Modes of Development- Forced Flow, Overpressured Layer Chroma

Figure 18 Anticircular chromatogram. (Reproduced with per-mission from Fenimore DC and Davis CM (1981) High perfor-mance thin-layer chromatography. Analytical Chemistry 53: 252A.)

with sulfuric acid containing naphthoresorcinol byspraying or dipping with this reagent and heating at1003C for 5 min. The spots near the origin are sym-metrical and compact but those further away are morecompressed and elongated at right angles to the direc-tion of development. The sample was also separated inthe same chromatographic system, but using lineardevelopment on a 1010 cm plate (Figure 15B).

If the sample is introduced in the mobile-phasestream, then separated bands form concentric ringson the chromatographic plate, as shown in Figure 16.This circular chromatogram demonstrates the separ-ation of lipophilic dyes on a silica gel 60 F254 highperformance TLC pre-coated plate, 1010 cm (E.Merck) with a mobile phase of hexane}chloroform}NH3, 70 : 30; the distance of development (from en-try position of solvent to eluent front)"30 mm ina Camag U-chamber.

In the anticircular mode of development the mobilephase enters around the entire periphery of the adsor-bent layer which is usually formed as a circle byscraping unwanted adsorbent from a square plate.

The samples are applied on an outer circular startingline and development proceeds from the periphery ofthis circle layer to its centre (Figure 13B). This modeof development can be performed with a Camaganticircular U-chamber, shown in Figure 17.

Anticircular chromatography is seldom applied inpractice. An example of a chromatogram obtained bythis mode of development is given in Figure 18. Thespots are compact near the origin and elongated in thedirection of the mobile-phase migration.

Conclusions

Conventional modes of chromatogram developmentare often applied in analytical practice for both quali-tative and quantitative purposes. The most popularamong the modes described is linear development.There are several reasons which contribute to thissituation, including a simple operation procedure andlow cost and time of analysis per sample. These fea-tures will still determine a future use of the modes inthe analytical practice of planar chromatography inspite of increasing interest in the application of auto-mated and forced-Sow development.

See also: II/Chromatography: Thin-Layer (Planar): In-strumentation; Modes of Development: Forced Flow,Over-pressured Layer Chromatography and Centrifugal.Appendix 2 / Essential Guides to Method Developmentin Thin-Layer (Planar) Chromatography.

Further Reading

Geiss F (1987) Fundamentals of Thin-layer Chromatogra-phy (Planar Chromatography). Heidelberg: HuK thig.

Grinberg N (ed.) (1990) Modern Thin-layer Chromato-graphy. New York: Marcel Dekker.

Poole CF and Poole SK (1991) Chromatography Today.Amsterdam: Elsevier.

Sherma J and Fried B (1996) Handbook of Thin-layerChromatography, 2nd edn. New York: Marcel Dekker.

Zlatkis A and Kaiser RE (1977)HPTLC High PerformanceThin Layer Chromatography. Amsterdam: ElsevierScience.

Modes of Development: Forced Flow, Overpressured LayerChromatography and Centrifugal

S. Nyiredy, Research Institute for Medicinal Plants,BudakalaH sz, HungaryCopyright^ 2000 Academic Press

Introduction

Forced-Sow planar chromatographic separation canbe achieved by application of external pressure (over-

pressured layer chromatography }OPLC), an electricReld, or centrifugal force (rotation planar chromato-graphy } RPC). Figure 1 shows schematically thesuperior efRciency of forced-Sow techniques by com-paring their analytical performance with those ofclassical thin-layer chromatography (TLC) and highperformance thin-layer chromatography (HPTLC).Forced-Sow planar chromatography (FFPC) tech-

876 II /CHROMATOGRAPHY: THIN-LAYER (PLANAR) /Modes of Development

Figure 1 Comparison of the efficiency of analytical TLC and HPTLC chromatographic plates when used with capillary action andforced-flow planar chromatography (FFPC). NUS, normal unsaturated chamber; UM, ultramicrochamber; NUS, normal saturatedchamber.

niques enable the advantage of optimum mobilephase velocity to be exploited over almost the wholeseparation distance without loss of resolution. Thiseffect is independent of the type of forced Sow.

