

o

o

~176

tr

o t"q t",l

o

~

Chapter 24

~,D

0 I-4

0

0 0

d

o.

n~

o 0

Ob

Ob

L~ 0 ~k n~

n~ 0 o

~

n~

0 0 cxl

0

0

t-- ...~ ~

r,.)

0,,,~

0

tr

. ~

t--,l

n~ o

o

o

d ~n o

o o o

"~

o o o F~

co n~

~.

o~ ~ o

~ _ ~ -o

i ~

o'1 t ~

tr

~

~ ~

" o

~ ~ ~.~.~ o.o

o o

~

~ ~

.~ . '~ ~-~

~,

o.~

.~-~

Applications

~o

t~3

0

.~

o

,-2

..

N

9

r

~

cq

~

o'U

-~.~

.,..~

.o

~

o

r

.,..~

.2

0

~

.~

.2

0

oX~

9- ~

o"~

z ~~e o

0

o

o

References pp. 656-658

o ,..~

t~

o

0

f2~,X~

~.~

o

, ~

~o

o

0

N

o o r

g

t~

o

9

~4

~

o

o

0

~'=o

an

c',,l

o

~

~6

o

n~

o

. ,-q

o ,..~

~

o

9,..~

o

649

~0

650

0 o

.<

c,l

.'d .,.i

X 0

0 o

,

o~

t'N

~o

ID

.S

.~ -~ .~

0

.,

~'~o r..)

~o ~

0

~

t",l 0

9

-~. ~ ."~

~.~

~-~ o

~

t'-.

=_

11

tt')

0

,~

"~

~x 0

o

0

0

zl. ~zl"

o ~

~

0

f~

~z

o~ cg

9,..~

0

9-~

0

aY

0

Chapter 24

Applications

>

.-c~

~

4

~

C',l

0

0

= 4

~

~>

t~

"~

x

~

0

r

tt~

,-.2,.0

~_~

-~

o

"~

0

""

0

0

0

<

o

o

~'~

~

=o o~~ ~'

~

eq .--, ..o

0

=o

< ~

0

'~"

eq..o

~

r~

,

=o ~

"~ :~.~~ ~.

0 N

.=.

~ ~i 0

0 0 0 Im

~

References pp. 656-658

651

652

Chapter 24

24.3 ALCOHOLS, PHENOLS, OXO COMPOUNDS, CARBOHYDRATES AND ORGANIC ACIDS This section may appear diversified with regard to the chemical nature of the separated analytes. However, the reason is twofold: first, in the respective practical applications these categories of compounds are frequently separated side-by-side and, second, there are so far not many papers describing the use of monolithic packings for the individual chemical entities listed in the heading. The main type of monolithic sorbents used for alcohol separations are sol-gel bonded ODS or ODS/SCX phases [3,11]. These have been used successfully for the separation of benzyl alcohol, dimethyl phthalate, benzophenone and benzaldehyde. Concomitant separation of hydrocarbons such as biphenyl is possible. The separations are generally effected electrokinetically in capillaries of standard length and i.d. at a voltage of about 20 kV. A number of separations using sol-gel bonded phases can be found in refs. [ 11] and [ 14]. The separation of alkyl phenones can be achieved on a number of other monolithic phases, typically using methacrylic acid-methacrylate sorbent containing 10% lauryl methacrylate as EOF-creating component. Such electrokinetic separations run in Trisborate buffer-acetonitrile or even water-acetonitrile mobile phases in standard dimension capillaries, typically 50-100 ~tm i.d., 20-50 cm long. The recommended pH is mostly within the alkaline range (8.2-9.1) [3,11]. However, for acetophenone and valerophenone the acetonitrile-HEPES buffer, pH 6.6 was used. Separation of phthalic acid derivatives has been obtained with monolithic sol-gel polymers of alkoxy silanes, on both the preparative and analytical scale, using 100 x 25 mm columns and methylcyclohexane-ethyl acetate as mobile phases [22]. Separation of phenols in the CEC mode can be achieved with poly(styrene-co-divinylbenzene-co-methacrylic acid) monoliths in acetonitrile-Tris buffer [2]. Reversed-phase chromatography on C 18 rod columns was reported for the separation of chlorophenols. Silica gel rods enable a wide range of applications. They can also be used in HPLC for the separation of carboxylic acids and their esters [7,25]. Alternatively, similar separations can be achieved in the electrokinetic mode using acrylic acid polymers with 10% lauryl methacrylate. Separation of carbohydrates using monoliths is seldom reported. CEC separation of malto-oligosaccharides (Glcl-Glc6) using a macroporous polyacrylamide matrix derivatized with C4 ligand was described [ 19].

