HPLC and Column Liquid Chromatography

8/12/2019 HPLC and Column Liquid Chromatography

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E.R. Adlard Fd .) , Chromatography in the Petroleum IndustryJournal of Chromatography Library Series, Vol. 56

1995 Elsevier Science B.V. All rib eserved 347

CHAPTER 12

HPLC and column liquid chromatographyA.C. Neal

Esso Research Centre, Milton Hill, Abingdon, Oxfordshire OX13 6AE, UK

12 1 INTRODUCTIONThe development of high performance liquid chromatography (HPLC) waspredicted in 1941 by Martin and Synge [l] In addition to pioneering liquid-liquid chromatography and the theoretical plate model of chrom atography, these

authors predicted that HPLC would be achieved by using very small particlesand a high pressure difference across the column. In fact, the origins of suchcolum ns can be traced back to the work of T swett in 1903 [2] and their use wasfurther extended by Kuhn and Lederer in 1931 [3].The advance of gas chromatography (GC) in the petroleum industry in the1950s was such that liquid chromatography was effectively overlooked through-out that decade. This rapid exploration and application of GC rekindled interestin liquid chromatography as a complementary technique which could open upregions of solute polarity, molecular weight and bulk separation alien to GC.Commercialization of HPLC columns, pumps and detectors during the 1960s andearly 1970s simplified operation of the technique and allowed potential users toapply it with relative ease. During this time, various terms were used to describethe new technique: modern liquid chromatography, high pressure liquidchromatography and high performance liquid chromatography and the latteris now universally used although it is not easy to define exactly what is meant byhigh performance. A comprehensive introduction to HPLC can be found inSnyder and Kirkland [4] although this excellent book is now almost 20 years old.More recently, Parris [5] and G ilbert [6] have written good general reviews onthe subject and the reader is referred to reference [4] for detailed and fundamen-tal information and the latter two books for general theory and instrumental de-scriptions.Referencesp p . 372-374


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348 Chapter 12The applications of HPLC in petroleum analysis has itself been reviewed byAmos in 1979 [7]. At that time, the number o f specific applications in the indus-

try were few and the major uses were in hydrocarbon type analysis and the de-termination of hindered phenol antioxidants in fuels, most notably aviation tur-bine fuel. By com parison with paper chromatography and thin-layer chromatog-raphy, which had independen tly com peted with HPLC during the earlier years ofdevelopment, Am os concluded that the choice between PC , TLC and HPLC isnow fairly clear cut and that HPLC should be used for all routine high-speedquantitative analysis.Since that review w as written, HPLC pump, column and detector designs haveadvanced and expanded markedly, such that a far wide r field of app lications nowexists. HPLC has also diversified into aqueous/ionic systems (ion chromatogra-phy) and high performance size exclusion chromatography (gel permeationchromatography), and been hyphenated with spectrometry including induc-tively coupled plasma emission spectroscopy (ICPES ), nuclear m agnetic reso-nance spectroscopy (NMR), Fourier transform infra-red spectroscopy (FTIR)and most importantly mass spectrometry (MS). The n umerous attempts to inter-face HPLC with M S have resulted in a variety of LC-MS systems with each in-terface type having its own specific limitations and applications.

12 2 APPARATUSA typical HPLC system (Fig. 12.1) is still composed of a pump, sample injec-tor, column, detector and data recorder much as described by Amos [7]. How-ever, considerable improvem ent and development of each component has takenplace. Advances in colum ns and detectors have resulted in a w ider range of sepa-rations and detection strategies being available.

12 2 1 Solvent reservoirsSolvent reservoirs consist of purpose built glass bottles with a helium inletand filter (for degassing) and a solvent outlet composed of a fritted particulatefilter and PTFE outlet tube. Som e systems even allow for a slight helium over-pressurization of the reservoir to assist pump priming and prevent cavitation inthe solvent inlet tubing.

12 2 2 PumpsPumps have progressed from single isocratic systems delivering premixedsolvent, to purpose built binary, tertiary and even quaternary mixing systems


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HPLC and column liquid chromatographySolvent Reservoirs

349

Detectors Data SystemFig. 12.1.Elements of an HPLC system.

which premix the solvents to the desired composition and deliver them at therequired flow rate, com pensating automatically for pressure and viscosity effectswhich may occur during the mixing process.During the early 1980s, much debate took place over the relative merits ofhigh pressure mixing (after the pump outlet) and low pressure mixing (at thepump inlet).High pressure mixing suffers from a number of drawbacks: chief among theseis the need for more than one pump with the concomitant expense. In addition,imprecision in the solvent composition may occur if one or more of the solventsis present as less than 5 of the total.Low pressure mixing requires only one pump with the solvents proportionedand mixed before the pump head. Control by microprocessor or computer datasystems allows for almost any shape of gradient (and flow) profile to be deliv-ered. For these reasons, low pressure mixing, under either of the remote controlsystems given above, has come to dom inate the market for LC gradient systemsbut some caution is still necessary in use. Firstly, for complete mixing, somesystems rely on a fairly large volume mixing chamber on the outlet side of thepump. In certain applications, such as backflushing, a sharp gradient profile isdesirable and this may be compromised by the hold-up volume of the chamber.In o ther words, if it is necessary to m ake sudden step changes in the gradient, thestep may actually be a slope. Low volume ( 1 0 ~ 1 ) ynamic mixers, such as theReferencespp. 3 72 3 74


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350 Chapter 12LEE micromixer, are of considerable use in eliminating this problem. Secondly,efficient degassing of the solvent used to be a prerequisite for accurate mixing inorder to eliminate cavitation in the mixing system and the detector noise and in-accurate flow rates that could result. In practice, improvement in the design ofthe solvent reservoirs and pump head geometry have reduced the occurrence ofthese problems, provided that the manufacturers advice is heeded. Gradient elu-tion may also limit the choice of detector to be used, especially if the detectordepends on changes in a physical property of the mobile phase itself. This issueis discussed in detail in the section on detectors.Three main types of pump, reciprocating piston, syringe and diaphragm, wereall applied in the early days but the reciprocating piston pump now dominatesthe standard HPLC market where flow rates are at or above, 1 ml min-*. Sy-ringe pumps are best suited to lower flow rates and as such find more use inmicroboreHPLC where flow rates are typically well below 1 ml min-l.Reciprocating piston pumps operate by means of a rotating eccentric camwhich drives a piston. The piston draws solvent into a cylinder through the inletcheck valve during the return stroke. During the delivery stroke, solvent is ex-pelled through the outlet check valve and hence ultimately to the column. Thesepumps are relatively inexpensive, simple to maintain, and deliver a constant flowof solvent over a wide range of flow rates. The piston drive is usually controlledby solid state pulsing circuits and a stepper motor. This allows for rapid refillingof the cylinder followed by swift repressurization of the solvent in the pumphead and then a smooth, constant volume delivery until the end of the deliverystroke. The design of the cam and its eccentricity determine the smoothness ofthe flow profile. This is now so well defined that accurate, rapid refilling hasbecome commonplace and methods for smoothing the profile such as large vol-ume pulse dampers and dual or triple stage pump heads are largely redundant.Lower volume dampers may still be used and are often an integral part of thepump, invisible inside the box.

