THIN-LAYER CHR(lMlA TOGRAPflY
The term "chromatography" denotes a procedure in which a solution of substances to be separated is pnssed, in a direction determined hy the arrangement nf the appm~ttus, over a more or less finely divided insoluble, organic or inorganic solid, resulting in retaining of the individual components to different :;;xients. Chrornatography is by far the most useful generai group of techni,pe~ available at present for the separation of a component from a mixture of closely related compounds. Chromatography is essentially a separatior: l.(~chnique, although the apparatus for carrying out some types of chromatcgrnphy, t:.g. gas-liquid chromatography and high performan~e liquid cim.:matography, also have a detector system as an intergraJ paii of the i11stnm1ent v,rhich enables anaiysis of components to take place immdiately ;titer rhey have been separated by chromatography. The underlyir:g mechanisms aJe the partitioning of the moving compounds between two liquid phases and also their being reversibly bcund on the surface of the adsorbent ff in the l2ttcr instance physical surface forces are mainly involved, the pn)ced'.1rc 1:~ referred tu as adsorption chromatography; in ion-exchange chrornuh'<£.rJrmy on the other hand, true heteropolar chemical bonds are forn·,,:d reversib!y between the moving compound and the adsorbent. In this ,Ticthud, their exi::,t as ions of opposite charge. In practice, a combinaticn 1.Jf ndsPrption, ion exchange and pa11ition chromatography 1s usua1l:y involved, with one or other of the types predominating.
A chromatographi,; systern i..''Onsists basically of tvvo different phases, one termed the "station.uy phase" the other the "mobile phase 11 • The principle of the method exploits differences in the degree of interaction of each of the components of the mixture with each of the two phases, The speed at which each component p.:isscs along the chromotographic system depends firstly on the rnte of flow of the mobile phase, and secondly on the degree of retardation brought about by the interaction of the component \Virh the stationary phase. Sn1r1e of the components ,Nill be relatively little retarded and will pass rapidly through the chromatographic system \Vith a \/elocity approaching that 0f t\.._, mobi '.e plrn.se, whereas others will be held up more ir; the siationary pLasc and pass along the system more slowly. The separation may be stopp 'd at a p2r(1cuJar time and the relative positions -, , " t~ J t, -t ,., ,.,.. . t1 • 1 i t L th"1s 01f' ti· uie L:Cmpo:rh.n ;s o.e ,ec e-._1. i-1:-' •n paper or nm··JE!Jcr C!lroff:a ograp1"y~ 1
is often termed "development cbrom,atography". The chromatographic process may alternatively be continued untd all components have pa:::sed through and been eluted from the systen1, ns in gas-Liquid chromatography and the several kinds of column chromatography; this is termed "elution chromatography". There are a number of interactions betv.1een the sample and the stationary phase which can be exploited in order- to effect the separation. These include adsorptive properties such HS are used in thin-layer chromatography, partition between the phr;ses as is used in gas-liquid chromatography, antigen-:mtibody interacrions and enzyme-substrate interactions as .are used in affini1y chromatography, molecular shape and size as is used m gel filtration, and ionic charge as in ion-exchange chromatography. ln this context, electrophore~;is can be thought of as a specialized form of chromatography in \vhich the main property of the substance which is exploited is its electrical charge. No separation is brought about solely by, for instance, adsorption or electrophoresis or ionexchange interactions. By selecting the mo::t appropriate physicochemical property for the mixture under investigation it is possible: to find suitable chromatographic techniques for the separation of a wide variety of chemical compounds. The stationary phase rnay be ? solid, as m thin-layer chromatography, or a liquid as in gas-liquid chromatography and paper chromatography. (NB. The main princip1e of paper chromatography is that of liquid-liquid partition, tn ,vhich the :!:tationary phase is a liquid, immiscible with the mobile phase, vvhich is Lapped in the interstices of the supporting paper matrix). The mobile phase may be a gas or a liquid. The combination between the possible mobile phases and the possible stationary phases give rise to four major types of chromatographic syst:::ms, gas-liquid chromatography,
gas-solid chromatography, liquid-liquid chmmawgraphy, and liquid-solid chromatography. In general adsorption is the basic principle employed where the stationary phase is solid and piJtrtition is the basic principle employed where the stationary phase is liqui,,; Partition chromatography is us1..1 :11ly n1o;c effective at separating a number of closely related compounds. If the ,.:omponcnts are volatile, or can be made so by derivatization, gas-liquid ch, Jmatography usually gives . superior separntions to thost" obtaint:d by i1qwd·-liquid chromatography.
3
Gas chromatography i:, also likely to be superior to liquid-liquid chromatography if quanfrative analytical results are required. The use of high--performance liquid chrornat0graphy however is now being shown to produce quantitative results 0.rd efficient se:parations which are comparable with those obtained by gn:: chl·omatography. Liquid-liquid chromatography is usually superior to ga:::-li1.-;;u;d chromatography if the purpose of the separation is to isolate and p1.Tit:-' 1hc components in a mixture. Adsorption chromatography is oflen more appropriately used for separating different classe:; of ccrr:.pounds from each other or for separating molecules with widely diiforing physicochemical charncteristics. Thinlaycr and paper chromatography a.r{: widely used to effect rapid qualitative or semi-quantitative sep,~rations either for routine screening programmes or as a preliminary to further, more sophistkated separations. Chromatographic separation can be used for the qualitativ(! screening, preparation, identification and quantitative analysis of a ,vide variety of chemical compounds.
The way in which n mixture <"'f compound is separated into its components lies in the ability of the stationary phase to interact reversibly with the individual components in the mixture in such a way that the migration of each component ;s retarded to a different degree from that of each of the other components. The major interaction in thin-layer chrmnatography is adsorption. This type of interaction is also fundamental to the separations achieved by a number of Liquid-solid column systems and by gas-solid chromatography Although pcilrtition is the major interaction involved in liquid-liquid and gas-liqcid chromatography. Adsorption is the fundamental process underlying thin-layer, liquidsolid and gas-solid chromatography. The stationary phase, or adsorbent, is a particulate matrix, containing, on the surface of each particle a number of reactive groups. The molecules of the moving solvent phase compete for these reactive groups with nioiecu1ec; of the solute components in the mixture being separateJ. E;ich type of molecule will interact with the reactive adsorbent grvups to di 1Te1 :nt degrees, those molecules which internet less readily wi!l be carried ~·i!cng rnore rapidly by the mc,bile phase.