Applications

6 53

24.4 STEROIDS

Owing to the nearly general applicability of sol-gel bonded ODS/SCX monolithic packings, it is not surprising that steroids were also separated using this type of stationary phase [3]. Alternatives are macroporous acrylamide polymers [26] and poly(AMPS-co-IPAA) hydrogels [27]. Acetonitrile-water-phosphate (or ammonium phosphate) buffers are used as mobile phases at acid pH. Hydrogels can be operated also at alkaline pH (typically at 8.1). The separated analytes comprise both free and conjugated steroids (selected androstanes) and their dansyl derivatives. 24.5 AMINES

Poly(styrene-co-divinylbenzene-co-methacrylic acid)- and sol-gel bonded ODS

columns have so far been used for the separation of amines such as positional isomers of phenylenediamines [2] and aniline-related compounds [3]. Separations were achieved in the CEC mode at alkaline pH (8.0-8.8) using the acetonitrile-aqueous buffer system [2]. The buffer concentration in the mobile phase can vary. The use of negatively charged polyacrylamide monoliths represents another option [28]. 24.6 AMINO ACIDS, PEPTIDES AND PROTEINS

Reports on the separation of amino acids using monolithic columns concern practically all typical amino acid derivatives. For example, dansylated amino acids were separated on a positively charged polyacrylamide monolith containing 13-cyclodextrin [29]. Phenylthiohydantoin amino acids are best separated on sintered Zorbax ODS monolithic columna. A tetraethyl orthosilicate column with embedded silica particles was used for NBD-F derivatives [31 ]. Buffer-acetonitrile mixtures were used as mobile phase. For example, the separation of NBD-F derivatives was achieved in 5 mmol/L phosphate, pH 2.5-acetonitrile (30:70). This approach also offers the possibility of separating amino acid enantiomers. Separation of amino acid enantiomers can be effected even without derivatization, using a methacrylamide-piperazine diacrylamide copolymer containing vinylsulfonic acid, that drives the electroosmotic flow, and N-(2-hydroxy-3-allyloxypropyl)-L-4-hydroxyproline as chiral selector in the ligand exchange CEC mode. A 50 mmol/L dihydrogen phosphate buffer containing Cu(II) at pH 4.6 was recommended as the mobile phase for pressure assisted CEC separation [32]. For separations of DNB derivatives, monolithic polymers prepared from quinidine were reported [33]; 0.4 mol/L acetic acid containing 4 mmol/L triethylamine in acetonitrile and methanol in a ratio of 80:20 served as the mobile

References pp. 656-658

654

Chapter 24

phase. Alternatively, 0.4 mol/L acetic acid and 4 mmol/L ethanolamine in acetonitrile with methanol (80:20) can also be used for enantiomeric separations of DNZ-leucine and DNP-valine. Simple peptides such as gly-tyr, val-tyr, methionine and leucine enkephalins were separated by CEC on a methacrylate ester-based monolithic column [16]. Acetonitrile-buffer at pH 7.0 served as the mobile phase. For peptide mapping and more complex peptide mixtures, C18 COMOSS microfabricated devices were reported [381. C 18 does not represent the only reversed-phase used. Macroporous polyacrylamide monoliths with C 12 ligands or porous polystyrene monoliths with dimethyloctyl functionalities can be used as well. Buffer-acetonitrile mixtures optimized according to the nature of the separated peptides, mainly by varying their pH, are generally used. Monolithic microfluidic chips were reported for the separations of PTH peptides prepared from bovine serum albumin using 1 mmol/L carbonate buffer, pH 8.7, and electrokinetic operation [35]. A different microchip system enables separation of simple underivatized tripeptides containing aromatic residues and leucine- and methionine enkephalins in 5 mmol/L phosphate buffer pH 7.0 and acetonitrile. Detection was achieved either via fluorescence or UV absorbance at 215 nm [ 16]. Polychloromethylstyrene-based monoliths were used for the separation of basic proteins such as cz-chymotrypsinogen, ribonuclease, cytochrome c, and lysozyme [42]. As with the standard capillary electrophoresis of proteins, careful optimization of conditions is necessary for separation in the CEC mode. This regards the choice of the eluent, which almost invariably represents a mixture of acetonitrile and an aqueous buffer. Sometimes gradient elutions were also applied [43]. Admittedly, this represents a considerable complication in standard CEC systems. Step gradients can also be applied. This is particularly of advantage in operations with preparative disks, such as in the isolation of annexins from hepatoma 777 plasma membranes [44]. Affinity HPLC separations with monolithic beds bearing peptides capable of specific adsorption of compounds such as Factor VIII have also been reported [45]. Methacrylate-based DEAE CIM disks and tubes were applied for fast-, and even preparative, separations of proteins, annexins, czl antitrypsin, Factor IX, myoglobin, conalbumin, and soybean trypsin inhibitor [39,41,48,49]. 24.7 NUCLEIC ACIDS AND THEIR CONSTITUENTS