Although FFPC can be started with a dry layer, asin classical TLC, the forced-Sow technique also en-ables fully online separation in which the separationcan be started on a stationary phase equilibrated withthe mobile phase, as in high performance liquidchromatography (HPLC). The following FFPC com-binations of the various ofSine and online operatingsteps are feasible:

Fully ofSine process: the principal steps, such assample application, separation, and detection areperformed as separate operations

OfSine sample application and online separationand detection

Online sample application and separation and off-line detection

Fully online process: the principal steps are per-formed as nonseparate operations.

Overpressured LayerChromatography

In addition to capillary action, the force driving sol-vent migration in OPLC is the external pressure.Depending on the desired mobile-phase velocity, op-erating pressures up to 50 bar can currently be used.In OPLC (Figure 2) the vapour phase is completelyeliminated; the chromatographic plate is coveredwith an elastic membrane under external pressure,thus the separation can be performed under control-led conditions. The absence of any vapour space must

II /CHROMATOGRAPHY: THIN-LAYER (PLANAR) /Modes of Development 877

Figure 2 Schematic diagram of online OPLC. 1, Support block; 2, chromatoplate; 3, support plate; 4, spring; 5, casette system forfixing the chromatoplate between two Teflon layers; 6, Teflon layer; 7, Mobile phase inlet; 8, mobile phase outlet; 9, hydraulic system.

be considered in the optimization of the solventsystem, especially in connection with the disturbingzone and multifront effect, which are speciRc featuresof the absence of a vapour phase (see section entitledElimination of Typical Problems With Use ofOPLC).

Principle of Multi-Layer OPLC (ML-OPLC)

OPLC is suitable for the development of severalchromatographic plates simultaneously if the platesare specially prepared. With this multi-layer tech-nique, many samples can be separated during a singlechromatographic run. By connecting chromato-graphic plates in parallel (Figure 3) more HPTLCplates can be developed simultaneously. By circularOPLC, 360 samples of plant extracts can be separ-ated in 150 s. The rapidity and/or efRciency of theOPLC separation of complex samples can be in-creased by use of ML-OPLC, in which the same ordifferent types of stationary phase can be used for thedevelopment of more chromatographic plates.

Principle of Long-Distance OPLC (LD-OPLC)

Long-distance OPLC is a multi-layer developmenttechnique with specially prepared plates. Similar tothe preparation of layers for linear OPLC develop-ment, all four edges of the chromatographic platesmust be impregnated with a polymer suspension. Themovement of the eluent with a linear solvent frontcan be ensured by placing a narrow plastic sheet onthe layer or scraping a narrow channel in the sorbentfor the solvent inlet. Several plates are placed on topof each other to ensure the long running distance.A slit is cut at the end of the Rrst (upper) chromato-

graphic plate to enable the mobile phase to travel toa second layer. Here the migration continues until theopposite end of the second layer, where solvent Sowcan be continued to the next adjacent chromato-graphic plate, or the eluent is led away (Figure 4A) ifmigration is complete. Clearly, on this basis a verylong separation distance can be achieved by connect-ing one plate to another.

Figure 4B shows a typical combination of the sametype of chromatographic plate (homoplates). In thearrangement presented, the upper plate has an eluentinlet channel on one side and a slit on the other sidefor conducting the mobile phase to the next plate.The slit (width approximately 0.1 mm) can beproduced by cutting the layer; this enables readypassage of the mobile phase and individual sampleswithout mixing. The cushion of the OPLC instrumentis applied to the uppermost layer only, and eachplate presses the sorbent layer below. As a conse-quence of this, glass-backed plates can be usedin the lowest position only. The illustrated fully off-line separation is complete when the front (thefront of the Rrst solvent in an eluent solvent mixture)of the mobile phase reaches the end of the lowestplate.

The potential of the connected layers can be in-creased by use of different (hetero) stationary phasesduring a single development; this is shown in Fig-ure 4C, in which the different sorbents are markedwith various shades of gray. The eluatecan, furthermore, be led from the lower plate,similarly to the way in which it was led in. This givesthe possibility of online detection. For this fully on-line operating mode all layers placed between thehighest and lowest plates must have 1 cm cut from the


Figure 3 Schematic diagram of multi-layer OPLC (ML-OPLC).(A) Linear one-directional development; (B) linear two-directionaldevelopment; (C) circular development.

Figure 4 Schematic diagram of long-distance OPLC (LD-OPLC). (A) Principle of the method; (B) fully offline LD-OPLCusing homolayers; (C) fully online LD-OPLC using heterolayers.

length of the plate, to leave a space for mobile phaseoutlet.