4
Tbeor.yJlf.ArlsQrpilim. The kinetics of the reaction between a solute X can be summarized by an equilibrium reaction:
where
3J1d
an adsorbent A
the concentration of free solute Xf ==
the~ concentration of free binding site on the adsorbent X.A. ,cc concentration of so Jute bound to adsorbent The equilibrium constant, k is given by
k
___ _(XA)__ (Xf) (Af)
If the solute 1;oncentration is small, then the amount of solute bound to the adsorbent is much smaller than the total concentration of the adsorbent, which can therefore be assumed to be constant for a particular system. Thus. {"\Y A'\
-V..:.....a..;
- a constant
(XtJ The constant, K, is called the adsorption cceffi..:ient, and is given by,
K
_oon~~rnr?tim.1 o.f.Q.b.sm:h.cl solute c1.:1ncentra-tion of r~on--adsorbed solute
If a grnph is drawn of the concentn.tti.on of adsorbed solute against the concentration of the non-adsorbed solute, it will be seen to be a straight line. with slope K. This is the ideal adzwrption isotherm. In order to effect good chromatographic separation i.t i" necessary to choose conditions so that the adsorption isotherm is as chx:e w linearity as possible. In practice, providing that th.e solute concentration is low, linear adsorption isotherms can readily be achieved. Under this condition the concentration profile through a migrating zone can app1-:,);-.imate closely to a Gaussian distribution. At higher solute concentrattons however where there is an
5
appreciable fall in the number ot' binding sites available for solute molecules, the concentration of adsorbed solute molecules approaches a maximum and any further increase in total solute concentration will result in the value of K being rcdu1.'ed. This fall in the value of K at high solute concentration results in a convexly adsorption isotherm, which is associated with significant tailing of the :.rd.grating zones, leading to poor resolution between components. This situatinn can a:rise in thin-layer chromatography when the plate is overloaded, but;,;; less .likely to happen in column systems. It is a]so likely to be encountered when preparative chromatographic separations are being undertaken. Ihs; lineJ!.L cap.a..ci:tx of ;m adsorbent or chromatographic system is defined as the maximum solute concentration which can be achieved without he adsorption isofr1~:Trn deviating :~ignificantly from linearity. It is possible by controlling a number of fact<:irs to maximize the linear capacity of a given chromatographic system. This is of use practically as, in order to visualize the compone;1t:; in a mixtun; ar~d possibly to isolate the components in order to carry ou1 further studies, it is frequently necessary to apply as much of the mixture as possible. Any factors which can be manipulated in order to extend the concentration range and maximize the linear capacity will therefore he of some importance. The main factors ;;\ffec1ing the adsorption coefficient and the linear capacity arc : (a) the nature of the adsorbent, (b) the properties of the developing solvent and (c) the chemic2d structure of the solute. (K i5 a!sc" temperature dependent.)
The forces which are of importance m determining the adsorption interaction in Ghromatography are : (a) Hydrogen bends ( b) \Veak covalent bonds (c) Dipole forc,~s ( d) Dispersion forces In addition to these n:+tt1vely weak forces, stronger interactions can also exist b.etween the adsorbent and th 1: components being separated. These interactions are sometim~:'i referred to as "chemisorption" and result from strong covalent or ionic bondmg. ln general chernisorption is not desirable in chromatograpLic systems as the desorption process is frequently so slow that the iength of time required to complete a
chromatogram is impracticably :r.mg. Use is however sometimes made of chemisorption in separating compon.::mts, ,..vhich are strongly bound to the adsorbent and which remain at the ori:zin of the chromatrn2ram, from those which are held by weak forces only and which m;grate through the adsorbent bed. By changing a property c1f the eluting solvent, e.g. its oxidation state, its pH or its ionic strength. it is sometimes possible to hreak the chemiso:rption bonds and allow the bound components to pass along the chromatographic system. The technique is particularly used in column chromatograpny including ion-exchange chromatography, but its success does depend upon changing the nature of the eluting solvent, either by one or more discrete changes or by a continuous change in solvent composition as in gradient elution. For most chromatographic separations however, particularly when only one solvent system is being used, it is necessary to minimize the effects of chemisorption and to rely for separntjon of the weaker interactions listed above. Another undesirable property of certain adsorbents is the ability of their active groups to act as catalysts which promote chemical changes in the molecules of solvent or solute. The chracteristic properties of adsorhants which are exploited in adsorption chromatography are : I. The surface area of the adsorbant, which is proportional to the adsorbent volume and inversely prop:xtion.:il to the particle size, and 2. The nature, frequency and distribution of reactive groups - "active sites"-which should enter into a reversible adsorption reaction with solute and solvent molecules, but which shculd not bind such molecules irreversibly. These properties can be modified by deactivation processes, as described below, or by interaction v1ith the solvent or solute or wiih compounds deliberately added to the adsorbent in order to change it:; characteristics. In general, adsorbent can be polar Ol non--,polar; the polar adsorbents can be acidic, e.g. silica, or basic, e.g. alumina. Charcoal is a classical example of a non-polar adsorbent. Polar adsorbents will retain poiar compounds relative to non-polar ones; acidi1. adsorbents will preferentially retain basic solutes and basic adsorbents preferentially retain acidic solutes. Non-polar adsorbents tend to retain preforentially compounds of higher molecular weight and compounds with aro:'rrntic properties. The properties of an adsorbent can be deliberately modified in order either to minimize undesirable propert;c:,, such as the presence of chemisorptive groups or excessive activity, or to give a desirable property to •_.J
--;;
7
an. adsorbent, e.g. changing th,:· pH of' f:iiica by adding sodium carbonate to give a basic adt:orbent. T."olar adsorbents have ,: variable number of water molecules a(:isorbed onto the tmrface of the particles. These tend to de1ctivate some of the bind:ng ,:.,roups. For some separations this is an advantage; indeed it may Ix ni:v::ss;:lry to d,~activate still further by allowing further adsorption of \Vater. For other separations it may be necessary to increase the activity of the 2dsorbt:nt by h ~ating at between ahout I 00-150 1
0
C to drive off some of th(:se ,v:Jsorbed ,vater molecules. Activity can also be increased by reducing the· ni~::,.rn particle size. This has the beneficial effect of inciea.sing the efficiency of the adsorbent. Increasing the activity may however have the undesirable effect o :~ decreasing the linear capacity. Relatively fo,v adsor;-,cnts h'lve found widespread use in TLC, silica being by for the most popL.la.,·. Others v,hich are used include alumina, cellulose powder, Flori.sH ( irwgnesiu:-n silicate}, Kicselguhr, starch and Sephadex. The particle :;izc of rnost commercially avaiJable adsorbents is about 20-50 ,um. The adsorptive properti~·:s of silica depend upon the presence of hydroxyl groups linked to e '.,i!icon c1torns on the surfa.ce of the particles. These groups may exist as free hydroxyls or as groups hydrogen-bonded to each other. Both forms :1 1·e active adsorbent groups and interact with solvent or solute molecuk~: mainly by hydrogen-bonding. Silica is 1 frequently termed 'silica ;!~!" in TLC and "silicic acid" in column chromatography: it h::1s ~l gocid Iinear capacity, shows activity to a wide variety of compounds and docs nor catalyse sample decomposition. Silica does not however separ'.lk 1,,vel1 compounds which differ only in their content of ctouh1e ( olefinic) bonds. lt can neverthelass he used for this purpose if silver nitrate is added V:, the adsc,rbent.. The structure of alumina is similar t0 that of silica, chromatographic separation probably depending rnainly on hydrogen bonding between the solute molecules and hydroxyl groups on th~ alumina. Some workers have however suggested that clectrc-static forces also have a significant influence. Alumina is an adsorbent -.:vLich has a \Nide range of applications, but also has a tendency to cataiysc s::1mple decomposition and to chcmisorb acidic comp1..1t:nds ln order to bind the adsorbt:nt partic.les together and to the supporting glass plate, a binding agent U5tial Jy cJJciurn suiphate, is frequently added to the adsorbent when thin-layer plates nre being prepared. \Vhiie inclusion of binder gives some advantages dH~re is no doubt that some- modification to tl10 propertie:, of tht: 1dsud cnz layer ,'.re introduced, -::;orne of which are · '· "JV1ost. • ' \hCG. ac,sor I l·..Jents can 'rn practice ' i,)e app J'te d un d e·arabie. comrnon!y 0
without binder, although if solv;::~n1s of h; gh strength are used tht>re 1s tendency for pure adsorbents to peet off the .Plate.