The separation of nucleic acids, their fragments, and constructs, represents a category of separations using monolithic columns that is widely explored today. The applications range from the analysis of oligonucleotides [50,51] up to intact nucleic acid [52]. Disk operations appear attractive for isolating fragments from a complex

Applications

655

mixture as a pre-separation step. The separation mode is purely chromatographic, and employs gradient elution [50,51 ]. Methacrylate copolymers appear suitable for the separation of oligonucleotides and DNA using Tris-borate buffers [51,53,61]. Successful separations can also be obtained with Tris-HC1 buffers. Polymethacrylate 50 x 8 mm monoliths were applied for the separations of oligodeoxyadenylic and oligothymidylic acids in the HPLC mode [53]. In order to achieve good separations of the individual species, gradient elution was applied. The eluent was 20% acetonitrile-80% phosphate buffer (20 mmol/L) superimposed with a gradient of NaC1. Poly(norbornene-co-hexahydrodimethanonaphthalene) monolithic columns were reported also for HPLC separations of oligodeoxynucleotides. In this case, separation is effected using an acetonitrile gradient in triethylammonium acetate buffer, pH 7.0, containing in some cases 4% glycerol [55,56]. PS-DVB columns were successfully used for the separations of poly(adenylic) acids, both phosphorylated and dephosphorylated DNA digests or ribosomal RNA [54,57-59]. UNO Q1 columns appear suitable for HPLC separation of mononucleotides and synthetic oligomers using a gradient of NaC1 in Tris buffer, pH 8.2 or 7.4 [60,63]. Some rather exotic applications can be mentioned at the end of this section. A Sartobind Q-20K-60-12 unit operated with a sodium chloride gradient in 20 mmol/L sodium hydroxide was reported for the preparative separation of antisense nucleotides [64]. For preparative separation of proteins, oligonucleotides, DNA, and DNA-RNA complexes, DEAE membranes operated in a step gradient may represent the method of choice [62]. 24.8 ALKALOIDS

In our literature search we came across only a single paper dealing with capillary electrochromatographic separation of pteropodine, isopteropodine and metraphylline [3]. A sol-gel ODS-particle-loaded CEC column and acetonitrile-water system were used to achieve the separation. 24.9 FLUORESCENT DYES

To our best knowledge there is only a single report regarding this category of compounds. Rhodamine and fluorescein were separated using a microfluidic chip with collocated monolithic structures [35].

References pp. 656-658

Chapter 24

656 24.10 DRUGS

The reports on drug separation with monolithic packings reflect the diversity in the chemical structures of these compounds. The elution conditions in HPLC separations can be isocratic for drug intermediates, or a wide variety of gradients can be used for established drugs and their metabolites [68-71 ]. The chemical nature of the monoliths used for these separations reaches from those including acrylic acid, vinylsulfonic acid, and alkoxysilanes, to norbornene polymers along with commercially available Chromolith Speed Rod columns [5,69,70]. A less typical approach is the normalphase separation of cyclosporin A using heptane-methyl ethyl ketone as eluent [25]. The CEC separation mode is clearly preferred for chiral separations of drugs [24,45,66]. Negatively charged polyacrylamide gels with bonded cyclodextrin also appear very promising. 24.11 CONLUSIONS Obviously, this survey cannot embrace all of the chromatographic separations achieved using monolithic separation media published during the previous years. In fact, this was not even the aim of this Chapter. The extensive Table, which is its major part, is supposed to illustrate the multiplicity of targets, a wide variety of monolithic structures, and a plethora of conditions that have been demonstrated to work in this area. The data shown in the Table may also help the potential user to accelerate the method-development for a specific separation that is facilitated by monolithic columns. 24.12 REFERENCES

