PETROLEUM PRODUCTS - Thin-Layer (Planar) Chromatography

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especially suitable for nonvolatile samples. A betterapproach to qualitative and quantitative analysis ofvolatile samples is through methods based on anon-column interface. However, the retention gapmethod uses long uncoated pre-columns and onlyallows modest volumes of HPLC fractions to betransferred. The partially concurrent evaporation

method, where only a part of the HPLC eluentis evaporated, works with larger fraction volumes(approx. 200 L) and with shorter uncoatedprecolumns.

Future Trends

The development of new, more accurate techniquesbased on liquid chromatography (especially HPLC,TLC, and new multidimensional or hyphenated tech-niques) will be increasingly important owing to legis-lation calling for the reduction of aromatic content infuels. Therefore, these techniques will continue toplay a crucial role in the petroleum industry for thechoice of process conditions and the evaluation offuel quality.

See also: II/Chromatography: Liquid Chromatography-

Gas Chromatography. Chromatography: Gas: High

Temperature Gas Chromatography; High-Speed Gas

Chromatography. Chromatography: Liquid: Detectors:

Ultraviolet and Visible Detection; Large-Scale Liquid

Chromatography; Mechanisms: Normal Phases; Mecha-

nisms: Size Exclusion Chromatography; Multidimensional

Chromatography. III/ Bitumens: Liquid Chromatogra-phy. Crude Oil: Liquid Chromatography. Flame Ioniz-

ation Detection: Thin-Layer (Planar) Chromatography.

Flash Chromatography. Geochemical Analysis: Gas

Chromatography. Liquid Chromatography-Gas Chro-

matography. Medium-Pressure Liquid Chromatogra-

phy. Petroleum Products: Gas Chromatography.

Further Reading

Adlard ER (ed.) (1995) Chromatography in the Petroleum

Industry. Amsterdam: Elsevier.Annual Book of ASTM Standards, vols 05.01 and 05.02.Petroleum Products, Lubricants and Fossil Fuels.ASTM, Philadelphia PA 19103-1187, USA (issued an-nually).

Cagniant D (ed.) (1992) Complexation Chromato-graphy. New York: Marcel Dekker.

CorteH s HJ (ed.) (1990) Multidimensional Chromato-graphy. New York: Marcel Dekker.

Drews AW (ed.) (1989) Manual on Hydrocarbon Analysis,4th edn. Philadelphia: American Society for Testing ofMaterials.

Herod AA (1994) A review of the uses of planar

chromatography in the coal and oil industries. Journalof Planar Chromatography 7: 180}196.Kelly GW and Bartle KD (1994) The use of com-

bined LC}GC for the analysis of fuel products: a review.Journal of High Resolution Chromatography 17:390}397.

Pauls RE (1995) Chromatographic characterization ofgasolines. Advances in Chromatography 35: 259.

Ranny M (1987) Thin-layer Chromatography with FlameIonization Detection. Dordrecht: D. Reidel.

Standard Methods for Analysis and Testing of Petroleumand Related Products 1992, vol. 2. Institute of Petro-leum, London, 1992.

Yau WW, Kirkland JJ and Bly DD (1979) ModernSize-Exclusion Liquid Chromatography. Practice ofGel Permeation and Gel Filtration Chromatography.New York: John Wiley.

Thin-Layer (Planar) Chromatography

A. A. Herod and M.-J. Lazaro,

Imperial College, London, UK

Copyright^ 2000 Academic Press

Introduction

Thin-layer chromatography (TLC) has strengths notapplicable to other chromatographic methods. Thesecan be summarized as cheapness of materials, lowvolume requirement for solvents, the ability to useany mixture of solvents, and, most important, the