'1
As can be seen from the above di.s,:·u'.:sion, a. rather small range of adsorbents b normally used in adsorption chromatography and the modifications which can be made to the adsorbents are relatively limited and designed to increase the e.fficieney of the separation and maximize the linear capacity rather than effect the Gp?;~"ificity of the separation. This limited variability of adsorbent, cannot account for the wide range of specificity c{ separation which can bt: achieved by adsorption chromatography. The adsorbent can 1hen:,f:.re he looked at as a reactive: but relatively non-specific matrix which will lntcract with a wide variety of molecules, both of the solvent and of the component solutes. The specificity of these interactions vviU de.mend on the molecular structure of the solvent anJ, especially, of the solute. Each compound (solute) in a mixture subjected to chromatographic separation will interact with the adsorbent to a different degree, which is described by a solute parameter, S, terrned the "solute adsorption energy". The value of S wilI depend upon the p£~.rti(~uiar chromatographic system used, and especially upon the nature of the :::;cb/cnt and the activity and type of adsorbent. It is custornary to define a standard solute adsorption ()
energy, S for each adsorbent under defined conditions (usually with pentane as solvent and with an activated frnm of the adsorbent). The value 0
0
of S can be e:stimated experimentally. By e::c:timating S for a wide variety of known compounds it has been found rh:i.i., to a first approximation, the adsorption energy of a molecule is equal to che surn of the adsorption energies of each of the constituent groups and atoms in the molecule:
Vlhere
Qi
==
the adsorption energy of the group i.
This is primary effect of rnoiecular structure on adsorption energy is modified by the presence and relative p\)Sttions of the other groups in the molecule. The adsorption interaction d. on:: group with an active site on the adsorbent wiE obviousiy be affected by ;nternctions of neighbouring groups with the adsorbent. The group adsorption energy. Q~.' is dependant upon the type of adsorbent. although, as can be 5ecn from Tabie 1, Qf values are generally
9
similar on alumina or silic,). The rnaj0r exceptions to this generalization occur v1hen the group is s,:id;c or basic, acidic groups having larger Q0 I arger va l ues on1. • . • vaI ues on a Jumma t han on ~~,.,.11ca, a.no1 .basic groups 'nav mg silica than on alumina.
L411l.E...1 Q 01 VALUES FOR SOAfF' ·UJPHATlC CHEMICAL GROUPS ON AL UMIN,! AND SILICA r--------(-fr_o_u_p_________________( -------·····----·-· --Q~~-v-a-lu-e ----------]
I
I1---methyl
,- ---A1 u:m1-;;·; ___i_._____snI~~-
- - - - 1------
methylene I methene ! chloro Ii UHO ' . l
0
aldehyde Iester
-CHO - ,1.J,,... 1 -OH
I (..,,..,()
1
hydroxyl
amino
!
amide
i
J
I
1
--·--------·-·r··· ---------------t-----------i -0 03 / 0.07 i 0.02 I 0.05
~CH 3 -CH::-CH ,· -Cl ~-£-1. -,j
i
--
I
1 1
Ij
4.73
I
1'
-
j
6.50
I
5.60
'
8~0_J 9.6
I
J
6 :74 8.90
1
i
0.25 I.74 ,i · 70 4.97 •• 17 ::J ·-· ·
'5.00
1
-1"-'1l I -CONf 1:,
0.31 l.82 ?. - 80
!carboxylic acid______lcunr L __ j ______ ~21.0 ------·-
I /
_____ 7.6
The value of assessing the affects of indjvidual groups in a molecule on the adsorption energy, acd hence on the chromatographic properties of that molecule, lies in assistin1:o in the identification of unknown compounds and in predicting chromatographic chractcdstics. Jhe..s..ffi.Q_iency of .ILC. ~5..~s is n..
strlliall_r~..__bJJ_t __.5-u~h_1_.~QJJ:ii.d:;;nuiQJ1,'i.._Q.flrL....l1.e.rro.:w__tM_JieJd and .-1ldd -~.mwl~m~D1flI~: __e_Ylde.~_JQ___othQL_i.d_entif1~.ati.QJl._Luv_f:.s..tigati..0.11.S.
(Similar considerations apply in ether fields of chromatography, although the
pr1ramcters involved ln µanihm chrnnici.tography are not stfr.:tly the same. The much greater efficien•_'.> \vhich can bl'.~ achieved in GLC, vvhere retention vol.umes can be measured very accurately; en.ables a more sophisticated interpretation of mo!eculat -.Jnh.'turc to bi: made). However, it is unwise to make a11 identification of a :.~ompound merely on its chromatographic prope1iies however accurately tho::ie properties can he measured. Before a frill asse3srnd1t ,Jf the interaction between solute and
adsorbent can be rnade, the efCect of the compc·~,ition of the mobile solvent 1 C'Oi"')i ()n. tl,-,,, ·.i.i.;,,.; a· 0 .-;:, ;- ,. i ')"'"I f)tOCP·SS '1Yl! c; ::· .1 l ,: •'" ll,e C0\1 ~ i Jered .a.L :l
........
!-.1.\..;..._.,
r, .•. 1 •. , , ;
.
;
.,-?
