Download Thin Layer and Glass Paper Chromatography

149
T H I N I A Y E R A H 0 GLASS P A P l R CHROMATOGRAPHY
Cm 0. W I L L I A M S
Thin-layer
--------.
chromatography i s an a n a l y t i c a l method applying
techniques of paper chromatography t o a t h i n l a y e r of adsorbents normally
used in colwnn-chromatography. -This i s a r e l a t i v e l y new method of adsorption and p a r t i t i o n chrcjmatozraphy on a micro scale. Thin-layer
chromtography i s an excellent supplenent t o t h e previously known methods
of column, paper, ion-exchange and gas l i q u i d chrorcatography.
Historically, the first attempts t o use a t h i n adsorbent l a y e r
f o r chromatographic separations were described by Izmailov and Schraiber
(8) over twenty years ago. These workers separated mixtures of organic
compounds on layers of adsorbent powder, examining the rings produced
under u l t r a v i o l e t l i g h t . After i t s introduction, very l i t t l e work was
done with thin-layer chromatoGraphy u n t i l t h e period of 1949 t o 1951, A t
t h i s time several vorkers (Meinherds and Ea11 (14); Kirchner, e t a l . (9) )
described the use of binding agents t o f i x t h e adsorbent [email protected] t o t h e
glass p l a t e , Actually the method remained i n obscurity u n t i l 1956 when
Stahl described equipment and procedures f o r the preparation of chromatop l a t e s , and demonstrated the potential usefblness of thin-layer chromatography in t h e f r a c t i o n a t i o n of a wide variety of coupounds. Since equipment has become commercia1l.y available, thin-layer chroinatography has
suddenly gained vide recognition. Very f e w laboratories, e6peciaU.y those
engaged i n l i p i d research, a r e without t h i s valuable t o o l .
I- I
Apparatus. The g l a s s p l a t e s a r e usually about 20 x 20 cm, b u t
can be any s i z e depending on t h e dimensions of your applicator and
developing tank. Thickness of the p l a t e i s very important with most types
of applicators except the one based on the design of I h t t e r and Hof‘steSter
(22)
The uniform coating of glass p l a t e s with a thin-layer of adsorbent requires t h e use of a s p e c i a l applicator. A t the present t h e r e
a r e a t l e a s t three types of applicators on the marketa7D’C The equipment
described by S t a h l i s probably the most widely used, but the applicator
described by Xdtter and Hofstetter (22) i s advantageous i n a t l e a s t one
l
t h a t permits the
respect. These l a t t e r workers have designed ~ lapparatus
__
I^
.
-
aCamg, A. G., Eomhurger S t r . 24, Nut’ienz, B. I,.
Switzerland;
,
U.S.
representative : Arthur H. Thomas Company, Philadelphia 5 , Pa.
bC. Desaga h. b. H. Hauptstrasse 60, Heidelberg, Germany; U . S . representat i v e : C. A. BrinlaPann and Co., Inc., 115 CutterWll Road, Great Neck,
Long Island, New York.
CResearch Specialties Co., 200 South Garrard Blvd., Richmond, C a l i f ,
150.
regulation of t h e thickness of the adsorbent (about 250 microns desirable)
regardless of t h e thickness of t h e g l a s s plates. Thus, inexpensive, good
quality window glass may be used instead of p l a t e glass. Other applicators
require the glass p l a t e s t o be of uniform thickness i f the adsorbent layer
i s t o be t h e same on a l l plates. Preparative chromatograms may a l s o be
obtained with t h i s applicator. A more v e r s a t i l e model of S t a h l ' s applicat o r has been produced, which permits one t o obtain uniform t h i n layers of
any desired thickness between 250 U and ?.ma.
This modified applicator is
finding application in preparative work, but it also r e q a r e s t h e g l a s s
p l a t e s t o be of uniform thickness, i f layers from p l a t e t o p l a t e are t o be
uniform
.