intractable materials of a complex sample are re-

tained within the surface area of the chromatographicplate and may be recovered. In addition, chromato-graphic failures can be disposed of without damagingthe budget. Such considerations do not apply to gaschromatography or liquid chromatography, whereinvolatiles (in GC) or insolubles (in LC) are lost onthe column or inlet system with the possibility ofpermanent damage to the column performance. Thedifference in cost of thin-layer plates and chromato-graphic columns ranges from a factor of 100 for GCcapillary columns, up to a factor of 1000 for prepara-

tive HPLC columns. Whereas TLC has most often

3690 III/PETROLEUM PRODUCTS/Thin-Layer (Planar) Chromatography


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Figure 1 (See Colour Plate 109) Analytical development of coal tar pitch, tetrahydrofuran solubles, on silica developed in tetrahyd-

rofuran, chloroform/methanol(4 : 1 v/v), toluene andpentane. Pitch at lanes 3, 5 and 7; peryleneat lane 2; rubrene at lane 9; pyrogallol

at lane 6; coal extract at lane 1. Whatman K6 silica; solvent fronts THF 55 mm, chloroform /methanol 83 mm, toluene 153 mm and

pentane 178 mm.

been used to examine individual compounds, it hasfound use (as TLC with Same ionization detectionusing silica rods) in the examination of crude oils andmore recently as a preparative method to separate thehigh molecular mass fractions of coal and biomassliquids and petroleum vacuum residues for examina-tion free from the smaller, more volatile components.

Uses of Thin-Layer Chromatography

Thin-layer chromatography has been applied to tarsand oils in three ways: (1) to identify individual com-pounds or groups of compounds such as polycyclicaromatic hydrocarbons (PAHs); (2) to measure typesof compounds such as oils, maltenes, asphaltenes andpreasphaltenes in crudes by the TLC-FID method;and (3) as a fractionation method for examination byother techniques such as NMR or laser ionizationmass spectrometry. These uses are considered in moredetail below.

Identi\cation of Individual Compounds or Groups

of Compounds

Analytical TLC of coal tar has been achieved usingsilica gel plates and development by a manual methodusing the series of solvents } tetrahydrofuran, chloro-form/methanol (4 : 1 v/v), toluene and pentane. The

separated components have been recovered andexamined by probe mass spectrometry directly withno prior extraction from the silica. A typical analyti-cal separation is shown in Figure 1. The standardcompounds on the plate included pyrogallol, peryleneand rubrene, with a coal extract produced in a bombwith tetralin solvent. Mass spectra of recovered frac-tions are shown below.

Measurement of Compound Types

The separation and quantiRcation of compound types} oils, maltenes, asphaltenes and preasphaltenes(terms derived from solvent solubility fractionation)

III/PETROLEUM PRODUCTS/Thin-Layer (Planar) Chromatography 3691


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Figure 2 TLC-FID chromatogramscorresponding to the follow-

ing development sequences: (A) 38 min n-hexane, 3 min toluene,

30 s dichloromethane (DCM)-MeOH; (B) 38 min n-hexane,

20 min toluene, 5 min DCM-MeOH; (C) 45 min n-hexane, 20 min

toluene, 3 min DCM-MeOH; (D) 35 min toluene, 3 min DCM-

MeOH. Numbers correspond to area percentages. (TLC-FID re-

produced with permission from Cebolla VL, Vela J, Membrado

L and Ferrando AC (1996) Chromatographia42(5/6) March 1996,

^ Friedr. Vieweg & Sohn Verlagsgesellschaft mbH.)

} in oils and coal derived liquids may be achievedusing the combination of TLC on rods with Sameionization detection (FID). The normal equipmentused for this is the Iatroscan Chromatograph withChromarods to effect the separation. Rods arecleaned and prepared by passage through a hydro-gen/air Same, sample is deposited near one end of the

rod and development proceeds as for a plate, using anappropriate solvent sequence. The developed anddried rod is passed through the Same detector toevaluate the separated fractions; calibration of theresponse factors of the different types of fraction isessential to give quantitative information. The sampleis destroyed by the detection method but, as onlysmall quantities of sample are used, several develop-ments can be made at the same time to allow repeata-bility checks. Figure 2 shows details of differentdevelopment sequences to obtain adequate separationof a coal tar pitch. The initial sequence was notsatisfactory owing to the aromatic character of thepitch. The Rnal sequence indicated that the hexanedevelopment had no effect on the separation of thepitch, since hexane would separate aliphatics, whichare absent from the pitch.