.. ,
-
,
lU
The Solvcn_t Although the major factor in dctcnnining th~ chromatographic properties of a compound i:; its niJilecular structure, important modifications ·are brought about by changes in the coir.qJosition of the solvent system. Discussion about the simplified adsor-pt on reaction between solute and adsorbent, must be complemented b) ~:onsideration of the competition between the solvent and solute molecules f(Y free sites on the adsorbent. During a chromatographic proct>ss solvent passes through the adsorbent bed ahead of solute moI~cules. Tlm:::, when the solute reaches a particular zone of adsorbent an equilibriurr tvill already have been set u.p between the molecules of the solvent and th: bi!h.iing site: on the adsorb,:·nt:
EA
Ef+ Af where
the concentration of free solvent molecules the concentration of free binding sites on
and
the adsorbent EA = tbe concentration of
solvent/adsorbent
complexes As the soJute molecules tht::n enter rh.~ :zor c a second equilibrium will be set up bet;,veen solute molecules and adsorbent: 1
xr+Ar
XA
This equilibrium ,vill be affected by th1:' av'.1ilability of free binding sites, and henc:e by the tenacity with v:hich solvent rnolecules are bound to these sites. The propmtion of the solute vvhich is bound, and hence the value of the adsorption coefficient, K, will i:L:pend cin the relative affinities of solute and solvent molecules for the adiwrbcnt binding sites. This leads to a competitive equilibrium as follovvs:
XA + Ef A pararneter describing the affinity
of solvent molecules for . so l vent w ...h.1cn' adsorbent is the "solvent strength", e . I\ '..::h:1nge c· f· cIutmg causes an increase in solvent strength will increa:3,2 the rate of migration of a • ' tue !,., • b •le nue;u:r . . L o f ;'free l.omomg • • • particular solut,: by re,:l unng availa sites. (I
.
.
11
If there is a disparity k;t,,. ecn the size of a relatively large solute molecule and a relatively small S,)ivent molecule it 1s possible that the above relationship will not be a one-to-one molecular equilibrium but that one molecule or solute wouid displace more than one molecule of solvent. In the following discussion i he ;nolecu lar area occupied on the adsorbent surface by a solute molecule ls represented by Ax. The total number of binding sites available will also depend on the total volume of adsorbent, Va, a:1d er: its ai::;tivity a. The factors described above are related to each oth.::r and to the adsorption coefficient, K, as fc!knvs: , ,.. ~ -,; , .. 0 G log K .-., tcg va + uiS -Axe) In the case of a gi~, u.i soh~te passing through a given adsorbent, Va, 0
A>., a, and S will be const,Jnt and equation becomes:
Where J\11 and P are t:onst.nts This is the equation tci ~: straight !ine and shmvs that log K is inversely proportional to the solvent :,tn:ngth.
TltUL.E_Z.
ELUOTROPJC SERJES or A. JVHJMBER OF CO:l\1MON SOLVENTS 0 TOGETHER Vv'ITH THEIR SOLVENT STRENGTH VALUES(e ) FOR /\ POLAR ADSORBENT
fri:::~e----- Solve!1t____________..,_.____
t ______ £6~__
--·--------------·-------·--- -----·-------·------------- -- -r-----·-------------
j carbon tetrachloride
1
isopropyl ether I benzer1e I chloroform I ethylene dichloride I l methyl ethyl ketone acetone
11
i
I
j diethylamine
I pyridine ! e:thanol
lmethanol
i
I:
; /
I I
--- ~-----
'
1
?· 18-
1
0.28 0.32 0.40 0.44 0.50 0.55
!
0.63
I
0.71 0.88
--·-·-·----------l----------·---~:95 ___________
1j
lj
I
J
'
The tmportanc:e of solvent ~:trength in the µractical application of chromatographic procedures has !ed to ~he listing of solvc:nts tn the 1'a bl · ' usea . . t,cnes. . e luo t rop1c , e 2 11st~ an c,1 uotrup1c sen• es o f comn1on1y chromatographic solvents. The above discussion on solvents is umfine
,,J
..,.
J
Separation P1n.ra.m~.rs The term "separation" can be used strictly in chromatography to describe the disengagement of the centre of the zone of each component substance from that of each other co:rnpc,ncnt during a chromatographic process. \\/hen used in this way it is not interH.fr:d to give any indication of the degree of dispersion and widening of Lie zones nor of the degree of overlap benve:::n adjacent zones. l'he tenn which is used to describe all these functions is tbe "resolution", R. The main function of a chromato,,sn::phic system is to effect a satisfactory resolution of two or more substaI1ces initially present in a mixture. As discussed above, such resoluiion i? dependant both upon an adequate diseng2.gement of the zone-centrr
l:n order to obtrdr.1. :1.1:1.xunum resolution m a particular chromatographic system it is useful to .have a quantifiable measure of resolving power so that changfs can be made in the conditions used for the chromatographic nm and the:s-:~ changes can be correlated with changes produced in the resolving po\v,;:r. In this \VilY optimum conditions can be achieved. As might be expech!d, however, the optimum conditions for the best resolution of one pair of co1 t1ponents may not be strictly the sarne as those for the best resolution of anou1er pair. In practice therefore it is usual tu study rhe resolution of one prw: icuiar pair of components, chosen because of their similar Rr values in u. variety of conditions or because of the importance which is r:;bcrd ,.m achicvin.g their separation. If the distances mi11;r.:::.tcd by these t'No components, ! and 2, are 1 1 and 12 respectively, and. the respt::ctive standard deviations of spread of their zones arc s 1 ands~> theri th(: resolution, R 1, 2, is given by:
,r
Resolution is regurded as adeq:mre :.f R i. : is greater than 1.0. The standard deviation of the 2:oL,; wioth is not a directly measurable parameter and, in practice, use is mark of tht fact that the total width, w, of a zone is approximately equal to 4s. Thus: R : . ,.'.. --
I ;
~
-J. I •')
- -.1.-LL·-
ZS(W1
+ W;)
The art of thin-lay1~r chromatography is to modify particularly the solvent system, and also other factors, such as the type and pore size of the adsorbent, the temperature, the pH of adsorbent and soivent, in order to produce an optimum scp,n-at;rm of all the components in a mixture in a reasonable time. To some t:xtent this can be achieved empirically and by reference to the extensive Jiterature:, but in order to obtain maximum benefit from the technique a planned 1ffvcstigation of the factors involved, based on the theoretical principles outhned :1bove, shouid be undertaken when each new separation is attempted.
a
r·-·
c
!
l
.
.. ----.-----.I
!
I!
s
I
c==--:J [
0
fig._L The progressive separation (15.