12 2 3 Sample injectorsSample injectors are almost exclusively of the six-port valve type, althoughon-column syringe injectors were initially used. Injection valves are connectedbetween the pump and the column and as close to the top of the column as prac-tically possible. An interchangeable sample loop of discreet volume is connectedto the valve and isolated from the flow of mobile phase. The loop is filled with

sample solution and the valve is then turned manually or electronically so thatthe loop is connected into the flowing mobile phase and the sample is therebyinjected onto the column.


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HPLC and column liquid chromatograph y 351Various designs of injectors exist, depending on the volume of sample avail-able and the physical scale of the HPLC system. Internal microlitre-sized loop

injectors are available for microbore applications, whereas for standard andpreparative work, interchangeable sample loops up to 5 ml in volume can beused with standard Rheodyne or Valco valves. Loops are filled by a standardglass syringe with a Luer fitting, with or w ithout a flat-ended needle according tothe design of the sample inlet on the valve.Remote actuation of injection valves or flow switching valves is usually ac-com plished pneumatically or e lectrically. Typically the solvent delivery systemor da ta station allows for timed events, one of which is the activation of the in-jection valve. In pneumatic actuation, the valve is turned by a supply of highpressure gas, usually air, in a purpose made pistodcylinder type actuator. Thegas supply is itself delivered to the actuator by a solenoid valve. The solenoidvalve opens or closes under the control of the solvent delivery system or datastation, in order to pressurize or depressurize the actuator and hence operate theinjector.Electrical actuators tend to be m ore expensive but are faster than pneum aticactuators. They use a synchronous, high torque electric motor, directly con-trolled by a relay closure or TTL switch. The valve may be coupled directly tothe motor, minimizing the number of moving parts. Although faster and poten-tially more reliable than the pneumatic actuator, the extra capital cost is often thesole factor which m itigates against their use. In our experience, pneumatic actua-tors seldom give any cause for concern and the ex tra cost of electrical actuatorsis rarely justified.

12 2 4 ColumnsSelection of the appropriate column is, of course, entirely dependent on theparticular separation desired. Over the last 15 years, the technology of columndesign and m anufacture has advanced markedly, as has the range and reliabilityof packings available. In the late 1970s, columns were almost exclusively250 mm long 316 stainless steel with an internal diameter of 4 6 mm. End fit-tings were of solid 3 16 stainless steel and packings typically amorphous silica oralumina, or else silica with an octadecyl bonded phase, commonly referred to asODS or C18. At that time, intermediate polarity stationary phases were begin-ning to excite interest but only amino (-NH,) and nitrile (XN) phases werereadily available. Since then the range of HPLC applications has broadenedconsiderably and advances in colum n chemistry and design have been fundamen-tal to that progress.HPLC analysis can be placed in one of four categories largely by virtue of thecolumn type used.

References pp. 3 72-3 74


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352 Chapter I2(1) Reversed phase: where the phase is of spherical silica with a non-polarhydrocarbon chemically bonded onto the surface or less commonly sty-

rene-divinylbenzene beads. The mobile phase is polar and is most often amixture of methanol and water, acetonitrile and water or tetrahydrofuranand water. One important variant of reversed-phase HPLC is reversedphase ion-pair chromatography (RP-IPC) where the analyte is ionizable orprotonizable, and the mobile phase consists of a buffered aqueous mixturecontaining a counter ion of opposite charge to the analyte.(2) Normal phase: where the column is packed with spherical silica or withsilica with a polar phase chemically bonded to it. Typical bonded phasesinclude amino (-NH,) and nitrile (-CN) already referred to (and whichmay also be used in reversed phase mode) and phenyl, nitro or diol. Somespecific phases such as dinitroanilinopropyl are also finding considerableuse. The mobile phase is non-polar, typically heptane with or without theaddition of small amounts of more polar solvents such as methylene chlo-ride or ethyl acetate.

(3) Ion exchange: consisting of sulphonate or quaternary ammonium func-tional groups chemically bonded onto silica or styrene/divinylbenzenepolymer particles. Weak cations or anions can be separated without theuse of buffer solutions as mobile phase, whereas strong cations or anionswill require them.(4) Size exclusion or gel permeation: where solutes are separated by virtue oftheir size in solution. This technique has many petroleum applications forthe determination of the molecular weight of polymeric lubricant addi-tives but is not considered in detail in this chapter.

The range of columns currently available is, therefore, extremely wide, suchthat the separation of hydrocarbons, functional groups, ionic compounds, poly-mers and even enantiomers can be achieved. Column design has advanced fromconventional columns to include disposable cartridges, radially compressedcolumns, metal-free columns made from polyetheretherketone (PEEK), and col-umns with adjustable end fittings which recompress the packing if voids de-velop, prolonging column lifetime.Most stationary phases are also available in microbore columns with internaldiameters of 1-2 mm, which offer the advantages of reduced mobile phase con-sumption and greater mass sensitivity. By contrast, preparative scale columnspacked with any of the aforementioned stationary phases (with the exception ofdiol) are also available off the shelf. These have internal diameters (i.d.) of 9or 21 mm and can be used to recover larger quantities of analytes, either for fur-ther purification or for identification.petroleum HPLC laboratory can serve a diverse group of needs includingthe analysis of fuels, lubricants, additives, waste water and refinery process


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HPLC and column liquid chromatography 353samples. As a consequence one could easily expect to find silica, C18, C8,-NH,, -CN, ion exchange and size exclusion stationary phases in routine use,each in one or more of the column designs described above.12 2 5 Detectors

All detectors, be they for HPLC or any other analytical technique, must beprecise, sensitive and stable. In addition, HPLC detectors should have a largelinear dynamic range, be insensitive to temperature and eluent composition, ex-hibit low noise and drift, and be simple and easy to maintain. Since the earlydays of HPLC, no single detector has been able to fulfil all these criteria as theflame ionization detector (FID) has done so admirably for GC. Instead, a rangeof detectors has evolved, based either on changes in the bulk properties of themobile phase, or upon a selective property of the analyte(s). The subject hasbeen frequently reviewed and descriptions of the main detector types can befound in any general HPLC text [5 6]. The reader is directed to Scott for a moredetailed and mathematical treatment [81. The treatment here will be restricted toa brief discussion of those detectors which have found application in the petro-leum industry. Even with this proviso, the majority of detector types currentlyavailable are still included.12 2 6 Selective property detectors12.2.6. W- v i s ib l espectrophotometers

Ultraviolet detectors have been used since the early years of HPLC and re-main the workhorse detector in the majority of laboratories today. Early exam-ples were effectively converted spectrophotometers with the only modificationsbeing associated with inclusion of a flow cell rather than a cuvette holder. Theseinstruments were therefore based on prism diffraction or grating interferometry,such that the specificA of interest could be selected in order to achieve maxi-mum solute sensitivity. The quantitation principle is the Beer-Lambert Lawwhich states that the amount ofUV or visible light absorbed will be directly pro-portional to the solute concentration. Sensitivity and limits of detection willtherefore vary from solute to solute as a function of the individual compoundsextinction coefficient. In extreme cases, where no U V or visible light chromo-phore is present in the solute, no absorption will take place and such solutes willnot be detectable. This is the chief limitation of UV detectors and it is especiallyapparent in petroleum analysis because saturated hydrocarbons have no chromo-phore. A second major limitation is that mobile phases which themselves absorbReferences pp . 3 72-3 74