Separation of Fractions for Examination

by Other Techniques

In this mode, TLC has opened a route to the isolationof fractions of coal-derived liquids and tars frombiomass as well as petroleum vacuum residues. Theessence of the method is the application of sampleeither in solution or as a suspension or slurry ina volatile solvent. Pyridine has been used for slurryingor dissolving all of these sample types; 1-methyl 2-pyrrolidone (NMP) is capable of dissolving coal andbiomass tars, but is involatile (boiling point 2023C)and cannot be removed easily from the plate. Afteraddition to the longer edge of a plate as spots or asa band along the bottom of the plate, usually10 cm;20 cm coated with silica gel, the pyridine isallowed to evaporate. Figure 3 shows a typical prep-

arative development of a synthetic naphthalenemesophase pitch using tetrahydrofuran and toluene,with application of sample in a pyridine slurry. Afterseparation the colours of the bands were black (im-mobile), brown (mobile in tetrahydrofuran but im-mobile in toluene) and orange (mobile in bothsolvents).

Development solvents used include pyridine fol-lowed by acetonitrile, pyridine followed by N,N-dimethylformamide and tetrahydrofuran followed bytoluene. In each case, theRrst solvent used is the morepolar of the two and development is not more than

half way up the plate, less than 5 cm. After drying, the

plate is developed in the second solvent to a distanceapproximately twice that of the Rrst, but less than10 cm. In some cases, the development has beenachieved using a manual multiple development tech-nique in which the plate is removed from the tankafter the solvent front has passed the sample applica-tion zone (or the solvent front of the previous sol-vent), dried and reinserted into the same solvent tocontinue the development. By this method, the possi-bility of the mobile material being partially retainedby the immobile fraction is reduced.

To avoid contamination of the tar fractions, the

plates are usually washed before use with the Rrst



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Figure 3 (See Colour Plate 110) Preparative development of a synthetic naphthalene mesophase pitch applied in pyridine slurry and

developed in tetrahydrofuran and toluene.

solvent to be used in the development, either pyridineor tetrahydrofuran. This is achieved by placing thefresh plate into a development tank containing thesolvent and allowing the solvent front to rise almostto the top of the plate. In the subsequent preparativeor analytical development, the Rnal solvent front isnot allowed to reach the washing-solvent front and,in consequence, the height of the plate used for theseparation is probably not more than two-thirds ofthe available plate height, with allowance for thesample application zone being above the initial levelof the solvent when Rrst placed into the developmenttank.

As indicated earlier, after development three zonesare normally visible (black, dark brown and orangeor yellow. These zones are recovered by scraping thecoated silica into a glass vial and extracting withNMP at room temperature, with ultrasonic agitationif necessary. The extract may be recovered either bydecanting the solvent from the silica, using a syringe,or by adding the slurry to a glass syringe equippedwith a Rlter tip (0.6 m). In this case, the physicalpressure necessary to force the solvent through theR

lter may cause theR

lter to be blown off the syringe,with loss of sample. The complete removal of black,low mobility material from the silica is difRcult toachieve and the residual silica may be dark inappearance; however, the recovered material isunlike the other fractions in molecular mass andspectroscopic behaviour (see below). The solu-tions derived by solvent extraction may be concen-trated by vacuum evaporation or used as recovered.NMP is a difRcult solvent to remove completely,but water washing of the almost dried fractionmay achieve removal since NMP is very soluble in

water.