I
Zl
_=:]
_
_J
components, z 1, and z 2 , during a TLC run. (a) soon ofter the stan, (b) at the halfway stage and (c) at the end of the run. S is the position of the: solvent front and O is the origin, 11 and l 2 are the distances moved from the origin by z 1 and z 2 . t\VO
Classically ·1 LC is carried out on 20 crn Y 20 cm or 5 cm x 20 cm glass plates ,:overed with a uniform :?50 vn thick layer of adsorbent (usually silica gel). Sampies are applied Js spots about 1-2 cm apart in a line across thi! lower part of the plate, the "origin", about 1.5 cm from the bottom edge. The plate is placed into a suiuble tank with a close fitting lid (Fig. 2). In the bottom of the tank is a solvent, or mixture of solvents, the "mobile phase". to a depth of 0.5-1 cm, so that the lower part of the adsorbent layer is immersed in the solvent but the app]ic:d spots are a short distance above the solvent level. The solvt:,nt rises up the plate by capillary attraction and the piate is removed frmn The tank when the solvent front is 15 cm or so above the origin. This wili usually take about 30 minutes. The solvent is allowed to evaporate from the pla.:e which is then sprayed with a colour reagent to elucidate the positions of ,.he separated components from
I.',
ea(;h applied sample. TheL-.:. ::re the basic methods introduced by Stahl in the !ate l 950's, when he \Vas 'Hr[:dy responsible for popularizing the general use of a technique which i1::td previow;ly been employed by only a few chrornatography expert.:;. Since then, inevitably, many modifications have been introduced to foci tit2.te pJ;ti~ular sepa.ratiions and to meet specifi<.: needs., but the basic techn\p1e rcnlains remarkably similar to that instituted almost two decades ago .
.Eig,_2. Developnicnt 'c2nk fr:;,r use with standard thin-layer plates.
Although the 250 ;:rn thick adsorbent fayer has proved to be about the optimum thickness for mo.·:;: sev1rntons, thicker layers, up to 2 mm, are frequently used for prepir:::iti ''C purposes. A number of manufacturers have , , I • " " • . pro(j uce d a vanety o f forms. c,1i' ;1ppr-iratus, <•·app,1cators , ror preparmg th.inlayer plates of standard 1 50 1-uL tLickness and most can be adjusted to produce the thicker prepaL:dY.::. plzlte'.~ The apparatus usuaUy consists of a · ' , i._,nger· . s 11gnt:v ' . rectangu l ar reservoir, than t11c norma l ~. .:'.._,n cm p Iate w1.dth , along the low.~r back edge o:-· \vhich is a slit of variable \Nidth through which the sluny of ,1dsorbent pass,.'S onto the ptnte beneath. In sorne methods, the app!ic:.1tor i;,: moved across ~1 Jir.,: ,-:,f elates, m oi·hers plates are pushed under +i '"·),··1·:cat·,J··· l.l 1p~, 'Af !_} .l .... J..
!6
12ntlv The lnlates lhemr.:elves ::re f',·.'-'u11 .,,,.,,.,.,· • ..... A,,.: ..,,, ·{Je of' rrlass a"lth<)u·gh ·1·n rP:r,.,.n~L years other more flexible materials :~uch as polyester, cellulose paper, 4-
r
.,,
...
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,
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aluminium foil and glass fibre have been used, particularly for pre-prepared plates obtainable from a number of 1rmni.1L1cturers. The important prope1ties of the plate are that it be planaL. srnooih and without blemishes. As mentioned above the standard size of plates is 20 x 20 cm or 5 x 20 cm, the former allmving application of about :i.o ::pots, the latter about 3 or 4; the author has found that window glass, Cd1. to size is satisL.1ctory for most routine purposes, although this may en.cl<. if heated rapidly to too high a temperature. For more exact work, panicu ls.rly if reproducibility of the Rr values is important, specially-made plar:eT plates of heat-resistant glass are widely available commerci:1ily. For quick q,Ja.litative screening of samples for a limited number of components, ffti'.:roscopic slides have been found useful. These need to be run for only about five minutes to give a crud<~ separation which is nevertheless satisfactory fm· these purposes. The plates should be scrupulously cleaned prior to use. Gl:Jss plates can be cleaned with water followed by acetone or alcohoL The adsorbent is usuaily pre.pared cs a slurry in water. As an example, 25 1; of silica gel G with 50 ml disiilied water will make sufficient slurry to produce five 20 cm x 20 cm plat•:;s on which t.he adsorbent is (1.25 mm thick. Once tbe slurry has been mixed it is necessary to use it with minimum delay, as it soon hecomes too thick to produce smooth layers, especially if the adsorbent contains calcium sulphate or some other binder, It is therefore necessary to have th~ apparatus prepc:rcd ,-(,r use before the sluny is made. After the layer has been applied, the wet plates are allowed to dry in the air for abom ~O minutes, and then stoi'ed, p1Tfr:rably in a desiccator, and if necessary actt\'ated by heating priur to v,c rhe side edges of the adsorbent layer on laboratory-madr~ plates are often irregular and of uneven thickm:~ss, It is advisable therefore, in the interest uf r,1inimizing edge effects to remove a strip of adsorbent, about 3 mm wide, each side edge~ this can be dor.e with a spatula or a finger. The most regular and reproducible plates are made with the types of manufactured apparatus described above, but other, more manual methods have been us.:!d with success. Simple applicators can be made from a glass or metal rod or straight·-edge, the central pt'ttion of which is raised above the level of the plates by the required amount by means of spacers laid along each side of the plate, or by wrapp:n,?, a number of turns of thin adhesive tape arc,und each end of the rod or straight .:::dge. Slurry can then be poured onto a rov1 of plates and levelled by dragging the "applicator" along the row. Some workers are able w produce iav,~,r~: to their satisfaction merely by
f,,,m
17
pouring a predetermined ;:.n1,JtWl of slurry onto a plate:, which is then manipulated until the slurry is evenly distributed. Plates can also be coated by dipping them into a slurry,. removing them and allowing tJ1,?.m to drain. If two plates, placed back to back, are treated in this way a relatively even layer of adsorbent can be p:·och,~:ed on one side of each. This technique has been particularyly applied tu sm?ll plates such as microscope slides. Plates can also be coated by srt:iy;;jg the ad:mrbent with a spray-gun. Some workers have successful!::' used plates on which the adsorbent has been applied as a dry powder: th;;'.sf: plares cannot of course be used vertically in the conventional ascendinf, diTumat.ography tanks as the adsorbent is not bound in any way to the :.upporting medium. Plates made by the relativeiy simple manual methods de:;c:;.ribed in this paragraph do not show sufficient reproducibility of results eitl1er \vithin each plate, or between plates for the purpose of accurate identi fic:ation of unknown spots. They are however cheaply produced and adeqtFd.e for many general purposes, especially when the nature of the comp>)nf.:nts in the components in the sample is well understood. For reproductivity of chromatographic properties it is better to use plates prepared by one of the specia.Hy-niade applicators or~ possibly better still, by using pre-preprr:-i:·d olates available commerdally. The manufacturing process can produce regular, homogeneous and lawless adsorption layers and e3(:fi pl.ate in a batch will have highly reproducible chromatographic propertii.~::, These plates are of course significantly more expensive than plates rnadc in the laboratory, but arc of great value in carrying out identification v..ro;)rk, or in those laboratories where TLC is not used frequently enough t(i innke preparation of plates worth-while. Special features are also sometir:1e.:-: il'iCorpornted into marmfactmed plates. Regular channels about l cm ::i.;;a:, c:1.;1 be :,cored in the adsorbent layer along the length of the plate: this technique minimizes edge effects and leads to more reproducible Rr values. LJ.bor:J.ory--made plates can also be scored in this vvay by mmiual me::in'\ b,.,t the resultant score-lines are frequently less regular and clear cut than tlH:,::c product:d by machines. Another feature of some comme:rcial plates i:; ,h,~ inclusion of a pre-adsorbent area in the lower 2 cm of the plate, on to wl1ich sarnple .:.:an be applied more quickly and less painstakingly. The pre--,:1.dsorbcnt area is rnadc: of a material which abosrbs relatively large volumes of :snrnple. but has minimal adsorptive properties. After the samples have bef.'n Jpplied to this area and the plate placed in the development tank, the sol"v(>lt front rapidly rises through the pre-adsorbent ·. ,.,-., ,..,_.,,,, · ·· -'a i,. · ,·, ·,.,;th .. -.. ·"r'" -- '1 ·1Jt,,.::; · •·,., ~ ,: 1··.,. • t1'"e •· ''"np'"" nv th;1.:;c •n1ea1 arc.:: u.i k'n11;:, ,v. ... n; • ··u11e. 0-.. 11,. , "",i ,.... 1.,.. -< 1..s , ,':l. ._;,·t· ,·.:,u 1 relatively tight, concenln.Jed h::md of ::a.mple is carried to the interface between prc·-adsorbenr 1d ,rnd the adsurbent proper, where the
chromatographic process starts. technique are therefore minimized.