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354 Chapter 12UV light effectively impose a wavelength cutoff on the system. Below thiswavelength the absorption of the mobile phase itself is so strong that soluteswith A in the same region cannot be detected.The principle of detection within prism or grating instruments relies on thetransmitted light of the chosen wavelength being cast onto a photomultiplier.Only one wavelength is observed that is often a compromise between sensitivityand selectivity.I2.2 .6 .2Diode rr y detectors DAD)

Diode array detectors (Fig. 12.2) effectively allow a much broader wavelengthrange to be acquired simultaneously, such that an entire spectrum (moretypically200400 nm) can be captured repeatedly throughout the analysis. Thesedetectors became commercially available in the early 1980s, have rapidly estab-lished themselves as reliable and sensitive, and have allowed ever more complexdetection strategies to be employed. Of course the solute still needs a chromo-phore and the mobile phase cutoff must be observed, especially in gradientelution.The principle of detection within the DAD relies upon an array of photodi-odes of typically 0.5 nm resolution, such that the transmitted light after the flowcell is dispersed by a holographic polychromator and directed onto the linearphotodiode array. Thus, by recording the signal output from one photodiode, theeluent is monitored at a single wavelength and by recording the output from all

Photodiode rraygg/gg Ellipsoidal MirrorElliosoidal Mirror - Deuterium Lamp

Fig. 12.2.Optical system of a diode array detector.


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HPLC and column liquid chromatography 355the diodes, an entire spectrum is obtained. The major disadvantage of this systemis that light of all wavelengths is present in the sample cell simultaneously and assuch, fluorescent light may well be present at the wavelengths being monitored.In practice this is such an infrequent occurrence as to be of little consequencebut it must always be borne in mind, especially if the mobile phase or so lutes canbe excited.The DAD can be used as a simultaneous multiwavelength detector to maxi-mize sensitivity to each solute in turn, or to record entire spectra in o rder to ex-amine peak purity, or to produce three-dimensional maps of wavelength versusabsorbance versus time. All three modes offer the user more accurate quantita-tion than would be possible with a single wavelength dispersive spectropho-tometer. For research use, the ability to record an entire spectrum of all theunknowns in a sample can give an early indication of solute identity. Coupledwith retention behaviour, this can yield hypothetical structural information, orsolute functionality, or carbon number, depending on the LC mode employed(norm al or reversed phase).12.2 .6.3 Fluorescence detectors

UV light can interact with some solutes by exciting delocalized electrons intohigher energy states above the norm al ground state. When these electrons relaxback to the ground state, the solu te will emit most of the absorbed energy as lightat a longer wavelength than that which excited it. In solutes where this decay isinstantaneous or where it ceases immediately upon removal of the incident light,the solute is said to be fluorescent. It is possible to m onitor the emission wave-length and filter out the excitation wavelength altogether, and this produces veryhigh sensitivity, some two to three orders of magnitude greater than absor-bance and is therefore a highly desirable method of detection. In order to takeadvantage of the phenomenon, non-fluorescent compounds may be derivatizedprior to analysis with a reagent to produce a fluorescent derivative. In petroleumanalysis, fluorescence detection is most useful when the solute itself is highlyconjugated and fluo resces naturally, as do many polynuclear aromatic hydrocar-bons. Fluorescent light emerges from the sample at random angles and most in-struments monitor the light emitted at right angles to the excitation beam. Somesolvents have the ability to quench fluorescence such that the process is effec-tively suppressed. In particular very polar or aqueous mobile phases and buff-ered or ionic eluents are not recommended due to this phenom enon.12.2.6.4Electrochemical detectors

Compounds which are electrically oxidizable or reducible can be detectedelectrochemically. In coulometric detectors the solute is completely electrolysed,whereas in amperometric detectors, the solute is only partially electrolysed.References pp. 372-374


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356 Chapter 12Amperometric operation is more suited to flowing systems and is the commonestmode of electrochemical detection. In amperometric detectors, the solute con-centration is directly proportional to the diffusion rate of the solute acrossthe boundary layer to the electrode surface. The electrode current is thereforedependent not only on the solute concentration but also on its diffusion coeffi-cient. A detailed treatment of electrochemical detectors can be found inScott [8]. Electrochemical detectors rely on the mobile phase being electri-cally conductive and the most direct method of assuring this is to use buffer so-lutions.12.2.6.5 Flame ionization detector

The use of the FID in HPLC necessitates the removal of the mobile phase,chiefly by selective evaporation. Much effort has been expended into making theFID compatible with HPLC in order to take advantage of its properties of sensi-tivity, known response and linear dynamic range. A number of mechanical trans-port systems have been developed originating with James in 1964 [9]. A movingwire was employed to carry the column effluent through a heated zone where themobile phase was evaporated off, and then to another zone heated to a highertemperature in order to evaporate/pyrolyse the solutes and carry them into theFID in a stream of nitrogen.The chief disadvantages of transport detectors all lie with the transportmechanism itself. The wire, chain or disk has proved to be difficult to coat uni-formly, different solvents evaporate at different rates, accumulation of remainingtraces of solute give memory effects. These factors all contribute to relativelypoor signal to noise ratios. Since only a small proportion of the solute is evapo-rated and detected, the sensitivity and large linear range of the F D are not util-ized. In conclusion the compromises inherent in transport FIDs have meant thatthis detector is not widely used and its early promise for HPLC use remainslargely unfulfilled.

12.2.6.6 Mass spectrometersThe interfacing of mass spectrometers to GC instruments(GC-MS) is possiblebecause both techniques are readily compatible. GC-MS is now one of the mostpowerful diagnostic tools available to analytical chemists.Interfacing HPLC to mass spectrometers (LC-MS) is much more difficult andhas largely hinged on the design of liquid phase separation systems and removal

of eluent until relatively recently. Work on LC-MS began in the late 1960s but itwas not until the work of Homing et al. [lo], Scott et al. [ l 13, and Arpino et al.[ 21 in the 1970sthat LC-MS was effectively achieved.Overcoming the relative incompatibility of the liquid phase eluent and thehigh vacuum required in the source of the MS has proved to be a severe chal-


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HPLC and column liquid chromatography 357lenge. In addition, the higher molecular weight, lower volatility and chemicalpolarity of many compounds separated by LC make them less easily ionized thanthe compounds amenable to GC-MS. Because of this, electron impact ionization(EI), which is so successful in GC-MS, has proved less so in LC-MS. More rele-vant ionization techniques such as fast atom bombardment (FAB) and atmos-pheric pressure chemical ionization (APCI) have been applied in order to sur-mount this problem. The mere modification of LC to make it more compatiblewith ex isting high vacuum, electron impact MS has not on its own proved suffi-cient, and the development of more compatible MS ionization and inlet system shas been necessary for the two techniques to merge successfully. The wholesubject has been well reviewed recently by Niessen and van der Greef [13].These authors list 26 distinct types of interfaces for LC-MS developed since1972. The reader should consult reference [131 in respect of therm ospray LC-MSand the particle beam interface, both of which have been successful in petroleumand coal-based applications.12 2.6.7Injured and NMR