Instrumentation

For TLC on plates as described here, the equipmentneeded is minimal: simple development tanks linedwith absorbent paper to produce an atmosphere inthe closed vessel that is in vapour equilibrium withthe solvent pool in the tank, to reduce evaporationfrom the advancing solvent front on the plate. Theplates themselves may be used as commerciallysupplied, requiring only a solvent wash to removeimpurities from the area of the plate to be used forthe separation.

More complex automated chromatographic devel-opment instruments may be used but the simple sep-aration of tars into three fractions can be achievedwithout them.

TLC-FID requires specialist equipment since thechromatographic rods require passage through thedetector Same by a controlled mechanical technique.The Iatroscan Chromatograph with Chromarod silicarods has been developed for this analysis.

Analyses by Other Methods

Material from analytical spots or bulk fractions re-covered from thin layer plates may be analysed bya variety of analytical techniques. In particular, massspectrometric methods can be applied since they re-quire very little sample. Also, several different massspectrometric techniques can be applied to either onerecovered spot or a bulk fraction. In this section, theanalysis of fractions obtained by preparative TLC byprobe mass spectrometry, matrix-assisted laser de-sorption mass spectrometry (MALDI-MS), size exclu-sion chromatography (SEC) and UV-Suorescence

spectroscopy (UV-F) are discussed.



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Figure 4 Probe mass spectra of TLC spots from an analytical development of coal tar pitch. Plots are normalized intensity versus

mass number (m/z). (A) Fraction 2, aromatics; (B) fraction 5, neutral nitrogen heterocyclic aromatics; and (C) fraction 9, basic nitrogen

heterocyclic aromatics. (Mass spectra reproduced from Herod AA and Kandiyoti R (1995) Fractionation by planar chromatography of

a coal tar pitch for characterisation by size-exclusionchromatography,UV fluorescence and direct-probe mass spectrometry. Journal of

Chromatography A 708: 143}160, ^ 1995, with kind permission of Elsevier Science NL.



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Figure 5 MALDI-mass spectra of coal tar pitch fractions from

development in pyridine/acetonitrile. (A) Immobile fraction; (B)fraction mobile in pyridine only; (C) fraction mobile in pyridine and

acetonitrile. (Reproduced with permission from Herod AA et al.

(1996) Matrix-assisted laser desorption/ionization mass spectro-

metry of pitch fractions separated by planar chromatography.

Rapid Communications in Mass Spectrometry 10: 171}177,

^ John Wiley & Sons Ltd.)

Figure 6 SEC profiles at 350 nm UV absorbance of pitch frac-

tions. (A) Fractions A immobile in pyridine, B mobile in pyridine

and C mobile in pyridine and acetonitrile; (B) fractions D immobile

in pyridine, E mobile in pyridine, F mobile in pyridine and partly

mobile in dimethylformamide and F mobile in pyridine and

dimethylformamide; (C) fractions G immobile in tetrahydrofuran,

H mobile in tetrahydrofuran and I mobile in tetrahydrofuran and

toluene. (Reproduced with permission from Herod AA et al.

(1996) Matrix-assisted laser desorption/ionization mass spectro-

metry of pitch fractions separated by planar chromatography.

Rapid Communications in Mass Spectrometry 10: 171}177,

^ John Wiley & Sons Ltd.)

Probe Mass Spectrometry (Probe-MS)

This method can be used without extraction of thefraction from the silica. The range of molecular mass

achieved depends on the volatility of sample in vac-

uum and the probe temperature; for fractions of pitchan upper mass of around m/z 600 is possible. Theexamination of spectra of spots in combination withRF values of standards can permit the identiRcation ofcompound types } aromatics, pyridinic and pyrrolicnitrogen heterocyclic aromatics. The absence ofsignal for the material left at the origin indicates that



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Figure 7 SEC profiles of pitch in NMP solvent of (A) fractions A, B and C from TLC in pyridine and dimethylformamide and (B)

fractions D, E and F from TLC in tetrahydrofuran and toluene; detection by UV fluorescence with excitation at 320 nm and emission at

380 nm (fractions C and F) or 480 nm (fractions A, B, D and E). Fractions A and D are immobile; fractions B and E are partly mobile;

fractionsC andF are very mobile. (Reproducedwith permission from HerodAA and KandiyotiR (1996) Fractionationof coal tar pitch by

planar chromatography for the characterisation of large molecular mass materials. Journal of Planar Chromatography 9: 16}24,

^ Research Institute for Medicinal Plants, H-2011, Budakalasz, Hungary.)

it contains large, involatile molecules rather thanaggregates of small polar molecules.