7 8 9 10

J.L. Liao, N. Chen, C. Ericson, S. Hjert6n, Anal. Chem. 68 (1996) 3468. B. Xiong, L. Zhong, Y. Zhang, H. Zou, J. Wang, J. High. Resolut. Chromatogr. 23 (2000) 67. Q. Tang, M.L. Lee, J. High Resolut. Chromatogr. 23 (2000) 73. C.K. Ratnayake, C.S. Oh, M.P. Henry, J. High Resolut. Chromatogr. 23 (2000) 81. K. Cabrera, D. Lubda, H.-M. Eggenweiler, H. Minakuchi, K. Nakanishi, J. High Resolut. Chromatogr. 23 (2000) 93. N. Tanaka, H. Nagayama, H. Kobayashi, T. Ikegami, K. Hosoya, N. Ishizuka, H. Minakuchi, K. Nakanishi, K. Cabrera, D. Lubda, J. High Resolut. Chromatogr. 23 (2000) 111. M.R. Schure, R.E. Murphy, W.L. Klotz, W. Law, Anal. Chem. 70 (1998) 4985. C. Ericson, L. Liao, K. Nakazato, S. Hjert6n, J. Chromatogr. A 767 (1997) 33. E.C. Peters, M. Petro, F. Svec, J.M. Fr6chet, Anal. Chem. 70 (1998) 2296. S. Constantin, R. Freitag, J. Chromatogr. A 887 (2000) 253.

Applications 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

6 57

Q. Tang, M.L. Lee, J. Chromatogr. A 887 (2000) 265. C.K. Ratnayake, C.S. Oh, M.P. Henry, J. Chromatogr. A 887 (2000) 277. R. Asiaie, X. Huang, D. Farnan, C. Horv~ith, J. Chromatogr. A 806 (1998) 251. J.D. Hayes, A. Malik, Anal. Chem. 72 (2000) 4090. Q.C. Wang, F. Svec, J.M.J. Fr6chet, J. Chromatogr. A 669 (1994) 230. C. Yu, F. Svec, J.M.J. Fr6chet, Electrophoresis 21 (2000) 120. E.C. Peters, M. Petro, F. Svec, J.M.J. Fr6chet, Anal. Chem. 70 (1998) 2288. C. Fujimoto, J. High Resolut. Chromatogr. 23 (2000) 89. A. Palm, M.V. Novotny, Anal. Chem. 69 (1997) 4499. R. Wu, H. Zou, M. Ye, Z. Lei, J. Ni, Electrophoresis 22 (2001) 544. D. Josic, A. Strancar, Ind. Eng. Chem. Res. 38 (1999) 333. M. Schulte, J. Dingenen, J. Chromatogr. A 923 (2001) 17. A. Maruska, C. Ericson, A. Vegvari, S. Hjert6n, J. Chromatogr. A 837 (1999) 25. T. Koide, K. Ueno, J. Chromatogr. A 893 (2000) 177. M. Schulte, D. Lubda, A. Delp, J. Dingenese, J. High Resolut. Chromatogr. 23 (2000) 100. A.H. Que, A. Palm, A.G. Baker, M.V. Novotny, J. Chromatogr. A 887 (2000) 379. C. Fujimoto, Y. Fujise, E. Matsuzawa, Anal. Chem. 68 (1996) 2753. T. Koide, K. Ueno, J. Chromatogr. A 909 (2000) 305. T. Koide, K. Ueno, J. High Resol. Chromatogr. 23 (2000) 59. E.C. Peters, K. Lewandowski, M. Petro, F. Svec, J.M.J. Fr6chet, Anal. Commun. 35 (1998) 83. M. Kato, M.T. Dulay, B. Bennett, J.-R. Chen, R.N. Zare, Electrophoresis 21 (2000) 3145. M.G. Schmid, N. Grobuschek, C. Tuscher, G. Gtiblitz, A. Vegvari, E. Machtejevas, A. Matuska, S. Hjert6n, Electrophoresis 21 (2000) 3141. M. L~immerhofer, E.C. Peters, C. Yu, F. Svec, M. Fr6chet, W. Lindner, Anal. Chem. 72 (2000) 4614. M. L~immerhofer, F. Svec, M. Fr6chet, W. Lindner, Anal. Chem. 72 (2000) 4623. B.E. Slentz, N.A. Penner, E. Lugowska, F. Regnier, Electrophoresis 22 (2001) 3736. I. Gusev, X. Huang, C. Horv~ith, J. Chromatogr. A 855 (1999) 273. F. Regnier, J. High Resolut. Chromatogr. 23 (2000) 19. B. He, J. Ji, F.E. Regnier, J. Chromatogr. A 853 (1999) 257. A. Strancar, P. Koselj, H. Schwinn, D. Josic, Anal. Chem. 68 (1996) 3483. Q.C. Wang, F. Svec, J.M.J. Fr6chet, Anal. Chem. 65 (1993) 2243. D. Josic, J. Reusch, K. Lester, O. Baum, W. Reutter, J. Chromatogr. A 590 (1992) 59. X. Huang, J. Zhang, C. Horwith, J. Chromatogr. A 858 (1999) 91. C. Ericson, S. Hjert6n, Anal. Chem. 71 (1999) 1621. D. Josic, Y.-P. Lim, A. Strancar, W. Reutter, J. Chromatogr. A 662 (1994) 217. K. Amatschek, R. Necina, R. Hahn, E. Schallaun, H. Schwinn, D. Josic, A. Jungbauer, J. High Resolut. Chromatogr. 23 (2000) 47. D. Josic, H. Schwinn, A. Strancar, A. Podgomik, M. Barut, Y.-P. Lim, M. Vodopivec, J. Chromatogr. A 803 (1998) 61. A. Podgomik, M. Barut, I. Mihelic, A. Strancar, in F. Svec, Z. Deyl, T. Tennikova (Editors), Monoliths, Elsevier, Amsterdam, 2002.