Analytical OPLC Separations

In OPLC, the most frequent modes of developmentare linear one- and two-directional (Figure 5A,B).Linear OPLC, however, requires a special chromato-graphic plate sealed along the edge, by impregnation,to prevent the solvent from Sowing off the layer.

The advantage of circular development, in whichthe mobile phase migrates radially from the centre ofthe plate to the periphery, is well known for theseparation of compounds in the lower RF range,

where circular development gives 4}5 times greaterresolution. The separating power of circular develop-ment is better exploited if the samples are spottednear the centre. As the distance between the mobile-phase inlet and sample application increases, the res-olution begins to approach that of linear develop-ment. No preparation of the plate is necessary forofSine circular OPLC (Figure 5C); for online circularOPLC (Figure 5D) a segment-shaped region must beisolated by removing the surrounding adsorbent andimpregnating its edges.


Figure 5 Development modes in analytical OPLC using20 cm20 cm HPTLC chromatographic plates. (A) Linear uni-directional; (B) linear two-directional; (C) circular with 8 cm devel-opment distance; (D) circular with 18 cm development distance,or online circular; (E) anticircular; (F) anticircular with 18 cm de-velopment distance, or online anticircular.

Conventional ofSine anticircular separation (Fig-ure 5E) is rather difRcult to perform because of thelarge perimeter of the mobile-phase inlet (ca. 60 cmfor a 20 cm20 cm plate). Fully ofSine and onlineanticircular separations can, however, be performedover a separation distance of 18 cm, after suitablepreparation of the plate by isolating a segment of thelayer (by scraping) and sealing the isolated segmentwith polymer suspension (Figure 5F).

In linear OPLC the maximum separation distanceis 18 cm for 20 cm20 cm chromatographic plates.In ofSine circular OPLC the maximum separationdistance is 10 cm, and only one sample can be ana-lysed. If the distance between the mobile phase inletand the point of sample application is 2 cm, a separ-ation distance of 8 cm can be achieved; this enablesapplication of more samples.

Micropreparative OPLC Separations

Instrumental methods such as OPLC increase prep-aration time and costs but also signiRcantly improveefRciency. As a rule of thumb, if the sample containsmore than Rve substances, up to 10 mg of sample canbe separated by micropreparative OPLC with lineardevelopment on an HPTLC plate. This can be in-creased Rve-fold by use of Rve HPTLC plates anda multi-layer technique; thus preparative amountscan be separated by means of a micropreparativetechnique. If the sample contains fewer than Rvesubstances, the amounts can be increased to 50 mg ona single chromatographic plate. Linear online OPLCis preferable if the structures of compounds to beseparated are similar. The circular ofSine techniquecan be used if the separation problem is in the lowerRF range.

Probably the most important application of layerswitching is in sample clean-up based on a new con-nection between the layers. A special clean-up effect,sample application and reconcentration, can beachieved simultaneously as shown in Figure 6A, inwhich the upper plate serves for clean-up. Needless tosay, these steps can both be performed in fully ofSineor fully online operating modes, or in freely chosencombinations of different ofSine and online steps.

The connection illustrated in Figure 6B is an ar-rangement suitable for a larger amount of complexsample. In this example micropreparative developmentcan be performed on pre-coated Rne particle-size ana-lytical plates. The mobile-phase inlet system with theslits is analogous to that for multi-layer development.In the example illustrated, the direction of mobile-phase migration is the same for each pair of plates. Thescraped channels are located at the beginning of theupper two layers and the slits are located at the endsof the adsorbent layers. On reaching the end of theRrst pair of plates the mobile phase passes through tothe adjacent pair of layers. Suitable location of chan-nels and slits ensures mobile phase transport throughthe whole system. The collector channel at the end ofthe lowest plate leads the eluate to the outlet.

Preparative OPLC Separations

Whether or not the use of OPLC for preparativeseparation is necessary depends on the kind of sampleto be separated. The potential of linear online OPLCon 20 cm20 cm plates with a separation distance of18 cm as a preparative method is considerable. Be-cause the average particle size of pre-coated prepara-tive plates is too large, not all the advantages of thismethod can yet be realized. Generally, preparativeonline OPLC can be used for separation of 6}8 com-pounds in amounts up to 300 mg.


Figure 6 Micropreparative ML-OPLC separations on analyticalHPTLC plates. (A) Schematic diagram of cleanup procedureusing fully online LD-OPLC; (B) schematic diagram of fully onlineLD-OPLC for a large amount of a complex mixture.