Figure 4 shows mass spectra for some spots re-covered from an analytical separation of a coal tar,following a separation similar to that shown inFigure 1. Fractions 1 (mobile in pentane) and 2}4(mobile in toluene close to toluene front) gave mo-lecular ions for polynuclear aromatic hydrocarbonsranging from Suorene (m/z 166) to m/z 482, corre-sponding to a dimethyl tetrabenzobinaphthyl type.Fractions 5, 6 and 7 correspond to the range ofmobility between aromatics in the toluene zone andthe chloroform/methanol solvent front and showevidence of the presence of nitrogen-containing het-

erocyclics. Fractions 8}

11 were taken from material

mobile in chloroform/methanol but not mobile intoluene and correspond to basic nitrogen heterocyclic

aromatics. The probe mass spectra of thin-layer frac-tions have allowed the identiRcation of isomer classesrather than individual isomers but have extended themass range of identiRed nitrogen PAH to nearly m/z500. The identiRcation of neutral and basic nitrogencomponents can be achieved during one rapid, simpleand inexpensive separation with the use of standardsto deRne the separation. Also, interference from the13C isotope peak of the more abundant polycyclicaromatics which have molecular masses one unit lessthan the nitrogen heterocyclics, is avoided, as op-posed to the situation in GC-MS where both types

elute together.



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Figure 8 SEC profiles in NMP of TLC fractions of Point of Ayr

liquefaction pilot plant coal digest. Fractions A immobile in py-

ridine, B mobile in pyridine, C mobile in pyridine and acetonitrile;

UV absorbance detection at 350 nm. (Reproduced with per-

mission from Herod AJ et al. (1996) Planar chromatography as

a method of fractionation of a coal liquefaction extract for

MoK

ssbauer spectroscopy. Journal of Planar Chromatography9: 361}367, ^ Research Institute for Medicinal Plants, H-2011,

Budakalasz, Hungary.)

Figure 9 SEC profiles of Stockholm tar (a commercial pine-

wood tar) fractions. (A) A immobile in pyridine, B mobile in py-

ridine and C mobile in pyridine and acetonitrile; (B) D immobile in

tetrahydrofuran, E mobile in tetrahydrofuran and F mobile in

tetrahydrofuran and toluene. UV absorbance at 300 nm. (Repro-

duced from the work of Lazaro MJ, Domin M, Herod AA and

Kandiyoti R (1999) Fractionation of a wood for pitch by planar

chromatography for the characterisation of large molecular mass

materials. Journal of Chromatography A 840: 107}115; not pre-

viously shown in this form.)

Matrix Assisted Laser Desorption Mass

Spectrometry (MALDI-MS)

The application of MALDI-MS to coal-derived frac-tions is at an early stage. Much of the published workcorresponds to laser ablation MS, where the fractionsare examined with no added matrix but with thesmall molecules of the sample itself acting as thematrix. In the absence of small molecules (indicatedby SEC) to form an effective matrix for kerogenextracts, no signiRcant mass spectrum can be gener-ated for the large molecules; addition of suitablematrix materials allows the generation of massspectra, however. The upper limits observed for coal-derived materials by MALDI are in excess of100 000 u but so far, it has not proved possible togenerate mass spectra from the TLC-immobile frac-tions containing the largest molecules. Similarly, thetechniques and matrix materials used to generatespectra for coal-derived fractions have not proved