IR photometers have found little use as HPLC detectors for two main reasons.Firstly, most solvents used a s mobile phases absorb in the m ost useful regions ofthe IR spectrum. Secondly, using absorption wavelengths away from solvent ab-sorption bands has invariably resulted in less sensitivity and higher backgroundlevels. The exceptions to this have been where the analyte contains a carbonyl(C=O) group and in size exclusion chromatography of polymers. The formercase is able to take advantage of the high extinction coefficient and hence h ighsensitivity of the carbonyl group. The latter application is able to overcome bothlow sensitivity and high background by virtue of the relatively high sam ple con-centration required by SEC.Many of the limitations have been overcome or greatly reduced by Fouriertransform infrared (FTIR) instruments. Modern FTIR spectrometers have signalto noise ratios over 100 times larger than energy dispersive instruments and a s aconsequence sensitivity is greatly improved. Their ch ief disadvantage is that ofhigh cost and another disadvantage is incompatibility with reversed phase elu-ents. The combination of water absorption and band broadening due to hydrogenbonding conspire to reduce sensitivity and to limit the usable part of the IRspectrum.Proton nuclear magnetic resonance spectroscopy ('H NMR) has also beenused as an on-line HPLC detector. This technique exploits the odd spin of thehydrogen nucleus, lH, in order to gain information on the environment of varioushydrogen atoms in the analyte molecules. In this way, the signals due to m ethyl,methylene and aromatic protons in various molecular environments can be sepa-rated and quantified. Once normalized, the proportion of various hydrogen typesReferencesp p . 372-3 74


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358 Chapter 12can be calculated and the alkyl, aryl and heteroatom substituents present in asample elucidated. Proton NMR will be unable to distinguish hydrogen atomsfrom the mobile phase from those of the analyte and these will be included erro-neously in the normalization if present. For this reason, static NMR experimentsor LC-NMR cannot use standard solvents but are required to use perhalogenatedor perdeuterated solvents. This is a severe limitation to on-line LC-NMR sincethese solvents are extremely expensive, especially if significant volumes of per-deuterated solvents such as chloroform-d (where 99.8 of the hydrogen is re-placed by deuterium) have to be used for the LC separation. Another consider-able limitation is the high capital and running cost of a modern Fourier transformNMR spectrometer. Nevertheless, this technique has found application in petro-leum analysis and is expected to find increasing use.12.2.7 Bulk property detectors12.2.7.1 Refvactive index detector

The refractive index detector remains the second most widely used LC detec-tor after the UV detector. It is universal, detecting all analytes whose refractiveindex (RI) differs from that of the mobile phase.The RI of a substance is a dimensionless constant which typically decreaseswith increasing temperature. Three types of RI detector are available and all aretermed differential refractometers, that is they measure the difference in R be-tween a sample cell and a reference cell containing mobile phase only. It fol-lows, therefore, that all refractometers are sensitive to temperature changes andto changes in eluent composition. Thus n order to use them for gradient elu-tion, the reference cell must always contain a mobile phase of identical compo-sition to that in the sample cell and this is often impossible to achieve. For goodbaseline stability, RI detectors are thermostatically controlled, either by a water

bath or by an insulated cabinet.Deflection or angle of deviation instruments have a split flow cell, with Sam-ple on one side, reference eluent on the other. Light from the source passesthrough this cell to a mirror behind it and is reflected back through the cell to aphotomultiplier. If a solute of different RI enters the sample cell, the light beamwill be deflected. The photomultiplier output is proportional to the magnitude ofthe deflection. Deflection RI detectors are simple and have a wide linear dy-namic range. Instruments manufactured by Waters Associates have typicallybeen of this design.Fresnel refractometers pass parallel incident light through a prism onto sam-ple and reference simultaneously. If the refractive index of the liquid in thesample cell differs from that in the reference cell, some light from the sample


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HPLC and column liquid chromatography 359cell will be diffracted, reducing the intensity of the beam reflected back out ofthe sample cell. The difference between the intensities of the sample and refer-ence beams is measured by a photomultiplier and recorded. The linear concen-tration range of this detector is less than that of the deflection instrument unlesstwo separate prisms are used to cover the en tire RI range. The optical cleanlinessof the system is also more critical than for the deflection detector. Fresnel refrac-tometers have been m anufactured by Perkin Elmer.Interference refractometers split the source beam, pass it through sample andreference cells simultaneously and then recombine it. Any difference in refrac-tive index between sample and reference cells will manifest itself as a d ifferencein optical path length when measured by an interferometer. This design is moresensitive than the previous types and additional sensitivity is possible if a laser isused as the light source as by Woodruff and Yeung [14,151.In summary, RI detectors are universal and can be sensitive under carefullycontrolled conditions. Their use in gradient elution is still far from straight-forward and base line drift is to be expected when the mobile phase com positionchanges even by relatively small amounts. Despite all these operational draw-backs, they are still the detector of choice when the solutes have no UV chromo-phore , especially in isocratic determinations of saturated hydrocarbons.12.2.7.2Evaporative light scattering detectors

The evaporative light scattering detector (Fig. 12.3), evaporative analyzer ormass detector was developed and patented in 1966 by Ford and Kennard [16,171.It was not until 1978, however, and the comprehensive work of Charlesworth[18] that its usefulness as an HPLC detector was fully realized. The theory ofoperation , construction and performance of what is now referred to as the massdetector can be found in that reference.In essence, this type of detector consists of a nebulizer, evaporation cham ber,light source, scattering chamber and light trap and a photom ultiplier set at 135

to the incident light beam. Column eluate is nebulized with a relatively high flowof nitrogen or air and the mobile phase evaporated as the solvenugas mixturepasses down the vertically mounted evaporation chamber. At the bottom of thechamber, all that is left is gas, solvent vapour and finely divided droplets or par-ticles of analyte. This aerosol passes through the light beam and the photomul-tiplier detects that portion of the incident light which is scattered by the analyte(at an angle of 135). At this angle, Charlesworth found the result to be e ffec-tively independent of the RI of the analyte.The true linear working range of this instrument is not extensive, typically 1.5orders of magnitude in concentration. Above and below this range, the size ofthe analyte droplets produced no longer promote the reflection and refraction ofthe light. Although this is a drawback, it is a relatively minor one, as the re-Referencespp. 3 72-3 74


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360Nebuliseraa

Chapter 12

n I8 ExhaustFig. 12.3. Schematic of an evaporative m a s detector.

sponse functions due to Rayleigh and Mie scattering in the non-linear regions arewell described and still make calibration possible.The chief disadvantage of this detector is the volatility limitations imposedupon the analyte. The solvent evaporation chamber is, in effect a mild blow-down apparatus which removes the mobile phase. If the analyte volatility or va-pour pressure approaches that of the mobile phase it will vaporize and give noresponse. In our laboratory, we have found hexadecane (b.p. 256C) to be par-tially evaporated when the detector is operating at ambient temperature withhexane (b.p. 68C). It is therefore likely that hydrocarbons below n-CI7will notgive full recovery. Even given this limitation, the detector finds considerable usefor intermediate and low volatility analytes.12.2.7.3 Dielectric constant detecto r

With few exceptions, the dielectric constant of a substance increases with itspolarity. As an LC analyte elutes from the column, the dielectric constant of theeluate will change. The dielectric constant of a non-polar or semi-polar sub-stance is a function of its refractive index and as such many of the practical con-siderations concerning RI detectors apply equally to the DCD. A more detailedtreatment may be found in Scott [8].