Figure 7 Elimination of typical problems in OPLC. (A) Break-ineffect } a consequence of improper impregnation of thechromatographic plate; (B) meniscus effect } a consequence ofimproper impregnation of the chromatographic plate; (C) lack ofthe appropriate inlet pressure for linear separation.

Elimination of Typical Problems with Use of OPLC

It is of practical importance to summarize the mostimportant distorting effects which arise in OPLC andto describe means of eliminating these problems.

Linear separations require specially preparedchromatographic plates with chamfered edges thatare impregnated with a suitable polymer suspension,to prevent solvent leakage at overpressure. For properpreparation of the chromatographic plate, the surfacefrom which the stationary phase has been scratchedmust be fully cleaned from particles. If this is notachieved, a narrow channel may be formed under the

polymer suspension, resulting in faster migration ofpart of the mobile phase, because of lack of layerresistance; the mobile phase then re-enters furtheralong the plate (break-in effect as shown in Fig-ure 7A). This reduces the value of the separation, atleast at the edge(s) of the layer.

If the area impregnated is too wide, i.e. the edges ofthe stationary phase covered by the polymer suspen-sion are wider than approximately 1 mm, themeniscus effect can occur (see Figure 7B). As a


Figure 8 Multi-front effect } a consequence of the use of multicomponent mobile phases. (A) The fronts occur between thecompounds to be separated; substances migrating with one of the fronts form sharp, compact zones; (B) the compounds to beseparated all migrate behind the lowest front, so the fronts do not influence the separation; (C) diagonal application of the samples (asbands) for linear separations to check the place of the different fronts; (D) eccentric application of the samples (as spots) for circularseparations to check the place of fhe different fronts.

consequence of this effect } which occurs either in theconcave or convex form, depending on the physicalproperties of the solvents used } the eluent Sowsmoreslowly or more quickly on both edges of the chrom-atographic plate, again distorting quantitative results.

Before starting the separation with the optimizedmobile phase, the mobile phase inlet valve is closedand the eluent pump is started to establish an appro-priate solvent pressure. The separation is then startedby opening the inlet valve; this ensures the rapiddistribution of the mobile phase in the inlet channelnecessary for linear migration of the mobile phase. Ifthe inlet pressure is too low and the mobile phasedoes not Rll the inlet channel totally, the start of theseparation is similar to that for circular development;the distorted linear separation obtained is shown inFigure 7C. No preparation of the plate is needed forofSine circular separations.

If multi-component mobile phases are used in un-saturated TLC, the fronts arising from the compo-nents can have a decisive inSuence on the separation.This effect can be substantial in OPLC; thesecondary fronts appear as sharp lines because novapour phase is present. Compounds of the mixturemigrating with one of the fronts form sharp, compact

zones whereas tailing or fronting can be observed forcompounds migrating directly in front of or behindthe front. With multi-component mobile phases themulti-front effect can appear in two forms. In theRrst (Figure 8A), one or more fronts can occur be-tween compounds to be separated. In the second, allthe compounds to be separated migrate behind thelowest front (Figure 8B), and the fronts do not inSu-ence the separation. As the position of the fronts isconstant, if the chromatographic conditions are con-stant, possibly undesirable effects of the multi-fronteffect can be monitored and taken into account byapplying the spots or bands stepwise. Thus for linearseparations the sample is applied at different distan-ces (s"1, 2, ..., n) from the mobile phase inlet chan-nel (Figure 8C). In circular OPLC the samples areapplied at points on concentric circles (or rings) withtheir centres at the mobile phase inlet (Figure 8D).Quantitative evaluation is usually made more difR-cult, but not impossible, by the multi-front effect,because the phantom peaks formed at the fronts canbe measured densitometrically in the substance-freezones at the sides of the chromatographic plates, andthus the values are taken into account. It must bementioned that the multi-front effect also has a


Figure 9 The disturbing zone as a consequence of differentair/gas volume ratios adsorbed by the surface of the stationaryphase and dissolved in the eluent.

positive effect in preparative separations, becausecompounds that migrate with front can be eluted ina very small amount of mobile phase.