successful with immobile fractions from either bio-mass tars or petroleum vacuum residues.One essential requirement for producing good

mass spectra is the reduction of polydispersity (ratioof mass average to number average molecular mass)of fractions. Figure 5 shows the MALDI-mass spectraof coal tar pitch fractions from development in py-ridine/acetonitrile: (A) immobile fraction; (B) fractionmobile in pyridine only; and (C) fraction mobile inpyridine and acetonitrile. Increasing the mobilityleads to shifts of molecular masses to smaller valuesand to changes in the shapes of spectra; the spectra

become narrower and sharper with increasing mobil-

ity. Comparing relative intensity scales, intensities ofimmobile fraction spectra were only 1}4%, whereasthe spectra of mobile fractions gave signal at full scale(100%). Smaller-mass molecules appear to be prefer-entially ionized and desorbed, thus skewing themolecular ion distribution in favour of the smallermolecules.

Size Exclusion Chromatography (SEC)

Size exclusion chromatography has been used exten-sively for the examination of oils and tars from coal,biomass and petroleum. Until recently, tetrahydro-furan (THF) was the solvent most used, for examplefor asphalts for road tars. In work with coal tar, wehave shown that the use of THF gives erroneousresults since the high-mass portion of the tar is lost tothe guard column, which gradually blocks. It can be



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Figure 12 UV-fluorescence synchronous spectra of coal tar

pitch fractions. (A) A immobile in pyridine, B mobile in pyridine,

C mobile in pyridine and acetonitrile; (B) D immobile in pyridine,

E mobile in pyridine, immobile in dimethylformamide, F partly

mobile in dimethylformamideand F mobile in dimethylformamide;

(C) G immobile in tetrahydrofuran, H mobile in tetrahydrofuran,

immobile in toluene and I, mobile in toluene. (Reproduced with

permission from Herod AA et al. (1996) Matrix-assisted laser

desorption/ionization mass spectrometry of pitch fractions separ-

ated by planar chromatography. Rapid Communications in Mass

Spectrometry 10: 171}

177, ^ John Wiley & Sons Ltd.

Figure 13 UV-fluorescence synchronousspectra of Stockholm

tar fractions. (A) B mobile in pyridine and C mobile in pyridine and

acetonitrile, the immobile fraction A did not show fluorescence;

(B) E mobile in THF and F mobile in THF and toluene, the

immobilefraction D did not show fluorescence.(Reproduced from

the work of Lazaro MJ, Domin M, Herod AA and KandiyotiR (1999) Fractionation of a wood for pitch by planar chromatogra-

phy for the characterisation of large molecular mass material.

Journal of Chromatography A 840: 107}115; not previously

shown in this form.)

mass or size. In SEC, the sequence of solvent-derivedfractions shows a trend towards increasing molecularsize from oil to preasphaltene. The combination ofevidence from SEC, TLC, solvent solubility andMALDI-MS indicates that the immobile fractions docontain the largest molecular masses present in thetars from different sources. The SEC proRle of the

whole pitch shows a relatively smaller peak of ex-cluded material compared with the immobile frac-tions. This points to the masking effect of the greaterconcentration of smaller masses and more mobilematerial, and emphasizes the utility of the planar

chromatographic separation.Calibration of the column separation is normallyachieved using polymer standards; in this work poly-styrene standards have been used. The calibrationthen appears to apply to polycyclic aromatic hydro-carbons and their N, S and O derivatives up to massesof approximately 1000 u. At higher masses, twoproblems apply: (1) there are no standard PAH avail-able; and (2) the structures of the tar molecules aretotally unknown. Calibration curves based on poly-styrenes up to relative mass 1.84 million are shown inFigure 11 for two columns. The linear regions from

low mass (A) 20 000 u or (B) 200 00 u correspond to



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Figure 14 UV-fluorescence synchronous spectra of petroleum

residues. (A) Sample 1 and (B) sample 2 (A immobile in pyridine,

B immobile in acetonitrile and C mobile in pyridine and acetonit-

rile).(Reproduced from Deelchand J-P, NaqviZ, DubauC, Shear-

man J, Lazaro MJ, Herod AA, Read H and Kandiyoti R (1999)