Effects ,·auscd by poor application
Samples may be applied as discrete spi:)ts, as short streaks (about l cm long) or as D continuous streak extcn.ding a..:ross almost the whole width of the TLC plate. This last method is of ~peciaJ value in preparative TLC. The former methods can each be used to apply ,.;. number of different samples and workers are divided in their views 011 ':.b~: merits of each. Ideally spots should be applied so H1::-J they cover as small an area of adsorbent as possibh~. and should certa,nly not be more than 5 mm in diameter. In order to achieve this, 1t it, necessary to use relatively concentrated solutions, preferably in a volatile solvent. By using such solutions, re.latively rapid applic;:1tion of spots can be effected, thus reducing operator tim·~ and minimizing sample decomposition. Up to about SO µg of a sample mixture can be applied to each spot on a 250 µg thick layer; the volume of the sample should be about 1- l Op;. A l O µl syringe or a syringe driven by a microrneter are the most acce,Jtable manual instruments for applying samples~ both will probably be fitted with a steel needle. Less expensive, small volume, glass pipettes rm;)' also be used but are in general less satisfactory and require more skilfol operation. Glass capillaries, as are used in blood gas analysis, are also useful tor applying samples. A number of mechanized. multiple-sampk applicators are also available commercially. In applying the spot it is necessary th::ri the integrity of the adsorbent layer be maintained. Scratches made by th: syringe r.eedle will interfere with the normal chrornatographic developmcr,'. of the sample and will lead to abnormally shaped component spots Rr.:n1oving the pointed end of the syringe needle with a file will help to prevent inadvertent piercing of the adsorbent layer. Because the layer is fairly fragile it is advisable to use some sort of spotting template which wd: both protect the surface of the adsorbent and indicate the positions at w},ich TO apply the samples; effective templates can readily be made in the lahorntory. Even with the best apparatus and v,1 id1 readily applied samples, it is inevitable that it will take some minutes to apply up to 20 samples on one thin-layer pl:ate. The components of those sample spots which are applit::d first \}Vill therefore have to spend a few rnlnutes in a dry state on a finely divided, activated adsorbent 1 condi1 ;nns '
19
such a size that a thin-layer piate can be placed inside it, Along one of the inside edges of the tube, to p:>,\~ cm through holes in its side wall, and to blow over the spots which :ire being r.:ippiied to the TLC plate. Nonoxidizing conditions are thus achieved and the stream of gas hastens solvent evaporation. Covering the applica1ion tank with UV--adsorbcm material fnrther prevents sample decomposition. The application of samples as short streaks help to reduce di ffus.ion of tbe sample aiong the axis iii \vhich the chromatogram will be run. In contrast with spot application the sample applied as a short streak is not ail applied at the same point. 'T'hi:s enable-; one portion of the sample to dry as another portion is be.ing appLed a litti;~ distance away. In spot application it is frequently necessary to \?/::'Lit f;:,r some se~onds in order to allow already applied sovent to evap::\rntc before adding more sample to the spot. Continued appljcation of s·'.inpk; :~olvtion to an already moist spot will cause unacceptably large spots, lo the pcrir:neter of which much of the sample will be eluted, resulting in rin~:; 1 J:her than homogenous spot:;,. One minor disa,Jvantage of applying sampJcs in short streaks is that fewer samples can be nm on each plate. Such .: , dcfree of care in sampk application is not required when using the :m:.nufactured plates \Nhich have a pre-adsorbent spc•tting area. Along with the sample :-;pot3 a number of standard reference mixtures should also be applied. 11 i:-~ irnportant that these should be applied between unknown samples and no1 ail 1n one part of the plate or. for instance, at the edge. It is sometimes useful to run two or more different concentrations of reference mixtures in case the Rr values change slightly with increasing concentration.
R1umingJ:b~ Pia te In order to minimize: decomposition of sample components it is necessary to begin the chromatographic nm as soon as possible after sample application. A suitable solvent rnixture ir,ay be chosen by reference to the literature, by trial and errnr, ?.nd by refo;·ence to the eluoiropic series. The volvme of solvent is such that it \Nill. immerse the lower 0.5-1.0 cm of the adsorbent layer on the thin-Lwer plate, hut vv·ill not reach the level of applied samplE':5. It ;s important that tht: solvent mixt:1re is allowed to equilibrate with the air in the tank prior to \.Le :,;tart of the chromatographic run. It is also important that this equilibration i::; neirher unduly disturbed when the plate is placed in the tank Hor subji~ct t:) •hange during the nm due to leakage of vapour thro~igh an ill-fitting lid. :my chrnmatographic run proceeds the
'.-'O
compositi0:1 of the solvent rnixtur~ bf:,:ui"'.1cs richer in the less volatik solvents and poore1 in the more volatile cmc.'{_ If the atmosphere vvirhin the t anl< ..
is L.