If OPLC separation is started with a dry layer,distorted substance zones can sometimes be observedin different RF ranges, depending on the mobile phaseused and the velocity of the mobile phase. This effectappears during the chromatographic process as a zig-zag zone across the width of the plate, perpendicularto the direction of development as a result of thedifferent refractive indices of the solvents in front ofand behind this zone. This phenomenon, termed thedisturbing zone, is depicted in Figure 9. The extentof this phenomenon depends on the interrelationshipbetween gas physically bound to the surface of thesorbent and gas molecules dissolved in the mobilephase. Because modiRcation of the location of thedisturbing zone is possible within a very narrowrange, the only solution to this problem is to conducta prerun. For separation of nonpolar compounds thiscan be performed with hexane; for separation ofpolar substances the prerun can also be performedwith hexane or with any component of the mobilephase in which the components are unable to migrate.The selection of this solvent might be consideredduring optimization of the mobile phase.

Advantages of OPLC

The advantages of the different OPLC methods aresummarized as follows:

1. All commercially available chromatographicplates can be used, irrespective of their size andquality; stationary phases prepared from smallerparticles can be used without loss of resolution asa result of the overpressure.

2. Mobile phases optimized in unsaturated analyti-cal TLC can be transferred after a suitableprerun.

3. Circular development can be performed withoutspecial preparation of the plates; for linear andanticircular development specially preparedplates are necessary.

4. Many samples (up to 72) can be separated rap-idly on a single analytical plate and evaluationcan be performed densitometrically.

5. Multilayer OPLC is applicable for ofSine separ-ation of many (up to 360) samples, again withdensitometric evaluation.

6. The separation time is relatively short and scale-up for preparative work is simple.

7. All linear separation methods (analytical, micro-preparative, preparative) are online; removal ofthe separated compounds by scraping off thelayer is unnecessary.

8. Online determination of a single analyticalsample on Rne particle-size analytical plates, andonline micropreparative and preparative separ-ations can be recorded with a Sow through de-tector.

9. Online preparative separation of 10}500 mgsamples can generally be performed in a singlechromatographic run.

10. The development distance can be easily increasedby use of long-distance OPLC.

11. Combination of different adsorbents can be usedin long-distance OPLC so that each part ofa complex mixture can be separated on a suitablestationary phase.

Rotation Planar Chromatography

The term rotation planar chromatography (RPC) }irrespective of the type and quality of the stationaryphase} embraces analytical, ofSine micropreparativeand online preparative forced-Sow planar chromato-graphic techniques in which the mobile phase mi-grates mainly with the aid of centrifugal force, butalso by capillary action. The centrifugal force drivesthe mobile phase through the sorbent from the centreto the periphery of the plate. The mobile phase velo-city may be varied by adjustment of the speed ofrotation.

The different RPC techniques can be classiRed asnormal chamber RPC (N-RPC), micro chamber RPC(M-RPC), ultramicro chamber RPC (U-RPC) and col-umn RPC (C-RPC); the difference lies in the size ofthe vapour space, an essential criterion in RPC. Foranalytical separations many samples can be applied.For micropreparative and preparative purposes onlyone sample is applied as a circle near the centre ofthe rotating stationary phase. The separations can beperformed either in the ofSine or online mode. In thelatter, the separated compounds are eluted from


Figure 10 Principles of RPC. (A) Fully offline analytical separation; (B) fully offline micropreparative separation; (C) fully onlinepreparative separation.

Figure 11 Schematic diagram of preparative M-RPC. 1"lower part of the stationary chamber, 2"collector, 3"motor shaft withthe rotating disc, 4"glass rotor, 5"stationary phase, 6"quartz glass cover plate, 7"mobile phase inlet, 8"eluent outlet.

the stationary phase by the centrifugal force andcollected in a fraction collector (Figure 10). Allmethods can be used for online preparative separ-ations; M-RPC and U-RPC can also be used for ana-lytical and ofSine micropreparative separations.

Principles of N-RPC, M-RPC and U-RPC

In N-RPC the layer rotates in a stationary chromato-graphic chamber; in M-RPC } which uses a co-rotat-ing chromatographic chamber } the vapour space isreduced and variable; in U-RPC the layer is placed inthe co-rotating chamber fromwhich the vapour spacehas been almost eliminated. A schematic drawing ofpreparative M-RPC is shown in Figure 11; the layerthickness is approximately 2 mm. When the ultra-microchamber is used, the chromatographic layer isthicker (4 mm); the quartz cover plate is placed dir-ectly on the layer so there is almost no vapour space.In N-RPC the quartz plate is removed; this results ina large vapour space.