Planar chromatographic separation of petroleum residues and

coal-derived liquids. Journal of Chromatography A 830: 397}414;

Copyright Elsevier Science.

the working region in which solute moleculespenetrate the pores of the column packing andseparate by size. The linear relation for largerpolystyrenes at shorter elution times may correspondto separation in the space between the particles ofthe packing and molecules eluting in this regionare described as excluded from the porosity; the dis-

continuity is described as the exclusion limit of thecolumn.

UV-Fluorescence Spectroscopy (UV-F)

UV-Suorescence spectroscopy does not measure mo-lecular size directly, but shifts of Suorescence max-imum intensity to longer wavelengths indicate anincreasing aromatic cluster size since the Suorescenceoriginates from the largest aromatic system withina molecule, fed by energy absorbed by the smallerpendant aromatic groups. Such shifts to longerwavelengths point to increasing molecular size. De-creasing Suorescence quantum yields are a conse-quence of increased molecular size and complexitysince there are more pathways for the electronic exci-

tation to progress to vibrational and thermal energyrather than being lost as Suorescence.

UV-F spectra of coal- and biomass-derived liquidsand petroleum residues are shown in Figures 12+14.In coal-derived tars, the Suorescence intensity of thematerials showing reduced mobility in TLC (and be-ing largely excluded from the SEC porosity) decreases

and the maximum shifts towards red wavelengths,indicating that these fractions contain large mole-cules. With biomass tars, immobile fractions do notshow any Suorescence at all, indicating the presenceof very high molecular mass material.

The fractions mobile in both solvents showed rela-tively strong Suorescence intensities, the position ofthe peaks at lower wavelengths suggesting the pres-ence of relatively smaller polynuclear aromatic ringsystems and probably also the presence of lower mo-lecular mass material. Similarly fractions mobile inone solvent gave less intense Suorescence than frac-tions mobile in both solvents. TLC fractions of petro-leum vacuum residues show no similar shift to redwavelengths with increasing immobility, or markedlyreduced quantum yield but tend to cover the samerange of wavelengths with shifts of intensity of peakswithin that range. However, SEC of the immobilefractions indicates that the lack of mobility resultsfrom molecular size.

Conclusions

Several examples of the fractionation by TLC ofcoal- and biomass-derived liquids and petroleum resi-dues have been shown. TLC improves the isolationand characterization of large molecular mass frac-tions in oils and tars for examination by other tech-niques such as probe mass spectrometry, MALDI-MS, SEC and UV-F. The separation is relatively rapidand inexpensive and requires only small volumes ofsolvents. The fractionation has led to structural in-formation not readily available by direct character-ization of the original mixture. Molecular-massdistributions, determined by SEC and MALDI,

increase with decreasing mobility of the fractionsin thin layer chromatography. UV-F spectroscopyhas distinguished structural features by showing thepresence of large polycyclic aromatic systems thatincrease in proportion to decrease in mobility offractions. Detailed structures of the largest moleculesremain unknown. Probe mass spectra have allowedthe identiRcation of isomer class and extendedthe mass range of identiRed nitrogen PAH to nearlym/z 500, allowing the identiRcation of neutraland basic nitrogen types as well as the major compo-nents through one rapid, simple and inexpensive

separation.



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See Colour Plates 109, 110.

See also: II/Chromatography: Liquid: Mechanisms: Size

Exclusion Chromatography. III/Bitumens: Liquid Chrom-

atography. Crude Oil: Liquid Chromatography. Flame

Ionization Detection: Thin-Layer (Planar) Chromato-graphy. Geochemical Analysis: Gas Chromatography.