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HPLC and column liquid chromatography 361A typical DCD is a d ifferential, temperature controlled concentric cylindricalcapacitor through which the column eluent flows. The cell electrodes are made

of stainless steel and are connected electrically as one side of a W ein or ScheringBridge. If the m obile phase is less polar than the analyte, as in normal phase LC,the dielectric constant of the eluate will increase as the peak e lutes. The reverseis usually the case in Rp-HPLC, and in o rder to avoid negative peaks in reversedphase applications, DCD s allow for polarity reversal.Setting up and balancing DCDs can be a tedious business as each side of thebridge circuit needs to be balanced in an iterative fashion until the poten tial dif-ference across the bridge is zero.The linear dynamic range of the DCD is heavily influenced by the d ifferencein the dielectric constants of the mobile phase and the analyte, but has beenquoted as 3.5 X lo4which is comparable to the RI detector.

12.3 QUANTITATIONIn the majority of petroleum app lications of HPLC , calibration is by ex ternalstandardization and quantitation is by peak area. W here samples are analyzed asreceived or after dilution only, this approach is reliable and accurate. W here the

sample is worked up before analysis by liqu idliqu id or liqu idsolid ex traction, itis necessary to determine the extraction efficiency (or recovery) in order to becertain that a representative extract has been obtained. Where extraction effi-ciencies are low or where time does not allow the recovery to be determined, aninternal standard or a standard addition method should be employed, providedthe detector response to the solutes is linear in the range of interest.Peak a rea is most usually used for quantitation, as this is the most statisticallyprecise measure of analyte concentration. It does presuppose good resolutionhowever, and where this is not the case, a range of deconvolution methods oreven peak height measurement may have to be considered.Contemporary HPLC now has a vast range of competitive quantitation devicesand statisticaVgraphica1 software available. Stand alone benchtop integrators,microprocessor and PC data stations, local area networks (LANs), laboratoryinformation management systems (LIMS) and even m ainframe chromatographypackages are a ll available. Selection is a com promise between cost, specificationand, increasingly, compatibility with ex isting computer hardware. Any of thesedevices can take de tector output and convert it to a high quality graphical or nu-merical report, automatically labelled with peak identities according to previ-ously recorded retention windows.Caution is necessary, however, as any system will only act according to theway it is configured by the operator. At each stage of the data systems applica-References pp . 372-374


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362 Chapter 12tion, the user must be certain that each setting is sound in order to obtain a finalquantitative output of the highest possible integrity.

12 4 APPLICATIONSThe applications of HPLC in petroleum analysis are summarized in Table12.1. The wide variety of separation mechanisms, column chemistries and de-tection systems represented by HPLC offers the petroleum chemist a range ofdistinct systems. In general these fall into three categories:1 ) the separation and direct quantitation of individual compounds;(2) separation and characterization of compound classes such as, for example,

(3) preparative or semi-preparative fractionation of complex mixtures for de-saturates, olefins and aromatics in petroleum products;termination by other analytical techniques.Within each category, standard methods exist for particular determinations,which have been rigorously tested in terms of inter-laboratory precision. Suchstandard methods as exist within the Institute of Petroleum handbook, Standard

Methods of Analysis and Testing of Petroleum and Related Products, 1993 [19]are discussed in the following sections.

12 4 1 Individual compounds12.4.1.1Polycyclic aromatic hydrocarbons (PAHs)

These compounds have attracted considerable interest due to their role aspollutants and, in some cases, their carcinogenic properties. Amos [7] cites someearly W L C applications. Katz and Ogan [20] have used partition and size ex-clusion columns in series to effect the analysis, and a combination of normalphase amino and reversed phase C 18 columns has been used to determine PAHsin crude oil by Grimalt and Albaigks [21]. Further LC-LC methods, chieflyaimed at benz[a]pyrene, have been employed by Tomkins and Griest [22] andFielden and Packham [23]. In the former case, Partisil silica and analytical scaleVydac 201TP reversed phase columns were used and in the latter case cyclodex-trin and ODS silica. In both cases, the selectivity and sensitivity of fluorescencedetection was used to determine the PAH directly.Symons and Crick [24] have determined PAHs in refinery effluent after clean-up and preconcentration using a Radial-Pak CIS column with 75:25 aceto-nitrilelwater eluent and UV and fluorescence detection. Recoveries were vari-


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HPLC and column liquid chromatography 363TABLE 12.1APPLICATIONS OF HPLC IN PETROLEUM ANALYSISCrude oilha - a r e ne sDibenzothiophenePhenolsPolynuclear aromatic hydrocarbons (PAHs)Preparation of PAH fractionsSaturates/aromatic typesNaphthdgasolineAromatic nitrogen compoundsBenz[a]pyreneSaturateshromatic types

Saturates fractionsSaturates/olefindaromaticsAviation fuelAromatic nitrogen compoundsCoumarinPAHSSaturateshrornaticsSaturateshromatic types

Saturates/olefindaromatics2,4-Dimethyl-6-tertiarybutylphenol

DieseUdistillate fuelsAlkyl nitratesAromatic nitrogen compoundsMono/di/triaromaticsOlefinsPAHSPhenalenonesSaturatedaromaticsSaturatedarom atic types

Grimmer et al.Rebbert et al.Christensen and WhiteMacCrehan and Brown-ThomasGrimalt and AlbaigesOstman and ColmsjoWelch and Hoffman

Nondek and ChvalovskyTomkins and GriestApffel and McNairCookson et al.Munari et al.Hayes and AndersonASTM D 2002,2003ASTM D 1319

Nondek and ChvalovskyIP 374Fielden and PackhamIP PM-ATWelch and HoffmanCookson et al.Hayes and AndersonHaw et al.Davies et al.ASTM D1319Hayes and HillmanIP 343

Schabron and FullerNondek and ChvalovskyLienne et al.Fielden and PackhamDavies et al.MarshmanApffel and McNairCookson et al.

IP 391, M-AY

IP PM-AZ

References p p. 3 72-3 74


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364TABLE 12.1 continued)

Chapter 12

Diesel exhau st particulatesNaphtho[8,1,2-abc]coroneneNitrated PAHs

Fuel oilBenz[a]pyreneLubricating oilsAdditives (over 50)Aromatichon-aromatic fractionsRenz[a]pyreneFurfuralNaphthalene/phenanthrenePAH fractionsPolychlorinated biphenylsSaturates/aromaticsSaturatedaromaticsipolars

SulphonatesSulphurized alkylphen olsV1 improverZinc dialkyldithiophosphatesHeavy oilsOlefinic fractionsPAH fractionsSaturates/aromatics/PAH/resins/asphaltenes,tc.Saturates/naphthenes/alkylaromatics/thiophenes

BitumenPAH fractionsRefinery effluentPAHs

Davies et al.Hazlett el a1

Jinno et al.Paputa-Peck et alMacCrehan e f al

Tomkins and Griest

Musha et al.Musha et al.ASTM D 2549SaitoDeSanzo et al.Mazzeo et al.Palmentier et al.Ostman and ColmsjoDeSimone et al.IP 368ASTM D2007Pei et al.Pei and HsuBearASTM D3712Chen and NeroBlanco-Gomis et al.Fodor and Newman