In all three methods circular development is alwaysused for preparative separations. The sample is ap-plied near the centre of the circular layer, and the

mobile phase is forced through the stationary phasefrom the centre to the outside of the plate (rotor). Theseparated compounds are eluted from the layer bycentrifugal force and collected in a fraction collector.A detector and recorder can be incorporated beforethe collector.

M-RPC and U-RPC can be used not only for onlinepreparative separations, but also for analytical andofSine micropreparative purposes. Excellent resolu-tion is obtained on HPTLC plates.

Principles of S-RPC

For difRcult separation problems a special combina-tion of circular and anticircular development can beperformed with the sequential rotation planarchromatography (S-RPC). The mobile phase can beintroduced onto the plate at any desired place and,time. In S-RPC the solvent application system } a se-quential solvent delivery device } works by centrifu-gal force (circular mode) and with the aid of capillaryaction against the reduced centrifugal force (anticir-cular mode). Generally the circular mode is used forthe separation, the anticircular mode for pushing the


Figure 12 Schematic diagram of preparative C-RPC. 1"lower part of the stationary chamber, 2"collector, 3"motor shaft withthe rotating disc, 4" rotating planar column, 5"stationary phase, 6"quartz glass cover plate, 7" mobile phase inlet, 8"eluentoutlet.

Figure 13 Development modes in analytical RPC on 20 cm20 cm HPTLC plates. (A) Circular; (B) linear; (C) anticircular.

substance zones back to the centre with a strongsolvent (e.g. ethanol). After drying the plate withnitrogen at a high rotation speed, the next develop-ment with another suitable mobile phase can bestarted. By combination of the two modes of opera-tion the separation pathway in S-RPC becomes theor-etically unlimited.

Principles of C-RPC

In column RPC (see Figure 12) there is no vapourspace } the stationary phase is placed in a closed cir-cular chamber (column). The volume of stationaryphase stays constant along the separation distanceand the Sow is accelerated linearly by centrifugalforce, hence the name column RPC. Becausea closed system is used, there is no vapour space andany stationary phase can be used } Rne particle size,with or without binder. The rotating planar columnhas a special geometric design described by eqn [1]

h" K(a#br#cr2) [1]

where r"radius of the planar column, h"actualheight of the planar column at radius r, a, b, c, andK"constants.

This design eliminates the extreme band-broaden-ing which occurs normally in all circular developmenttechniques, and so combines the advantages of bothplanar and column chromatography.

Analytical RPC Separations

In analytical M-RPC there is a vapour space (1 or2 mm) between the chromatographic plate and thequartz glass cover plate. In analytical U-RPC a softcrepe rubber sheet is placed underneath the analyticalplate so that vapour space between the layer and thequartz cover plate is almost eliminated.

In analytical RPC (M-RPC and U-RPC) three de-velopment modes are available and the separationdistance and number of samples depend on whichmode is used. 20 cm20 cm plates can be introduceddirectly into the instrument. In circular mode (Fig-ure 13A) the most commonly used, up to 72 samplescan be applied to an HPTLC plate as spots; theseparation distance is usually 8 cm. Despite the cen-trifugal force, the mobile phase direction of Sow canbe linearized (linear development mode) by scrapingchannels in the layer (Figure 13B); this reduces thenumber of samples. The anticircular mode can also beemployed with special preparation of the analyticalplate (Figure 13C). Although the solvent is delivered


at the centre of the plate, in anticircular mode theamount of stationary phase available during develop-ment is reduced according to a quadratic relationshipby preparation of the layer.

Micropreparative RPC Separations

Amixture of components (5}15 mg) can be applied inthe form of a ring near the centre of a single analyticalHPTLC plate for isolation of relatively pure com-pounds by use of U-RPC or M-RPC. The operatingprocess is similar to that used for analytical separ-ations, with the difference that only one sample isapplied. Continuous development is possible if a ringof radius 9.8 cm is scraped from the stationary phase,to ensure the regular migration of the mobile phaseafter it has reached the outskirts of the plate. Whenthe Rrst compounds of interest reach this ring, theseparationmust be stopped, and either with a station-ary rotor or at a rotation speed of 100 rpm the separ-ated components can be scraped out and then theremaining substances can be eluted by the usual pro-cedures, similar to preparative TLC. The separationof ultraviolet (UV)-active compounds can bemonitored with a UV lamp during the separation.