In addition t o t h e g l a s s p l a t e s and applicator, a chromatographic
devgloping chamber, spotting template, desiccated storage cabinet and a
100 C drying w e n a r e e s s e n t i a l equipnent.
Adsorbents used a r e about t h e same a s those used i n column
chromatography, except in most cases a binder has been added. Adsorbents
commercially a v a i k b l e contain 5 t o 15$ binder (usually celcinated calcium
s u l f a t e ) , however, adsorbents can be o b t a b e d t h a t contain no binder. Work
in our laboratory indicates t h a t a binder i s necessary i n the adsorbent
only when t h e t u c k e r , preparative type, p l a t e s a r e being used.
I
*
-
P a r t i c l e size and uniformity of the adsorbent powder a r e much
more c r i t i c a l in thin-layer chromatography than in standard column
chromatography. A standardized 200 mesh adsorbent i s preferable, The most
widely used adsorbent is s i l i c a , s i l i c a g e l or s i l i c i c acid. These terms
a r e often used synonymously i n the l i t e r a t u r e . They r e f e r t o powdered
s o l i d s of t h e general formula Si02. X 0 which consists of porous, t h r e e
dimensional siloxane ( t e t r a h e d r a l Si.0. i structures with surface silanol
(Si-0H)group. There i s usually a mono o r multimolecular layer of adsorbed
water on the adsorbent p a r t i c l e s . During t h e a c t i v a t i o n process, which is
merely heating t h e chranatoplates a t about 105°-1200C, t h i s adsorbed "free
water" i s reverslbly removed, S i l i c i c acid p l a t e s should not be exposed t o
temperatures above 170%', because a t t h i s temperature t h e silanol groups
Will begin condensing, l i b e r a t i n g t h e "fixed water", The degree of thermal
degradation of the s i l a n o l groups increases with r i s i n g temperatures until,
a t about llOO°C only SiOz remains. This thermal degradation i s r a t h e r
i r r e v e r s i b l e , because it destroys pore s t r u c t u r e and permanently impairs
adsorbent properties by reducing t h e number of active sites available for
hydrogen bonding (26),
%I
S i l i c i c acid i s most frequently used f o r the separation of n e u t r a l
and acidic lipids. The s i l i c i c acid is s l u r r i e d with d i s t i l l e d water in a
r a t i o of 1:2 W/V and applied t o t h e p l a t e s in a uniform layer. S t a h l (21)
prepared a c i d i c s i l i c i c acid chromatoplates by using aqueous 0.5 N oxalic
acid solution instead of water i n making t h e slurry. This same worker a l s o
prepared alkallne chromatoplates in a similar manner u t i l i z i n g aqueous 0.5
N KoE, Mangold and Kamereck (13) used chromatoplates of s i l i c i c acid
containing about lo$ ammonium s u l f a t e f o r t h e separation of phospholipids
and strongly a c i d i c f a t t y acid derivatives. These layers prepared in basic
OT a c i d i c solutions do not adhere w e l l t o t h e glass plates.
It i s recommended that these p l a t e s be dried a t room temperature f o r several hours
before they a r e placed in t h e drying oven.
151.
Diatomaceous e a r t h or Kieselguhr i s especially suited for p a r t i t i o n separations of substances which a r e strongly hydrophilic or
Amphoteric. After impregnation, i’k can a l s o be used a s an i n e r t c a r r i e r
f o r the separation of hydrophobic substances. Like s i l i c i c acid
diatomaceous e a r t h i s applied t o t h e p l a t e s a s a s l u r r y (1:2 W/Vj made
with d i s t i l l e d water. Weill and Hanbe (24) used t h i s adsorbent f o r the
fractionation of Malto-oligosaccharides.
A l u m i n u m oxide (alumina) i s r a r e l y used for the chromatography
of complex mixtures because it causes hydrolysis of e s t e r linkages and
isomerizations of double bonds. However, alumina i s superior t o any other
adsorbent for t h e chromatographic fractionation of t h e Vitamin A group,
hydrocarbons and various classes of l i p i d s , especially the basic ones.
It i s also often used for t h e chromatography of s t e r o l s (16).
Other adsorbents such as magnesium oxide, magnesium carbonate,
calcium hydroxide, calcium carbonate, dicalcium phosphate, f l o r i s i l
cellulose and many others have been described i n t h e l i t e r a t u r e (l2j
Almost any adsorbent used i n column chromatography may be adapted t o t h i n layer chromatography
.