Yamamoto and AkutsuCoulom be and SawatzkyLancas et al.Hsu et al.Hsu et al

Coulombe and Sawatzky

Symons and Crick


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HPLC and column liquid chrom atography 365able and less than 87 for 4 6 ring PAHs. Saito [25] determined benz[a]pyrenein lubricating oils and greases with fluorescence detection after an aluminaclean-up; precision was reported as 6 7 RSD with a detection limit of3.95 nglg.HPLC has a lso been used purely as a fractionation technique for PAHs. Cou-lombe and Sawatzky [26] applied this method to bitumens and heavy oils anddetermined PAHs in the various LC fractions by GC. Palmentier et al [27] em-ployed a semi-preparative scale fractionation followed by GC-MS. Ostman andColmsjo [28] prepared PAH fractions from crude oil and used crankcase oil byelution from short silica columns followed by an autom ated backflushed Bonda-pak-NH, HPLC system. Individual PAHs in the final fractions were quantifiedby GC. Detection limits were in the order of 1 ppm from a 10-15 mg sam pleusing GC-FID or 0.1 ppm by scaling up the initial silica clean-up.Mazzeo et-al [29] detected PAHs as quinones by oxidizing them with CeN .Reductive mode electrochemical detection was employed to achieve detectionlimits in the order of ppb. Chromatography was performed on an ODS columnusing propan-2-01 /phosphate buffer as eluent. These authors applied the abovesystem to the analysis of naphthalene and phenanthrene in a m otor oil.A proposed Institute of Petroleum standard method, IP PM-BN, also exists forthe determination of PAHs in petroleum, coal and shale oil products. Detectionlimits of 0.1 d m f total, and 0.1 mg kg-1 of individual PAHs are quoted. Themethod uses open, gravity feed silica columns to produce a PAH extract which isfurther separated by HPLC on a 5,um particle Spherisorb amino column orequiva lent. The isolated 4-6 ring fraction is then run on a Sephadex LH20 parti-tion column in order to separate alkylated PAHs from the parent PAHs. Theseparent PAHs are individually determined by GC. Precision has yet to be estab-lished.12.4 .1.2 O ther indigenous compounds

Nitrated PAHs in diesel engine exhaust particulates have been examined byPaputa-Peck et al [30] and MacCrehan et al. [31]. Paputa-Peck employed anormal phase HPLC fractionation of methylene chloride extracts. Determinationof individua l nitrated PAHs was by GC with a nitrogen-phosphorus detector orby G C-MS. MacCrehan separated methylene chloride extracts by RP-HPLC andcompared voltammetry, amperometry and fluorescence for direct detection ofindividual compounds. Diesel particulates have also been examined by Jinno etal [32] for naphth0[8,1,2-abc]coronene using reversed phase separations andmultichannel detection.High molecular weight heterocyclic nitrogen and sulphur compounds havebeen studied by Borra et al [33] and Andreolini et al [34]. These authors usedReferences pp . 3 72-3 74


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366 Chapter 12highly efficient capillary LC columns and a combination of direct diode arrayfluorescence detection or fraction collection and mass spectrometry to examinesolvent refined coal distillate, syncrude and shale oil samples.Polycyclic aromatic nitrogen compounds (aza-arenes) in Arabian Light crudeoil have been examined and identified by Grimmer et al. [35]. These authorsused a TLC/HPLC isolation scheme with separation and identification of indi-vidual compounds by GC. Aza-arenes, anilines and alkyl aromatic amines ingasoline, kerosene and diesel fuel have also been studied by Nondek andChvalovsky [36] using two different charge transfer columns, 3-(2,4-dinitrobenzene su1phonamido)propyl silica and 3- 2,4-dinitroanilino)propyl sil-ica. A comparison of five different charge transfer columns for the separation ofaromatic compound classes from a crude oil distillate sample and other fossilfuel samples has been made by Thompson and Reynolds [37].Phenalenones such as 7H-benz[d,e]anthranen-7-one, enzanthrone and methylphenalones have been quantified in middle distillates by Marshman [38] usingsilica reversed phase separation and UV detection at 400 nm. Detection limitsquoted are typically in the region of 0.2 mg I -* . Dibenzothiophene has beenquantified in crude oils by Rebbert et al. [39] and by Christensen and White[40]. The former authors employed HPLC to fractionate samples for GC/FPDquantification. In contrast, the latter authors used a novel LC-tandem MS systemto separate and unambiguously identify dibenzothiophene directly. Indigenousphenols in crude oil have been examined by MacCrehan and Brown-Thomas[41]with detection limits of less than 100ng/g. These authors used alkaline sol-vent extraction of the oil, solid phase purification of the extract and RP-HPLCwith electrochemical detection.HPLC has even been applied to asphaltenes in order to assist the determina-tion of an average molecule. Monin and Pelet [42] used size exclusion and arange of bonded phase columns to fractionate such samples after selective disso-lution in a number of solvents.12.4. .3Additives and contaminants

The anti-oxidant 2,4-dimethyl-6-tert-butylphenol has been quantified by nor-mal phase isocratic HPLC withUV detection and is the subject of an IP StandardMethod, IP343. The method allows a number of columdmobile phase combina-tions and, in our experience, is robust and precise. Published repeatability andreproducibility at the 200mg/l level are 2.61 and 6.56, respectively. Somehomologues and isomers of this compound may also be separated using varia-tions in mobile phase composition. The same compound has also been quantifiedwith electrochemical detection by Hayes and Hillman [43].Alkyl nitrate cetane improvers in diesel fuel have been determined bySchabron and Fuller [44]. Normal phase LC on silica coupled with variable


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HPLC and column liquid chromatography 367wavelength IR detection was used to separate and quantify amyl, hexyl and octylnitrates. Recovery, accuracy and precision quoted were good and detection limitsof 0.05 and 0.01 vol. are given for amylhexyl and octyl nitrates.Up to 50 lubricating oil additives have been separated and retention times de-termined by Musha et al. [45,46]. These authors used both normal and reversedphase columns with UV detection at the maximum absorbance for each com-pound.Furfural has been determined in lubricating oils by Di Sanzo et al. [47]. A5 pm ODs-silica column with an eluent of 70:30 watedmethanol and detec-tion at 280 nm gave a recovery >95 , good precision, and good agreement witha bisulphite extractionKJV method. Samples for HPLC were pre-extracted withmethanol and cleaned-up with a C18 silica cartridge prior to determination.Synthetic and indigenous sulphonates, including alkyl benzene sulphonates, havebeen separated and quantified by Bear [48]. This author evaluated the evapora-tive light scattering detector in the analysis of a wide range of surfactants andconcluded . a uniform linear response for each class of surfactant, with detec-tion limits in the low nmole range. In particular, the detector response was re-ported to be independent of the alkyl chain length and the degree of aromatic-ity with respect to alkyl benzene and alkylaryl petroleum sulphonates. Columnsand mobile phase varied according to the application and samples were analyzedafter dilution of the parent product. A diode array UV detector was also used inseries with the ELS detector. Standard deviations of all the analytes were lessthan 1 .Sulphurized alkylphenols have been separated from reaction side products andbase oil on a normal phase, y-cyclodextrin silyl column with gradient elution andevaporative light scattering detection by Chen and Nero [49]. Individual fractionfrom the separation were also characterized by mass spectrometry. Fingerprintcomparison between samples which passed and failed engine test specificationsare presented. The advantages of the ELSD over RI detection were stated bythese authors to be freedom from ambient temperature variation effects, minimalbaseline drift with multiple solvent gradients and a response which was massdependent rather than concentration dependent.To illustrate the breadth of HPLC applications in the field of lubricating oiladditives, normal phase and reversed phase methods have even been applied tothe characterization of poly styrene-alkylmethacry1ate)co-polymer viscosity in-dex improvers of molecular weight up to 300 000 Da by Blanco-Gomis et al.