Preparative RPC Separations

Because all preparative RPC separations are per-formed online and no removal of the zones byscratching is necessary, the separation efRciency forthe last eluting compounds even increases during therun.

Because M-RPC and U-RPC can be used not onlyfor online preparative separations, but also for ana-lytical purposes, direct scale-up is possible for bothanalytical methods. From TLC separations using un-saturated or saturated chromatographic tanks, themobile phase can be transferred via analytical U-RPCand M-RPC to preparative U-RPC and M-RPC, re-spectively, if the solvent strength and selectivity arekept constant. For scale-up the sample may beapplied as a circle to a 20 cm20 cm analytical TLCplate and the amount of sample can be increasedstepwise in subsequent separations. The adverse ef-fects of different particle sizes and separation distan-ces in the analytical and preparative methods almostcancel each other, so only layer thickness has to beconsidered in the scale up procedure. The mobilephase Sow rate must be adapted to preparative separ-ation, so that the migration of the front is as fast asin the analytical separation.

Elimination of Typical Problems in RPC

In RPC extra evaporation of the mobile phase occursowing to the rotation of the chromatographic plate;

this can have undesirable effects. In analytical RPCthese are the surface effect and the effect of thestanding front. The optimum velocity of rotationdepends on the particular separation problem. TheSow rate is limited by the amount of solvent that canbe kept in the layer (layer capacity) without Soatingover the surface. The greater the amount of solventapplied, the higher the rotation speed must be to keepthe mobile phase within the layer. The parameters,rotation speed, perimeter of solvent application anddevelopment mode must be considered when settingthe pumping speed, otherwise the mobile phase Sowsover the top of the applied sample and the layer(surface effect) distorting the separation. A standingfront can occur if after a certain time, the well-separated compounds mix back again because the front becomes stationary owing to the amount ofmobile phase evaporating becoming equal to theamount being delivered. When N-RPC is used forpreparative purposes, the effect of the change ofmobile phase composition is a typical negative effect,which has to be considered during the optimization ofthe mobile phase.

Advantages of RPC

The advantages of the different RPC methods, can besummarized as follows:

1. Depending on the properties of the compounds tobe separated, the effect of the vapour space, andthus the extent of saturation of the chromato-graphic system, can be selected freely.

2. All commercially available stationary phases canbe used, irrespective of their size and quality;smaller particle size stationary phases can be usedwithout loss of resolution because of the centrifu-gal force.

3. Mobile phases optimized in saturated or un-saturated analytical TLC, or in HPLC, can betransferred to the various RPC methods.

4. All three basic modes of development (circular,linear, and anticircular) and their combinationscan be used for analytical separations.

5. For analytical purposes up to 72 samples can beapplied to a single analytical plate, and den-sitometric quantiRcation can be performed in situon the plate.

6. The separation time is relatively short and scalingup to preparative methods is simple.

7. All preparative methods are online, no scratchingout of the separated compounds is necessary, andthe preparative separation can be recorded witha Sow-through detector.

8. Because of the theoretically unlimited separationdistance, the separation power can be increased


Table

1Co

mpa

rison

ofth

edi

ffere

nta

na

lytic

ala

nd

pre

para

tive

FFPC

(OPL

Can

dR

PC)m

etho

ds

Met

hod

OPLC

N-RP

CM

-RP

CU-

RPC

S-RP

CC-

RPC

View

poin

tAn

alyt

ical

Prep

arat

ivePr

epar

ative

Anal

ytica

lPr

epar

ative

Anal

ytica

lPr

epar

ative

Prep

arat

ivePr

epar

ative

Cham

ber

type

Ultra

-mic

roUl

tra-m

icro

Norm

alM

icro

Mic

roUl

tra-m

icro

Ultra

-m

icro

Norm

alPl

anar

colu

mn

Pla

te(co

lum

n)

TLC/

HPT

LCpr

e-co

ate

dPr

e-co

ated

Self-

prep

ared

TLC/

HPT

LCpr

e-co

ate

dSe

lf-pr

epa

red

TLC/

HPTL

Cpr

e-c

oate

dSe

lf-pr

epa

red

Self-

prep

are

dSe

lf-fill

ed

Sta

tiona

ryph

ase

Alla

vaila

ble

Silic

aSi

lica

Alla

vaila

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signiRcantly by employing the sequential tech-nique.