Reversed-phase p a r t i t i o n TIC has been used i n resolving a l i p i d
c l a s s i n t o i t s individual constitueats. T h i s i s done by impregnating t h i n layers of adsorbent with s i l i c o n e (11)or sane other hydrophobic substance.
Ion exchange TIC has not been used a s extensive a s adsorption
TLC, however, I can see no reason whyr the present ion exchangers could not
Ecteola cellulose bound t o glass p l a t e s by
collodion was used by Randerath (20) t o separate various purine and pyrimidine
nucleotides.
be adapted t o this technique.
Selection of the solvent i s based upon the w e l l known principles
of adsorption chromatography. Polar solvents cause a greater migration than
sa%le-spo”ctraveg
) of a
non-polar solvents or t h e Rf value
Distance solveni; front t r a v e l s
substance increases with increasing p o l a r i t y of the solvent. Frequently, it
i s convenient t o adjust t h e p o l a r i t y of t h e solvent by adding small amounts
of a polar solvent t o a non-polar solvent. Generally this i s the case, b u t
it i s recommended t h a t these solvent mixtures be kept as simple a s possible
f o r b e t t e r reproducability (12)
P
I
-
The solvent required f o r good separation depends on t h e type and
number of functional groups i n t h e compounds being separated. Hydrocarbons
a r e adsorbed very l i g h t l y and require a low p o l a r i t y solvent for t h e i r
separation. Ir“ functional groups a r e introduced i n t o a hydrocarbon, t h e
adsorption a f f i n i t y increases i n t h e following sequence: R
cH?, R-O-alkyl,
R-C = 0, R
NHz, R OH, R COOH. Within each of t h e above c asses,
saturated compounds a r e eluted e a s i e r than unsaturated compounds. Polyunsaturated compounds with i s o l a t e d double bonds a r e more e a s i l y eluted than
those containing a conjugated system of double bonds. Cis-isomers a r e more
e a s i l y eluted than t h e i r t r a n s counterparts.
-
-
-
-
The a c t u a l selection of a solvent system t o nee% a p a r t i c u l a r need
may be accomplished by using a simple spot test and a thorough knowledge of
152
.
the order of solvents. The spot test is carried out by placing a drop of
sample on t h e layer and a f t e r drying, slowly adding solvent fran a micropipette. Tbe movement of t h e sample i s then observed. In the case of an
unknown mixture, the spot test is s t a r t e d by using a solvent of medium
polarity. If t h e sample remeins in the v i c i n i t y o f t h e s t a r t i n g spot, then
a stronger solvent must be used. E migration is t o o f a s t then a weaker
solvent mixture must be employed. By t h i s method one can s e l e c t the proper
solvent mixture with a minimum of extra work.
xisualization and I d E t i f i c a t f a n . Many chromagenic spray reagents,
In
commollly employed i n paper chrwt-phy,
can a l s o be applied t o TIL!.
addition, a number of corrosive sprays such a s concentrated
which cannot be used on paper, can be u t i l i z e d on these
A f t e r spraying with these oxidizing acids, the chromatoplate i s heated and the
coanpounds present can be see0 as dark spots OD a l i g h t background. Spray
reagents such a s 1, 4 dichlorofluorescein or Rhodamine 6 G may be used i n
conjunction with an u l t r a v i o l e t lamp. Fluorescent chemicals and oxidizing
acids may be incorporated i n t h e adsorbent layer f o r visualizing spots, and
this eliminates t h e necessity of spraying.
Autoradiographs a r e readily obtained when chrmatoplates cont a i n i n g radioactive labeled substances a r e exposed t o No-Screen Medical
X-Ray Safety Film.
Documentation of Chrcaaatopams. Many people consider documentat i o n t o be a major disadvantage with this technique. We have not found t h i s
t o be true. We-record chromatograms by t r a c i n g them onto acetate paper or
tracing them d i r e c t l y i n t o a laboratory manual. This gives a permanent
record Of' ell chromatograms.