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composition is minimal and does not 1.>:1u~i{' practicai problems. Ii~ however, the system is not totaIIy enclosed, va1_:i:)ur v1ill escape which is particularly rich in the :more volatile solvent; in un:Ji;l to maintain equilibrium some of this more volatile liquid will cvaporntc· froT.i the plate and leave a changed composition of the eluting mixture. This wiH result in abnormal chromatogram development, non--repn:•chicibl.e Rr values and tmi:v,::=:n migration of sample components in diH(::rent areas of the chromatoplate. Edge effects leading particu1ar!y to faskr migration of sample near to the edge of the plate will also be exaggera,.cd if the system is not enclosed. These edge effects can be noticed even ;n properly enclosed systems, but <1re minimized by removing narrow strips of" aosorbent from each edge of the plate, or by scori!1g straight lines in the C:idsc•rbent along the length of plate. about 0.5-1 cm from each side edge. Sarnp!e~ shouJd then be applied within the limits of these lines. In order to assist equilibration in the relatively large tank shown in Fig, 2 the walls of the tank can be lined with shet::ts of filter paper. It is also possible to comple:e i:·quilibration of the system after the plate has been introduced but before the development of the plate is started. Thus if the tank containing the rf~quired amount of solvent is allowed to lean over at an angle :o th;.; \'\:'iti'>•l 1t is possible to introduce the plate witlwut immersing any of the ?d~c,bent layer. After a period of equilibration the tank can be restored tn the verticuJ, the solvent mixture wiil cover the Iov.:er part of tb.e adsorhcnt hyer, and the development wil ! commence. Tanks specially designed fi)r this purpose are availablr' commercially, but with a little ingenu:t_y rn,.:,r.:;c standard thanks can also be adapted. Anothier way of ensuring. that rapid, c, 'Iiiplete and stable equilibration is achieved by making thr: internal volurru:> -.Jf rhe tank as small as possibJe. This can be done by placing a second pL(tc, which rnay be either of plain glass or a second chromatoplat~, on top of the adsorbent layer but separated from it by a thin spacer of air-tight m::lteriai placed in between the plates along th~ir side and top edges. A small gap is left at the !ower edge and this ex.tends internally between the pl .. ::te:~.- The· Iower edge is then immersed into the solvent mixture, which should 2.l.so be enclosed ·within the system. This is the so·calle
21
state must be developed at an angle of not more than 20° to the horizontal. Flexible plates can be bent into a cylindrical shape with the adsorbent on the inner surface, and developed in cylindrical tanks (thereby reversing the historical evolution of TLC which was initially envisaged as a chromatographic column which had been opened out to form a flat sheet). A standard chromatographic run is complete when the solvent front has moved about 1.5 cm above the the origin. A line can be scored across the plate through the adsorbent layer at this sort of distance, prior to development; this will arrest further development of the plate and enable the exact length of the chromatographic run to be standardized. Whether or not this technique is adopted it is important not to leave the plate in the tank for long periods after development, otherwise excessive diffusion of spots and abnormal Rr values will be experienced.
Detection of Components Except under the fortuitous circumstance that all the separated components from each sample are coloured, the next stage in TLC is to detect the positions to which the spots have run. In order to do this it is necessary either to exploit an inherent property of the component, such as its colour, fluorescence or radio-activity, or to subject the whole or part of the plate to a reagent which will react with the components to produce coloured spots. Ideally such a reagent should be easily applied, produce a distinctive colour with each of the components, from which it should be readily removed leaving them chemically unaltered. Needless to say, no reagent available for this purpose fulfills all-these criteria and compromises must be made in each situation. A reagent can be applied to the adsorbent layer during the preparation of the chromatoplates, which procedure has the advantage of producing a homogeneous distribution of reagent through all parts of the adsorbent, or it may be applied after development of the chromatogram by spraying or dipping. Spraying is the more widely used method and is satisfactory for most purposes. However if quantitation of the spots, particularly by scanning densitometry, is to follow, spraying is less satisfactory than dipping because, even in expert hands, reagent will not be sprayed evenly across the whole of the layer. A number of dyeing reagents are available in the form of aerosol sprays; these are the reagents which are most widely applied and ·use of them saves a certain amount of time, but limits the operator's choice of conditions. A more generalized method is to use a spray gun consisting of a pressurized propellant gas container to which is fitted a glass bottle
containing the reagent in suitable ;_;;olution, This method is of more general used and enables the operator to use spray reagents made up to his ov,.;;:1 specifications. This apparatus cannot be used with strongly corrosive reagents st:ch as 50% sulphuric acid, LF which an all-glass spray gun is used and for which th\! pressure reguin:·d [<:, produce the spray is provided by . d er 01(' compresseu rl . . . a cy lm n1trogen or .:ur.. For quantitative estimations h is ttcr to incorporate the reagent in the adsorbent d,iring preparation of t:·k~ pJc,It~s, but if this is nor possible the dye can be applied by dipping the plat1.:, i.nto a solution of the reagent Tn order to dip the plates satisfactorily rt i'.', ncc-,:ssary that the adsorbent lay,~'r is firmlv adherent to the gdass. aluminium. ot r:Iastic support. In order to ascertain the positicn elf' Dpots in conditions when it is not desired to spray the sample spots the:n1s'clVi..::;, 1t it possible to spray only part of the plate, containing the separati01 1 of a known standard mixture, while keeping covered the rest of the plates containing the sample components. The standard "marker'' mixture will direc; :;,ltention to those unstained areas of the plates where the spots are situati~d. This technique is particularly useful in preparative TLC. On all occasions when it is necess:.rry to maintain the chemical integrity of the smnples care shouid be Uken to minimize the possibility c>f oxidation by carrying out the detcci;c:r pr\)cess swiftly in a nitrogen environment. A large number of specific colour reagents have been used in TI,C. Three widely used and ger1era1ly applicab!.e nu::,thods of detection v,rili be discussed here. The first is the rnethoc of charring thf;~ components with concentrated sulphuric acid: \Vater (1 :lv\') This reagent must be used v-.1 ith an all-glass :~pray-gun and great cai.e T reagent chars most organic components yielding spots of vmying shadfs ,yf grey which can be measured semi-quantitatively by den~jtomctry. With :,._:,rm~ substances, sterols, sterol esters, steroids tt.nd bile acids, the sulphuric acid reagent wiH produce a Liehermann··Btffchard reaction yieldir,J: ,:: '- t'.ciety of pink, purple or brown spots, the colour being dmractedstic cf the pmticular compound. A second general.iz.ed colour reagent is iodine vapoc;r. The plate is placed in an enclosed dry tank at the bottom of which some crystals of iodine are scattered. The spots on the plate take i:p the vapourized iodine and turn brmvn; mos:: compounds are not cben;ic"dly affected by this treatment. The metthod has \:vide applicability. The tbit"d general method is the use of a fluore:::rent :reagent, usually a 0.1 ~,,o :'cLu.ior; of 2, 7-dichlorofluorescein in cthanol:wakr (95:5 v/v), or a simihr :.cduti;Jn of Rhodamine G. These are particularly useful in the detection of lipid compounds. Plates sprayed with ..