658 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73

Chapter 24 K. Branovic, A. Buchacher, M. Bamt, A. Strancar, D. Josic, Chromatogr. A, 903 (2000) 21. A. Strancar, M. Barut, A. Podgornik, P. Koselj, D. Josic, A. Buchacher, LC-GC Int. 11 (1998) 660. A. Podgomik, M. Barut, J. Jan~ar, A. Strancar, T. Tennikova, Anal. Chem. 71 (1999) 2986. A. Podgornik, M. Barut, J. Jan~ar, A. Strancar, J. Chromatogr. A 848 (1999) 51. N.S. Brahmasandra, V.M. Ugaz, D.T. Burke, C.H. Mastrangelo, M.A. Burns, Electrophoresis 22 (2001) 300. D. S3~kora, F. Svec, J.M.J. Fr6chet, J. Chromatogr. A 852 (1999) 297. H. Oberacher, C.G. Huber, Trends Anal. Chem, in press. B. Mayr, R. Tessadri, E. Post, M.S. Buchmeiser, Anal. Chem. 71 (2001) 4071. S. Lubbad, B. Mayr, C.G. Huber, M.R. Buchmeiser, J. Chromatogr. A 959 (2002) 121. C.G. Huber, A. Premstaler, W. Xiao, H. Oberacher, G.K. Bonn, J. Biochem. Biophys. Meth. 47 (2001) 5. H. Oberacher, W. Parson, R. MiJhlmann, C.G. Huber, Anal. Chem. 73 (2001) 5109. C.G. Huber, H. Oberacher, Mass Spectrom. Rev. (2002) in press. T.L. Tisch, R. Frost, J.L. Liao, W.-K. Lam, A. Remy, E. Scheinpflug, C. Siebert, H. Song, A. Stapleton, J. Chromatogr. A 816 (1998) 3. R. Giovanni, R. Freitag, T.B. Tennikova, Anal. Chem. 70 (1998) 3348. N. van Huynh, J.C. Motte, J.F. Pilette, M. Decleire, C. Colson, Anal. Biochem. 211 (1993) 61. C.H. Lochmtiller, Q. Liu, L. Huang, Y. Li, J. Chromatogr. Sci. 37 (1999) 251. R.R. Desmuth, T.N. Warner, F. Hutchinson, M. Murphy, W.E. Leitch II, P. De Leon, G.S. Srivatsa, D.L. Cole, Y.S. Sanghoi, J. Chromatogr. A 890 (2000) 179. T. Koide, K. Ueno, Anal. Sci. 15 (1999) 791. A. Vegvari, A. F/51desi, C. Hetenyi, O. Kocnegarova, M.G. Schmid, V. Kudirkaite, S. Hjert6n, Electrophoresis 21 (2000) 3116. D. Wistuba, V. Schurig, Electrophoresis 21 (2000) 3152. A.M. Enlund, C. Ericson, S. Hjert6n, D. Westerlund, Electrophoresis 22 (2001) 511. G. Dear, R. Plumb, D. Mallett, Rapid Commun. Mass Spectrom. 15 (2001) 152. R. Plumb, G. Dear, D. Mallett, D. Ayrton, Rapid Commun. Mass Spectrom. 15 (2001) 986. F.M. Sinner, M.R. Buchmeiser, Angew. Chem. Int. Ed. 39 (2000) 1433. L. Kvasni6kov~i, Z. Glatz, H. St6rbov~i, V. Kahle, J. Slanina, P. Musil, J. Chromatogr. A 916 (2001) 271. P. Coufal, M. Cih~ik, J. Such~inkov~, E. Tesa~ov~i, Z. Bos~ikov~i, K. Stulik, J. Chromatogr. A 946 (2002) 99.