Other methods of documentation t h a t a r e being used include
photograpby, radiograpby and dipping the chrctlretagrams i n t o a solution of
label glaze. After dipping a chromatogram in l a b e l glaze the adsorbent Layer
i s pulled off the glass p l a t e as a film.
Thin Layer Chranatogr-.
Present methods f o r
of thin-leyer chromatograms leave much t o be desired.
Few of t h e methods a r e exact and others a r e impractical.
Rough estimations a r e obtained by e l u t i n g and weighing. This method
i s p r a c t i c a l only i f individual spots contain from 20 t o 50 mg. of material.
Spectrophotcmetry and c o l o r b e t r y may also be used f o r quantitative estiolstims a f t e r t h e sam@e is eluted from the adsorbent. When u s i n g these
techniques, care must be taken t o assure removal of a l l t h e adsorbed substance.
As t h e p o l a r i t y of a canpound increases elution becmes more d i f f i c u l t .
et
Photodensitmetry a f t e r charring a s applied by P r i v e t t
9. (18)
(19) gives good results in a range 5 t o 35 ug o f material, The spots a r e
measured in a Photovolt Pbotodensltcmeter with a stage attached f o r semiautanatic p l o t t i n g of curves. The quantitative estimation is made by measuring t h e area under t h e curve. Individual standard curves must be prepared
f o r a l l campounds t h a t do not give spots of the same d e n s i t m e t r i c a l l y
determined response (18)
153,
Measuring spot areas may be useful in supplementing t h e photodensitometric method, as it can be applied t o amounts of 30 t o 100 ug with
an accuracy of f 576.
Applications, Although it has been stated t h a t t h e greatest usefulness of TIC i s f o r t h e separation of l i p i d s and e s s e n t i z l o i l s , a b r i e f
review of the l i t e r a t u r e reveals that it has extremely wide scope.
In t h e separation of l i p i d s , T U has been investigated quite
extensively. Good resolutions have been obtained in the separation of
s t e r o i d s (7) (lo), f a t soluble vitamins (Z), phospholipids (23) and crude
preparations of various f a t s , o i l s and waxes. Basic fractionations between
t h e various classes (aldehydes, ketones, acids etc.) of compounds have
been obtained, These classes may be f u r t h e r separated according t o degree
of unsaturation, chain length, and isomeric forms.
Thin-layer chromatography has a l s o been employed i n t h e f r a c tionation of carbohydrates, amino acids, and various proteins. This
technique has been applied t o the stu&j of various sulfur-and nitrogencontaining compounds such a s sulfa drugs (25) and nucleotides (20).
Thin-layer chromatography i s a l s o being employed In the
detection of food additives such a s antioxidants and coloring agents.
Other f i e l d s such a s toxicology and pharmacology a r e using this technique
f o r t h e rapid detection of impurities and poisons.
S m a r y . Thin-layer chrcmatography i s achieved by f i r s t making
a s l u r r y of t h e adsorbent, The slurry i s usually made with two p a r t s of
d i s t u e d water and one p a r t adsorbent, but use could be made of various
other solvents. T h i s slurry i s then applied uniformly t o glass p l a t e s with
t h e a i d of a standard applicator. After the adsorbent has been applied,
t h e p l a t e s a r e allowed t o dry a t room temperature, usually t h i s requires 5
t o 15 minutes. The p l a t e s a r e then placed i n a drying oven (usually 105120°C) f o r activation. Degree of activation depends on the length of time
t h e p l a t e s a r e allowed t o remain i n the oven and t h e teuperature of t h e
oven. The activated p l a t e s a r e now stored i n a desiccated container, u n t i l
they a r e used. If t o o much moisture i s adsorbed, the p l a t e s may be r e a c t i vated. The materials t o be chromatographed a r e applied t o the adsorbent
with a micropipette or a microsyringe. The spotted p l a t e s a r e then placed
i n an equilibrated developing chamber containing t h e solvent. The solvent
ascends in the adsorbent layer and separates t h e compound mixture a s it
moves ,
The advantages of t h i s method a r e quite evident. Development i s
much f a s t e r f o r thin-layer chromatography than f o r column or paper chromatography, The separating time, depending on solvent used and layer thickness, ranges from 20 t o 40 minutes f o r most applications. Only i n r a r e
cases, such a s f o r amino acids, a r e longer developing times required.