Polycyclic Aromatic Hydrocarbons: Gas Chromatogra-

phy; Solid-Phase Extraction; Supercritical Fluid Chrom-atography; Thin-Layer (Planar) Chromatography.

Further Reading

Davison RR, Glover CJ, Burr BL and Bullin JA (1995) SECof asphalts. In: Chi-san Wu (ed.) Handbook of SizeExclusion Chromatography, ch. 8, pp. 211}247. NewYork: Marcel Dekker.

Herod AA (1994) A review of the uses of planarchromatography in the coal and oil industries. Journalof Planar Chromatography 7(3): 180}196.

Herod AA (1998) Azaarenes and thiaarenes. In: NeilsonAH (ed.) The Handbook of Environmental Chemistry,vol 3. I: PAHS and Related Compounds, Chemistry,ch. 7, p. 271. Berlin: Springer-Verlag.

Jork H, Funk W, Fischer W and Wimmer H (1990) ThinLayer Chromatography: Reagents and Detectionmethods, vol. 1a. Weinheim, Germany: VCH Verlags-

gesellschaft.Lakowicz JR (1983) Principles of Fluorescence Spectro-scopy. New York: Plenum Press.

Somsen GW, Morden W and Wilson ID (1995) Planarchromatography coupled with spectroscopic tech-niques, a review. Journal of Chromatography A 703:613}665.

Touchstone JC (1992) Practice of Thin Layer Chromato-graphy, 3rd edn. New York: John Wiley & Sons.

Wolfbeis OS (1993) Fluorescence Spectroscopy } NewMethods and Applications. Berlin: Springer-Verlag.

PHARMACEUTICALS

Basic Drugs: LiquidChromatography

B. Law, AstraZeneca Pharmaceuticals, Mereside,

Alderley Park, Macclesfield, UK

Copyright^ 2000 Academic Press

Introduction

High performance liquid chromatography (HPLC) isthe most important technique for the separation,analysis and quantiRcation of a wide range of drugtypes. Although there are a variety of approachesavailable for the chromatography of basic drugs,analysis of these compounds is still one of the

main challenges for the practising chromatographerin the pharmaceutical industry. The general ap-proaches have remained the same since the early daysof HPLC, but there have been many reRnements anddevelopments since the late 1960s. In the mainthese have involved modiRcation and improvementsto the stationary phase, which are still continuingtoday.

This article focusses on the main methods of separ-ation and analysis of basic drugs that are currently inuse. Consideration is given to the relative pros andcons of the different approaches, as well as the devel-

opment and evolution of the techniques.

Liquid}Solid Chromatography

Liquid}solid, or normal-phase chromatography(LSC) was one of the Rrst approaches employed forthe separation of bases in modern LC. Its use, how-ever, has decreased dramatically since the 1970s andit is now rarely employed for the routine separation ofbasic drug molecules.

LSC was originally carried out using native silica oralumina, with the former being preferred for theseparation of bases. Recently, there has been a grad-ual shift towards the use of polar bonded phases suchas cyanopropyl, amino or diol, the last two showingpreferential retention of bases compared with cy-anopropyl. These bonded materials overcome someof the problems associated with silica phases such asdeactivation by water and long equilibration times.

The problem of deactivation is particularly acute inthe area of bioanalysis, where it can be difRcult toobtain extracts that are totally dry. To a certaindegree this problem can be overcome by the inclusionin the eluent of a small amount (1% v/v) of water ora short-chain alcohol.

Eluents for LSC typically consist of mixtures ofa nonpolar hydrocarbon, such as hexane or iso-octane, and a polar modiRer, e.g. dichloromethane,2-propanol, methyl t-butyl ether or ethyl acetate.Frequently, the addition of an amine modiRer such astriethylamine may be necessary to give satisfactory

peak shapes.

III/PHARMACEUTICALS/Basic Drugs: Liquid Chromatography 3701

PETROLEUM PRODUCTS - Thin-Layer (Planar) Chromatography

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