I
~
these rea.g,::nts are viev-1cd under LiV ligh1 when the spots fluoresce against a ds.rL background. Radioactive spets can b,,, dct,c:·:teO: either by autoradiography or by means of a radio-active scanner.
The methods of identifi .:alion ;::vailabie in TLC are by compm·ison of R1 values and, in a limited nurnber of c,}ses, by the specificity of a colour 1 - wh1c . ·. h. can 1oe carneu -r• . l .d . • · ' · - I r,:action (it,~ on tne p,al.es. '1 neoretlca consF eratlons, '1~; d.J.scussed earlier m this diapi;;;:r, can lead to predictions of r··h7"()·,v,3t,,cn·ai_·)l'•., _,._; .< f i.'1 ,. 11 .". . at;HJ I' I l::. !.L • Jn c:mca J10cnem13try lno'>vever tite range ot cm!1ponents m most 1~,.s :,J(Pl•, tn h,:., 1'~1.,r()1·,p1,,,·r", i,, n<·,,v E,1·r·1·u ,veil ·1n,·1erstood ~"-r11n The past va . .,..,1"'"'· .... .._,, ) _ .. _. . u . ,J ' .• l experience of the laboratu('/ :,:taff supple.memed by information from the literature will enable rea,:,;nablc identification of most compounds to be made. Standard mi>~tures uf known composition should be run alongside unknown samples to as:;.;_;'-.t idcnt.ific;1tion and if there is any doubt samples should :)..:! nm in more du,· ,_me ,...:hrorr:atographic system. Too great a reliance placed upon prc:v: ..-:n.1.s 1,:::'..::7:,3rience may however occasionally lead to error; in identification: th:: cVinicaI biochemist must ah-...,ays be on the lookout fr,r p(,ssib!e anornab::c1. 1
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Quantitation of compor,er::s separated from a mixture can be carried out either on foe plate, or after the spots or streaks containing the component:; have b'.:cn scraped fro1n the plate 3.nd eluted from the adsorbent. 'J1i,;int;t,>t1" rJn 1"·1·, .,.;t1·, 1',J·.···~·-·· " l,p..:;t C"'r·r·,,·,d n11t by US"'· ()f an JIlStrurnen1 \.~!~w..,1.•~Cl.;..:,,.,_x~"-.-.··,.~··.., ..._,: 1specia!ly designed for scanning ci1rnmatcigrnphy spots. A number of such instn'r1•"1t·"', a1·e ::>V'~1·1a·b· , ..., ,~c·"'1n·:,·1r,,e;~ii,_.· ;n,.·lpc·,11'ng s•)me \,,l·1;c'n employ· the ..4 .. · ,, tcc ·11mquc. · w·1th each or" Lese h · more rm f.erao, 1e •• t·1 ymg-spot instruments 1',c{'C·t (('',tilt<: S:\fe ,:,cbif•\!f-'d ·r,~!_; '~('..'•,"
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• • • 1 .o · Cf1Ite rs important m tne Hyn:gspot technique. An even adsorbent background is also advantageous.. !\ "eries of ,,tand:c>rd sam1"1°1PS of 1-i°"" ,~nrcr·,-n1,,·,1·; 'o•'.ing rnr,:•-:u,··;,d ,.,.t.~ l,.,.,_~ ... ,,1·1,r'nki ~.'..._.,.. ......... be P.in on the same plate as the unknov,.rns :cind sca:d·,ed similarly. A stt..ndard curve can then be drawn from which conccntr<'.tiqns of unknown samples can be read. Thest 1nstrurnent8 depend on light r·;~flectcd from the surfoce of the plate: the opacity of the layer prech1des the i_ue of transmitted light. Severa! workers have cl?..imt:d tlrnt ihe area occupied by a spot is proportional to its concentration and ::;evcral methods in clinical biochemistry depend on this relationship There are however some theoretical objections to this view. fvfany fr1-cwrs influence the size of spots, as has been discussed previously: the'..;e dd not include the concentration or the component, except possibly vthen the plat,"; is somewhat ovedoaded. !l is possiible that this rnethod of qmmtita· ion v.,-orks empirically. Perhaps only those parts. of the spot which c:nntzin greater than a minimum concentration of compound \vill be detecli~d ir1 this way, and therefore, to a first approximation, it is pos:,ib!e that die: visible area vvill be roughly proportiona1 to the total con(;tntratiec.. J-fo\o..?:ver, one should not discard empirical r:nethods which produc·~~ rcasorn: c r(:sults, even though they are not backed up by theore!ic,d consideration: : :nov;ding that the accuracy and reproducibility of t:he method has been s;o.tistac:tmily evaluated there ~s no reason why a::1 empirical method should r:<< b:: used with confidenct;, The methods of quantitation of ccn,y-'o:ients after elution from the thin-layer IJiat<~ are let~ion, and limited ordy. t:w . the amount of the subsI.ance which c3n be isolated, by the degree contamination with other components and with the adsorbent. m,.J by l,::ss of the component because of incomplete elution from the adserb,:nt. Fruv!ding that a substance c.:an be eluted comu!ctelv, ' ., in suffficient an::ount and free from contamination there is no reason ,vhy the most sophisticated nL:,.:,ods of identification and quantitation cannot be applied,. 1,.,1'
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,... · · ·\ :· r·r~d ·. ('J 978) l. \Vi 11 iams, D.L .. Nunn, n1,_. !'. ,:. !'\,larks, v. ,L'- S.j.. ,l , Scientific Foundations of Ciinical B-:,:vJ1ern.:.lstry; Volume I; Londoff \ViEi21m Heinemann ~v1edical Books. 2. Randerath, K. (1963), Thin-l,ay:r '-~"hromatography. New York: Acadernic Press. 3. Smith, 1. and Seakins, J.\V. T. (Ed\.\ (1976), Chromatographic and Electophoretic Techniques; '.folurne 1. L., ,:,
.2)
4. Synder, L. R. ( 1975) in Chromatography - A Laboratory Handbook of Chrornatogn.1phic and Ekctrophoretic i\.1ethods, 3 rd edition, (Heftmann, E., Ed.). New York and Loi1don: Van Nostrand R.heinhold. 5. Synder, L R ( 1968), Prind of Adsorption Chronwtography. New York: ~1arcei Dekker. 6. Stock, R. and Rice,, C. B. F. ( l 4). Chromatographic Methods, 3 rd edition. London: Chapman and Hall. 7. Stahl, E. (ed.), (1969), Thin}ayer Chromatography. A Laboratory Handbook, 2nd edition. New Ynrk o.nd B~~rlin:Springer.