Simplicity i s another major advantage of t h i s technique. With a minimum of
orientation good r e s u l t s can be obtained by ine,xperienced personnel,
S e n s i t i v i t y and capacity a r e a l s o very important a t t r i b u t e s of
t h i s technique. A m i n i m u m of 0.5 ug and up t o 500 ug, can be applied t o
one spot,
154
Among the r e l a t i v e l y few disadvantages i s t h e f a c t t h a t Rf values
a r e not reproducible, which suggests the running of standards alongside the
sample for comparison purposes. The quantitative removal and extraction of
individual spots finom the p l a t e may a t times be more tedious than elution
from paper chromatograms,
Glass-Paper Chromatography
T h i s i s a technique t h a t combines paper chromatography and thinlayer chromatography i n t o one technique, It makes use of a glass fiber
f i l t e r paper irnprepated with an i n e r t phase such a s s i l i c i c acid. Development time i s much f a s t e r than paper chramatography and Rp values a r e more
reproduc3bl.e than those of TU.
Sheets of glass f i b e r paper* of a size
--C_hroma_to&rain fieparation.
suitable f o r available equipment i s freed of organic impurities by heating
i n an oven a t 600°C f o r one hour. Dieckert e t a l e (3) accomplished t h i s
by placing the paper i n a borosilicate glass container with a loose f i t t i n g
lid, This container was then placed i n an annealing oven and heated a t
6OO0C f o r one hour.
I -
The impre@ating solution i s a potassium s i l i c a t e solution, which
is prepared by the addition of concentrated KOH t o a slurry of s i l i c i c acid
(6) have successfully used sodium s i l i c a t e for
and water, Muldrey et
the separation of some phosphorus containing l i p i d s , and these workers
omitted the Hcl treatment, The Hcl treatment - t r i l l be explained l a t e r .
After the s i l i c i c acid has dissolved, t h e solution i s f i l t e r e d .L;hrough a
sintered glass filter t o remove insoluble debris, The f i l t e r e d solution i s
then diluted t o about 0.4% (15) potassium s i l i c a t e . This solution has a
If CO i s
useful l i f e of about one week, because it readily absorbs CO
absorbed, it i s held u n t i l the Hcl treatment of the chromato&arn and & t h i s
time i s released a s a g a s . T h i s gas w i l l form undesirable pockets In t h e
chromatogram (5)
e.
The sheets of glass paper free of organic impurities a r e then
dipped i n the 0.49 potassium s i l i c a t e solution and allowed t o dry i n an
oven. Some workers stroke the dipped glass paper with a glass rod t o
remove the excess impregnating solution, After t h e imgregnated strips a r e
thoroughly dry, they a r e treated Hitb 4 N Hcl for 5 minutes. The acid
treatment precipitates t h e potassium s i l i c a t e a s s i l i c i c acid. The impregnated chromatogram is washed with d i s t i l l e d water u n t i l a l l acid is
removed. The chromatogram i s then dried in 8 100°C oven and stored in a
closed container u n t i l used (3),
The basic principles of t h i s technique a r e quite similar t o those
discussed e a r l i e r f o r TLC, Selection of a solvent i s about the same as t h a t
for T U or paper chrmatograplqy, Visualization of the developed chromatogram
i s a l s o very similar t o T U , I ~ a ~ e v e rit
, has the d e f i n i t e sdvantage over
paper chromatography i n t h a t the use of corrosive sprays i s possible,
*E, Reene Angel and Cos,
52 Duene St., New York, M.Y.
155
Applications,
--Most l i t e r a h r e concerning the use of glass-paper
chromatography deals with the separation of l i p i d s , however, Dieckert e t a l .
(3)(4) have successfully separated sugars a s well a s l i p i d s with t h i s
technique. This same group of workers have reported t h e separation of
glycerides, cholesterol and cholesteryl e s t e r s by glass-paper chromatography
(5)(4). Ory (17) has a l s o reported the separation of glycerides of mixed
f a t t y acid composition by glass paper chromatography. Dieckert et
(6),
and Muldrey e t al . (15) have used t h i s technique foi: the fractionation of
phosphorus containing l i p i d s .