An Institute of Petroleum method exists for the determination of the coumarincontent of kerosene. This compound, 1,2-benzopyrone s often added to keroseneas a marker for excise purposes. The method uses a silica column, a mobilephase of 2 propan-2-01 in hexane or heptane and detection at 274nm.

1501.

References p p . 3 72-3 74


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368 Chapter 12Typical calibration range is 2-4 mg/l and at the 2 mg/l level, repeatability isquoted as 0.06 and reproducibility0.28.12.4.1.4 Compound classes

The inherent normal phase separation mechanism (adsorption) has the abilityto separate complex mixtures of hydrocarbons according to degree of unsatura-tion. As such, it has been widely exploited in the characterization of petroleumproducts with respect to the saturate, monoaromatic, diaromatic, triaraomatic,polar and (to a lesser extent) olefin content. Ongoing development of bondednormal phases has largely been aimed at achieving cleaner cut-offs betweencompound classes, most notably by the use of substituent groups which separateby charge transfer mechanisms with the aromatic nuclei of the sample compo-nents. Products are often quantified in terms of the mass or volume fraction ofeach compound class present, and further separation of individual componentswithin any class is either not possible or unnecessary.No fewer than five standard IP methods of this type exist covering aviationfuel, auto diesel, drilling mud oils, gasoils and lubricating oil base stocks. Twodistinct HPLC technologies and quantitation methods are employed, both withisocratic elution.Silica columns and backflushing are used to separate saturates from total aro-matics in basestocks (IP 368) and gasoils (IP PM-AZ) with gravimetric quantita-tion and in aviation fuel (IF PM-AT) with VUV detection and quantitation oftotal aromatics and naphthenes. In all three cases, saturates elute through thecolumns unretained and aromatics (with or without olefins) are backflushed off.In auto diesel and drilling mud oils, two amino bonded phase columns areused to separate mono, di and triaromatics with RI detection and external stan-dard quantitation (IP 391 and IP PM-AY). The main concern in these last twomethods is that the external standards chosen are individual compounds, whereasthe actual sample components present in each class are many and varied. Detec-tor response factors between sample and standard can therefore vary and will becomposition dependent.A wide range of petroleum products and crude oil have been characterized byHPLC and it is best to consider each one in order of product type.Crude oil has been characterized by Welch and Hoffman [SO]. These authorsused an on-line microbore LC-GC-MS system with a 2,4-dinitrophenyl mercap-topropyl silica LC column. The system employed a retention gap between the LCand the GC columns and no attempt at quantitation was made. This article alsoincludes the analysis of JP-4 aviation fuel, isolating and identifying alkylben-zenes, alkyltetralins, alkylbiphenyls, naphthalene and dimethyl naphthalenes.Gasolines have been characterized by Apffel and McNair [52], Cookson et al.[53], Mussi et al. [S4] nd by Hayes and Anderson [ S S ] on an aminopropyl silica


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HPLC and column liquid chroma tography 369column to separate alkene-free gasolines into saturates, monoaromatics, diaro-matics, tri/polyaromatics and polar groups. Each group w as quantified by an RIdetector into weight percentage abundance. Calibration samples were obtainedby fractionation of a fuel sample rather than by use of single pure com pounds inan e ffort to minimize compositional and RI response factor differences.Both Apffel and M cNair and M unari et al. used on-line HPLC -GCR ID meth-ods to analyse gasoline saturates, unsaturates, aromatics and polar com pounds.The latter authors employed a retention gap between the two chromatographicsystems and microbore HPLC columns. Hayes and Anderson used off-lineHPLC with a dielectric constant detector to achieve an accurate group type sepa-ration and quantitation of gasoline with uniform response factors from the detec-tor. T he m obile phase w as 2,2-dichloro-1, 1 -trifluoroethane (Envron 123). Theindividual fractions were then analyzed by G C M SD to identify components andGC/FID to quantify them. The au thors reported that spent Envron 123 can be re-used several times without purification or easily redistilled on a continuousbasis.Kerosenes have been characterized also by some of the authors previouslycited [51,53,55]. In addition, Haw et al. [56] used a propylamino silica columnwith on-line NMR as the detector. n this case, the mobile phase was l , l , l-trichlorotrifluoroethane with 2.5 deuterochloroform and 0.05 hexamethyld-isiloxane as N M R reference. Each compound class (monocyclic and dicyclicarom atics) could be given an average composition. The average composition ofthe saturate fraction was, however, limited by problems in accounting for qua-ternary carbon. Davies et al. [57] utilized the LC-retention gap-GCRID ap-proach to a kerosene sample with microbore amino and silica glass-lined LCcolum ns in series with pentane eluent and backflushing. Unfortunately, the lowdead volume of detectors required for microbore LC precluded conventional RIor dielectric constant detectors and thus direct quantitation of the saturate andarom atic fractions prior to GC was not possible. The system was automatic andclearly improved the analysis of the aromatic fractions.Diesel and distillate fuels have been studied by all the m ethods described forcrudes, gasolines and kerosenes [52,53,57]. Silica and amino columns have beenused to separate diesel into saturates, olefins and aromatics with RI and/or U Vquantitation by Felix et al. [58 ] . Davies et al. [59] used the LC-GC techniquepreviously described, but with specific reference to polynuclear aromatics in die-sel fuel. The chromatographic system described by these authors produced acom plete fractionation by com pound class but in this study emphasis was placedon the definition of a two-dimensional (LC versus GC) retention map for se-lected PAHs. By comparison, the retention indices of arom atic compounds fromthe diesel sample led the authors to conclude that naphthalene, phenanthrene andtheir alkyl derivatives were the predominant aromatics present.Refeences pp . 3 72 3 74


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370 Chapter 12The LC-NMR approach, previously applied to kerosene, has been extended todiesels and distillates by Hazlett et al. [60], again culminating in the determina-

tion of average composition for each compound class. From LC-NMR data,Caswell et al. and Swann found it possible to predict the physical properties ofdiesel and jet fuels [61,62]. Multiple regression analysis was used for the corre-lation of 13 LC-NMR parameters from each fuel with 17 physical propertiessuch as cetane number, distillation data, flash point, pour point, density, etc.Thirteen of the 17 properties in reference [61] had correlation coefficients inexcess of 90 and seven were in excess of 95 .Copper(li) and silver-modified silica columns have been prepared by passingammoniacal CuSO, through the column or by use of ammoniacal AgN03 duringpacking by Lienne et al. [63]. With pentane or Fluorinert FC72 as mobile phase,olefins could be separated from light and heavy distillates with RI and UV de-tection.Heavy hydrocarbons have been characterized by Hsu et al. [64,65] by on-lineLC-MS. It was reported that distinction could be made between naphthenoaro-matics and alkylaromatics and also between aromatic hydrocarbons and thio-phenes. The value of this kind of information for refinery processing is veryhigh.

12.5 PREPARATIVE HPLC AND COLUMN LIQUIDCHROMATOGRAPHY12.5.1 Standard methods

The 1993 Annual Book of ASTM Standards [66] published by the AmericanSociety for Testing and Materials lists six liquid column chromatography meth-ods of relevance to the petroleum industry.ASTM D13 19 is identical to the Institute of Petroleum, London method IPI 56entitled Hydrocarbon Types in Liquid Petroleum Products by Fluorescent Indicator Absorption. It is limited to samples boiling below 3 15C which are sepa-rated by it into saturates, olefins and aromatics by elution through a silica col-umn with 2-propanol under air or nitrogen pressure. Fluorescent dyes are addedto the top of the column which co-elute with the olefins and aromatics and serveto mark the boundaries of each zone. The saturates front coincides with the wet-ted front of the material passing down the column. The lengths of each zone aremeasured at the end of the separation and these lengths are proportional to thepercentage of each class present in the sample. This test has been in use withslight modifications for many years and is especially relevant to gasolines andaviation kerosenes. Its main drawbacks are that it is time-consuming and opera-tor intensive and that strict control of the silica gel quality is critical.