9. On line preparative separation of 50}500 mg sam-ples can generally be applied in a single chromato-graphic run.

Comparison and Outlook ofFFPC Methods

The various OPLC andRPC techniques are comparedin Table 1. Study of the data shows that OPLC is anexcellent technique for analytical separations andthat RPC is more ideally suited as a preparativemethod for isolation of compounds from biologicalmatrices.

The advantage of combining online and ofSineseparations and two-dimensional development canalso be exploited in OPLC. The advantage of multipledevelopment methods is the possibility of analyticalRPC separations. A realistic means of increasing theefRciency of the planar chromatography of complexsamples is the use of long-distance OPLC for analyti-cal separations and sequential RPC for preparativepurposes.Workingwith multi-layer OPLC, the rapid-ity of the separation can increase signiRcantly, pro-viding new vistas in screening and genetic work.

FFPC techniques will open up a new Reld of planarchromatography, particularly in the separation ofcomplex samples. It is expected that future researchwill concentrate on the positive effects (applied pres-sure in OPLC and higher centrifugal force in RPC) offorced Sow. As a consequence, smaller particle size,narrower distribution range, and spherical stationaryphases will be needed to achieve maximum resolution.

See also: II/Chromatography: Thin-Layer (Planar): In-strumentation; Modes of Development: Conventional;Preparative Thin-Layer (Planar) Chromatography; Theory

of Thin-Layer (Planar) Chromatography. Appendix 2 /Essential Guides to Method Development in Thin-Layer (Planar) Chromatography.

Further Reading

Botz L, Nyiredy Sz and Sticher O (1990) The principles oflong distance OPLC, a new multi-layer developmenttechnique. Journal of Planar Chromatography 3:352}354.

Geiss F (1987) Fundamentals of Thin Layer Chromatogra-phy (Planar Chromatography). Heidelberg: HuK thig.

Nurok D, Frost MC, Pritchard CL and Chenoweth DM(1998) The performance of planar chromatographyusing electroosmotic Sow. Journal of Planar Chromato-graphy 11: 244}246.

Nyiredy Sz (1992) Planar chromatography. In: HeftmannE (ed.) Chromatography, 5th edn, pp. A109- 150. Am-sterdam: Elsevier.

Nyiredy Sz and FateH r Zs (1994) The elimination of typicalproblems associated overpressured layer chromatogra-phy. Journal of Planar Chromatography 7: 329}333.

Nyiredy Sz, Botz L and Sticher O (1989) ROTACHROM:A new instrument for rotation planar chromatography(RPC). Journal of Planar Chromatography 2: 53}61.

Nyiredy Sz, Botz L and Sticher O (1990) Analysis andisolation of natural products using the ROTACHROMrotation planar chromatograph. American Biotechnol-ogy Laboratory 8: 9.

Sherma J and Fried B (1995) Handbook of Thin-LayerChromatography. New York: Dekker.

TyihaH k E and Mincsovics E (1988) Forced-Sow planarliquid chromatographic techniques. Journal of PlanarChromatography 1: 6}9.

TyihaH k E, Mincsovics E and KalaH sz H (1979) New planarliquid chromatographic technique: overpressured thin-layer chromatography. Journal of Chromatography174: 75}81.

TyihaH k E, Mincsovics E and SzeH kely TJ (1989) Overpres-sured multi-layer chromatography. Journal of Chromato-graphy 471: 375}387.

Preparative Thin-Layer (Planar) Chromatography

S. Nyiredy, Research Institute for Medicinal Plants,Budakala& sz, HungaryCopyright^ 2000 Academic Press

Introduction

Preparative planar (thin-layer) chromatography(PPC) is a liquid chromatographic technique per-formed with the aim of isolating compounds, inamounts of 10}1000 mg, for structure elucidation

(mass spectrometry (MS), nuclear magnetic reson-ance (NMR), Infrared (IR), ultraviolet (UV) etc.), forvarious other analytical purposes, or for determina-tion of biological activity. PPC is a valuable methodof sample puriRcation for preparative purposes andisolation. The scope for modifying operating para-meters such as the vapour space, development modeand for ofSine sample application is enormous inplanar chromatography.

In classical PPC the mobile phase migrates by capil-lary action, whereas if forced-Sow PPC (FFPPC) is

888 II /CHROMATOGRAPHY: THIN-LAYER (PLANAR) /Preparative TLC

Modes of Development- Forced Flow, Overpressured Layer Chroma

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