I
a,
Very l i t t l e work has been reported on the glass-paper chromatographic
separation of proteins. Arimura and Dingman (1)have reported quite good
success with the technique when it i s used a s an assay for vasopressin and
oxytocin.
References
-
1. Arimura, A. and J. F. Dingman, Nature 184, 1878 (1959).
2.
3.
4.
5.
6.
7.
8.
9,
10
.
.
11
12
13
14
15
Davidek, J. and J. Blattna, J. of Chromatog. 7, 204 (1962).
Dieckert, J. W., W. B. Carney, R. L. O r y and N. J. Norris.
Chem. 30, 1442 (1958).
Dieckert, J. W.,
Anal.
and R, Reiser, Science 120, 678 (1954).
Dieckert, J. W. and R. Reiser, J. of Am. O i l Chern. SOC. 33, 123 (1956).
Dieckert, J. W. and R. Reiser, J, of Am. Oil Chem. SOC. 33, 535 (1956).
Hofmann, A , F., J, of Lipid Res.,
3,
127 (1962)
.
Izmailov, N. A., and M. S. Shraiber, Farmatsiya, 31 (1938)
(Review by H. K. Mangold, J* of An, O i l Chem. SOC. 38, 708 (1961).
.
Kirchner, J. G.,
(1951)
-
J. M. Mfller and G. E. Keller, Anal. Chem., 23, 420
Mahadenan, V. and W. D. Lunderg, J. of Lipid Res.
5 106 (1962).
Malins, D. C , and H. K. Nangold, J. Am. O i l Chem. SOC. 37, 576 (1S60).
Mamold, H. K.,
J,
Of
Am. O i l Chem. SOC. 36, 708 (1961).
Mangold, H. K. and R. Kammereck, J. Am. O i l Chem. SOC. 39, 201 (1962).
Melahard, J. E. and N. F. Hall.
. Muldxey, J. .E.,
(1959)
Anal. Chem.
21,
185 (1949)
.
0. N. Miller and J. G. Hamilton, J. of Lipid R e s .
1, 48
156.
16.
17.
Neher, R.,
Ory, R. L.,
J. of Cbramatog. & l.22 (1958).
J. of Chromato&
5
W3 (1961).
18. P r i v e t t , 0. S. and M. L, Blank.
J. of Lipid Res.
5
37 (1961).
J. of Am. O i l Chem,
19.
Privett, 0. S,, M. L. Blank end W. 0 , Lunderg.
20.
Randerath,. K., Angewc Chem. 5 436 (1961).’
(Review by H. K. Mangold, J. of Am. Oil Chem. SOC. 38, 708 (1961).
Soc. SS, 312 (3.961).
21. Stahl. E., Arch. Phana. 292, 4ll (1959).
22.
23.
24.
25.
26.
Thanas S c i e n t i f i c Appamtus (Bulletin), March 1962.
Co., V i n e St. a t 3rd, Philadelphia 5, Pa.
Vogel, W. C.,
(1962)d
W. 14. Doizaki and L. nene.
Weill, C. E. and P. Hanke.
Arthur E. Thanas
J. of Upid Res.
Anal* Chew.
9, l38
1736 (1962).
Wollish, E. C., & Schmall and M. Hawrylysbyn. Anal. Chem. 33, u3B
(1961)
Wren, J. J., J. of Chromatog.
5
173 (1960).
MR. PEARSON: This morning our discussion so far has centered
upon chranatographic techniques. 3basauch as we want to break up the
morning somewhat more evenly, we would l i k e t o go ahead with the next paper
which is quite a d i f f e r e n t tqic--and probably not so new--in that it bas
been used for a great marly years. It deals with Spectrophotometric Methcds.
This paper i s prepared by Dr. Damel E, GoU. and Dr, Harry E. Snyder of
Iowa S t a t e University, and I am got o ask Dr. OOU- t o give the paper.
############