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372 Chapter 12A mixed heavy end sample was separated into fractions with a 50 x 1 1 cmsilica gel Si60 column by Lancas et al. [72]. Two grams of the sample were

mixed with silica in a precolumn and single solvents or binary/tertiary mixturesused to fractionate it. Six solvents in six mixtures of increasing eluotropicstrength gave saturates, monoaromatics, diaromatics, triaromatics, polynucleararomatics, resins, asphaltenes and asphaltols. Typical recovery is quoted a s bet-ter than 90 n m ost cases, with an RSD of 1.2 .

12 7 FUTURETRENDS

A number of more selective column mechanisms are beginning to find appli-cation in petroleum analysis. Most specifically, the range of selectivities nowcomm ercially available in normal phase charge transfer columns such as DNAP,TNAO and TNAP columns are allowing a more precise definition of aromatictype cut point. As the industry has a con tinuing need for more precise total aro-matic and aromatic type quantification, it is expected that the use of such col-umns will increase. Similarly, the separation of functiona lized bad actors froma range of hydrocarbon products may prove to be accomplished by anion andcation exchange colum ns which are now also comm ercially available.

Undoubtedly, the single most useful advance in detector design for the petro-leum industry has been that of the evaporative mass de tector. This detector willfind increasing use in the field for two reasons. Firstly, the operation of the de-tector necessarily results in vo latile sample matrices being evaporated along withthe HPLC mobile phases used in petroleum applications. This may actuallyprove to be an advan tage in the analysis of some gasoline additives. Secondly, asheavier products will not suffer the same fate as gasoline/naphthas, characteriza-tion of such samples can take advantage of the detectors true linearity and com-position independence. These characteristics are unique in such a robust andrelatively inexpensive device.Finally, LC-GC is still waiting for an enterprising manufacturer to develop atruly turnkey system. Numerous applications of this hyphenated technique al-ready exist which should be transferable. The analysis of oxygenates in gasolineat percentage and at trace levels may yet prove to be the application whicharouses a sufficient volume of interest to be comm ercially viable.

12 8 REFERENCES1 A.J.P. Martin and R.L.M. Synge, J Biochem., 35 1941) 1358.2 M. Tswett, Proc. Warsaw SOC.Natl. Sci., Biol. Sect., 14 1903) No. 63 R. Kuhn and E. Lederer, Ber. Deut. Chem. Ges., 64 193 1) 306


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L.R. Snyder and J.J. Kirkland, Introduction to M odem Liquid Chromatography, Wiley, NewYork (1974).N.A. Parris, Instrumental Liquid Chromatography, Elsevier, Am sterdam (1984).M.T. Gilbert, High P erformance Liquid Chrom atography, Wright, Bristol(l98 7).R. Amos in Chromatography in Petroleum Analysis (Altgelt and Gouw, Eds.), MarcelDekker, New York. (1979).R.P. W. Scott, Liquid Chromatography Detectors, Elsevier, Am sterdam (1977).A.T. James, J.R. Rav enhill and R.P.W . Sco tt, Chem. Inst., 746 (1974).E.C. Homing, D.I. Carroll, I Dzidic, K.D. Haegele, M.G. Homing and R.N. Stillwell, J.Chromatogr. 99 (1974) 13.R.P.W. Sco tt, C.G. S cott, M. M unroe and J. Hess Jr., J. Chromatogr., 99 (1974) 395.P. Arpino, B.G. Dawkins and F.W . McLafferty, J. Chromatogr. Sci., 12 (1974) 574.W.M.A. Niessen and J. van der Greef, Liquid ChromatographyMass Spectrometry, MarcelDekker, New Y ork (1992).S.D. Wood ruff and E.S. Yeung, Anal. Chem., 54 (1982) 1174.S.D . Woodru ff and E.S. Yeung, Anal. Chem., 54 (1982) 2124.D.L. Ford and W. Kennard, J. Oil Colour Chem. Assoc., 49 (1966) 299.D.L. Ford and W. Kennard, Aust. Pat. Appl. No. 33406.J.M. C harlesworth , Anal. Chem., 50 (1978) 1414.Standard Methods of Analysis and T esting of Petroleum and Related Products, 1993, Instituteof Petroleum and W iley, London .E. K atz and K. Ogan, 5th Int. Symp. on Chem. Anal. Biol. Fate Polynucl. Aromat. Hydrocar-bons, B attelle Press, Columbus, OH (1980) pp. 169-178.J. Grimalt and J. Albaiges, Afinidad, 40(385) (1983) 223.B.A. Tomkins and W.H. Griest, J. C hromatogr., 386 (1987) 10 3.P.R. Fielden and A.J. Packham , J. Chromatogr., 479 (1989) 117.R.K. Sym ons and I. Crick, Anal. Chim. Acta, 151 (1983) 237.T. Saito, Bunseki Kagak u, 39 (1990) 21 1.S. Coulom be and H. Sawatzky, Fuel, 65 (1986) 552.J.P.F. Palmentier, A.J. Britten, G.M. Charbonneau and F.W. Karasek, J. Chromatogr., 469(1989) 241.C.E. Ostman and A.L. Colm sjo, Fuel, 68 (1989) 1248.J.R. Mazzeo, I.S. Krull and P.T. K issinger, J. Chromatogr., 550 (1991) 585.M.C. P aputa-Peck, R.S. M arano, D. Schuetzle, T.L. Riley, C.V. Hampton, T.J. Prater, L.M.Skewes, T.E. Jensen, P.H. Ruehle, L.C. Bosch and W.P. Duncan, Anal. Chem., 55 (1983)1946.W.A. MacCrehan, W.E. May, S .D. Yang and B.A. Benner Jr., Anal. Chem., 6 0 (1988) 194.K. Jinno, J.C. Fetzer and W.R. Biggs, Chromatographia, 21 (19 86) 274.C. Borra, D. Wiesler and M. Novotny, Anal. Chem., 59 (1987) 339.F. And reolini, C. Borra, D. Wiesler and M. Novotny, J. Chromatogr., 406 (1987) 375.G. Grimmer, J. Jacob, K.W. Naujack and D. Schneider, Erdoel Kohle, Erdgas, Petrochem.,38(2) (1985) 8 2 (Synopsis 8504).L. Nond ek and V. C hvalovsky, J. Chrom atogr., 3 12 (1984) 303.J.S. T hom pson and J.W. Reynolds, Anal. Chem. 56 (1984) 2434.S.J. Marshman, Fuel, 5 (1990 ) 364.R.E. Rebbe rt, S.N. Chede r, F.R. Guenther and R.M. Parris, J. C hromatogr., 284 (1984 ) 211.R.G. Christensen and E.V. White, J. Chrom atogr., 323 (1985) 33.W.A. MacCrehan and J.M. Brown-Thom as, Anal. Chem., 59 (1987 ) 477 .


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HPLC and Column Liquid Chromatography

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